Bio 2 Flashcards

Question

How do you end up with less [Cl-]in?

Answer 1

Cotransporters that use the K+ gradient move Cl- out of the cell K+/Cl- cotransporter (KCC): transport 1 K+ and 1 Cl- out of the cell Also electrically neutral- not influenced by voltage If you want to inhibit (hyperpolarize) a neuron via Cl- channel activation, you need KCC to produce [Cl-]in << [Cl-]out, change expression of transporters, start expressing kcc, inhibit neuron Mature neruons Cotransporters that use k gradient to move cl out of cell

Answer 2

SLC12A4 (KCC1) SLC12A5 (KCC2) SLC12A6 (KCC3) SLC12A7 (KCC4)

Answer 3

Immature- expressing nkcc or ncc- moving cl inside Have a neuron with high cl conc inside, eq is -5, it wants voltage to be—5, but voltage is -60, when have glycine channel activated have to move out of cell to make it more positive Mature- start overexpress kcc transporter, causing low cl inside the cell, the glycine receptor will drive cl in when activating the glycine channels, making cell more negative

Answer 4

Na+-dependent Cl-/HCO - exchange system uses the Na+ gradient to move bicarbonate into the cell and protons out HCO - is part of a physiological buffering system crucial in the nervous system, were cells have little tolerance for fluctuations in pH CO2 + H2O H2CO3 H+ + HCO3- ↑ cytoplasmic [H+] promotes H+ efflux and HCO - influx ↑ in [HCO -] then shifts the equation to the left, further neutralizing cytoplasmic pH Recently- research explored neurons with ion channels activated by light Have animal and can expose brain, have neurons express these light activated ion channels Use genetic tools to express it, turn on light and activate it causing it to activate(potassium) or inhibit it (cl) Mature neurons apply light- will inhibit the neurons(cl rushes in to make more negative), but found them to become excited like an immature neuron, different parts of neurons have diff gradients Dendrites- cl is inhibotry Axons- cl depolarized- \\ Cl helps neurons deal with excess protons(low ph) Sodium bicarbonate transportes- transport into cell for exchange of one proton Borrws energy from sodium conc to move sodium bicarbonate into the cell, while taking out a proton Glycolysis- makes carbon dioxide Active neurons make lots of carbon dioxide, it mixes with water to become acid and lower ph This exchanger helps mitigate this problem by directly removing protons and by moving hco3 into cytoplasm, causing formation of carbonic acid, becomes buffer system that absorbs excess protons

Answer 5

Neurotransmitters are synthesized in the cytoplasm then actively transported into presynaptic vesicles After secretion, neurotransmitters are often taken back into cells in a process called re-uptake Extracellular transmitter clearance helps stop synaptic signals Replenishes/recycles transmitters Presynaptic ue calcium influc to release neurotransmitters that goes into postsynaptic, binds and causes confirmational change in postsynaptic cell Want to remove the neurotranssmiters- don’t have to constantly make it Reuptake- take neurotransmmiter back into vesicles

Answer 6

Vesicular transporters use proton (H+) gradients to move transmitters into the lumen of synaptic vesicles Vacuolar-type hydrogen ATPases consume ATP to concentrate H+ ions in vesicles The H+ gradient provides energy for transporting NTs: Transporters that package the nt use proton gradients This pump is under presynaptic vesicle and pumps nt into vesicles The conc gradient allows nt to move into the vesicles using proton gradient to push into vesicle Monoamines- have one amine group and have different roles, share transmitter- slc18 Glycine- inhibitory, glutamate, excitatory

Answer 7

Recently, the structure of the human V-ATPase was solved via cryo-electron microscopy Large complex with many subunits Used the toxin SidK from Legionella pneumophila (causes Legionnaires' disease), which binds and inhibits the pump, to isolate the protein from cell extracts for structural analysis Sidk- not rlly apart of atpase, it’s the pump that pushes nt into vesicles Legionella pneumophila- produces thw white protein that binds with high affinity and inhibits the pump Have macrophages- engulf pathogens via endocytosis Surrons them to trap cell \ Kill bacteria by using pump to pump protons into lysosomes- causing high acidic environment to kill them This bacteria prevents lysosomes from becoming acidic, lives in macrophage and reproduce there to keep infection going

Answer 8

Instead, membrane reuptake transporters use Na+, K+, and other gradients to transport transmitters from the extracellular environment into the cytoplasm Differnet neurons can be classified by which transporter they use Want to know where dopaingeric neurons are, has to be where recpetorsa are packaging serotonin into cell Neurons want to package vesicles and have to bring them back into cell Cellular proton gradient isn’t used by sodium potassium is Slc1- uses sodium and k gradient to bring glutamate back into cell During stroke- loose oxygen and loose gradient conc, neurons cant remove glutamate because the systems meant to remove it need the k/na gradient, cant remove it from the outside Sri- inhibit reuotakes of serotonin To keep the cell more negative use this pump This pump doesn’t juts address the sodium potassium Many other cells use this gradient, its trying to compensate for other exchnagers using the gradient to transport different materials, otherwisewould be trabsporting equal k and na

Answer 9

We have established that RMP is set by different ion gradients trying to pull Vm towards their equilibrium potentials Vm at rest can be calculated with the GHK equation The K+ gradient and EK wins because it has the larger conductance (GK >> GNa)... The cell membrane is packed with leak K+ channels (called 2-pore K+ channels)

Answer 10

Recall that the membrane is a capacitor, that can hold charges when a voltage is applied giving rise to membrane voltage Vm Capacitors can take up applied voltage, but cannot generate their own voltage Also recall that ENa and EK are voltage sources (i.e., batteries) that can deposit charges onto the inner and outer leaflets of the cell membrane Addition of positive charges (Na+) by the ENa battery onto the inner leaflet of the membrane makes Vm more positive Addition of positive charges (K+) by the EK battery onto the outer leaflet of the membrane makes Vm more negative Any current will exist as long as have voltage and current for it flow For ions- the movement dependent on membrane voltage and equilibrium Current will flow until vm= eq, then equals 0, there is no more current The ENa battery will continuously charge Vm until it has an equal and opposite voltage (i.e., Vm = ENa) Current flow from the ENa battery onto the Vm capacitor requires a conductance: GNa (Na+ leak channels) Similarly, the EK battery will continuously charge Vm until it has an equal and opposite voltage (i.e., Vm = EK) Current flow from the EK battery onto the Vm capacitor requires a conductance: GK (K+ leak channels)

Answer 11

Note that this type of circuit is referred to as an RC circuit Why? It has at least one battery, one resistor (R) and one capacitor (C) Also note that GNa and GK at rest are defined by the population of Na+ and K+ leak channels (NaLk and KLk) expressed at the cell membrane At rest, there are more K+ leak than there are Na+ leak channels (i.e., GK > GNa) Their drive depends on the number of leak channels they have Have two batteries that try to charge the membrane, whoever wins is the one with more leak channels During rest- sodium leak channels are low compared to potassium But during graded and action potentials- add more leak channels

Answer 12

In a sense, the GHK equation can be used to conceptualize what happens during cellular excitation (i.e., graded and action potentials) Sudden changes in GNa and GK (and other GIon) occur when different types of ion channels open and close e.g., ion channels that are voltage-gated, ligand-gated (NTs), mechanically-gated, heat-gated, pH-gated, etc. At rest we have certain number of sodium and potassium leak channels Other types of channels open, causing changes in sodium and potassium gradient At rest, only leak channels are open During excitation- add other channels into the mix, allowing the battery to drive the memrbarne closer to its equilibrium Their resting state is closed, after the stimuli, they close, returining membrane to normal However, there are two important caveats: 1) The GHK equation does not work when we are dealing with fast changes in Vm This is because capacitors, like the cell membrane, take time to charge!!! Thus, while conductances can change rather quickly, their effect on Vm does not happen instantly but takes time depending on the size/shape of a neuron 2) GNa and GK change very quickly during an action potential! Voltage doenst change immediately is a gradual experience

Answer 13

Excitatory potentials result from a temporary rise in GNa e.g., Synaptic glutamate or acetylcholine receptors/channels that conduct Na+ (NaLigC) Inhibitory potentials result from a temporary rise in GCl e.g., Synaptic GABA, Glycine receptors that conduct Cl- (ClLigC)

Answer 14

Temporary rise in GNa followed by a rise in GK All or none membrane response that occurs when graded potentials activate voltage-gated Na+ (Nav) and K+ (Kv) channels Describing the action potential as a function of changes in GNa and GK was the Nobel prize worthy discovery by Alan Hodgkin and Andrew Huxley

Answer 15

Recall the squid giant axon prep developed by JZ Young... Subsequently, Kenneth Cole invented the voltage clamp technique Can hold and change Vm to different levels during electrophysiological experiments! Early on, electrophysiologists realized that understanding the action potential required understanding how GNa and GK change during the course of the action potential As we stated, at rest GNa and GK are made up of leak channels However, when Vm is depolarized (e.g., due to an excitatory graded potential), voltage-gated Na+ and K+ channels open to contribute to GNa and GK Ena- voltage experiences different voltage levels- have to figure out what the channels are doing at eavch of tgose levels The problem: during the AP, Vm keeps changing, and therefore the conductance of voltage-gated Na+ and K+ channels that contribute to GNa and GK also keep changing Before the voltage-clamp, it was not possible to study and understand the properties of NaV and KV channels (GNa and GK) at different fixed voltages, since the voltage changes so quickly during the action potential

Answer 16

Alan Hodgkin and Andrew Huxley saw the potential in Cole’s voltage clamp technique as a tool for characterizing GNa and GK at different fixed voltages, and using this information to understand the action potential

Answer 17

The first triangle (A) is a voltage amplifier that reads Vm Amplifiers can read differences in voltage between two inputs Inputs are wires from either side of the axon cell membrane The outside wire is grounded, meaning it serves as a "zero" voltage reference The other wire goes inside the axon If the inside of the axon is -65 mV more negative that the outside (ground), then the amplifier reads -65 mV = Vm Amplifiers have two inputs, can tell you voltage between two inputs Oneworks it way into the axon The other it connected top outside and ground Tells you voltage across axon Amplifier a tells us voltage across membrane

Answer 18

A second voltage amplifier (B) receives one input wire from inside the cell, and the other from a grounded “command voltage” The experimenter sets the command voltage amplitude and duration (Vc) If Vc is the same as Vm, then the difference between the two inputs in amplifier B is zero, and hence the amplifier has nothing to output Has wire on inside of axon and has other wire connected to ground Between ground and input have command voltage- tells neuron what voltage to be Np difference in voltage = difference between vc and vm If Vc - Vm is positive (e.g., -9 mV - -65 mV = +56 mV), amplifier B injects positive depolarizing current into the axon, raising Vm This process continues rapidly and dynamically until the two inputs into amplifier B are equal (i.e., Vm = Vc = -9 mV) Amplifer b will inject current into axon, making votage= to command volytagwe If Vc - Vm is negative (e.g., -85 mV - -65 mV = -20 mV), amplifier B injects negative hyperpolarizing current This process continues rapidly and dynamically until the two inputs into amplifier B are equal (i.e., Vm = Vc = -85 mV)

Answer 19

A third voltage amplifier (C) measures the current injected coming from amplifier B Has a resistor (R) between its two inputs... Use Ohm’s law to read the current exiting amplifier B into the axon (V=IR): There is a known resistance (R) The C amplifier measures the voltage across its two inputs (V) When amplifier B injects current into the axon, there is a voltage drop across the resistor: we can thus record the current amplitude over time by converting voltage to current using Ohm's law: I = V/R Want to know what is happening to soidum and potassium Record their currents at the voltage Amplifier c records it Charges from b are passing through resisitor (known resistance)into measured current c Apply current across resistor will have voltage drop C records voltage drop as current passes through Recording voltage- can trabsfporm into current

Answer 20

A third voltage amplifier (C) measures the current injected coming from amplifier B Has a resistor (R) between its two inputs... Use Ohm’s law to read the current exiting amplifier B into the axon (V=IR): There is a known resistance (R) The C amplifier measures the voltage across its two inputs (V) When amplifier B injects current into the axon, there is a voltage drop across the resistor: we can thus record the current amplitude over time by converting voltage to current using Ohm's law: I = V/R Want to know what is happening to soidum and potassium Record their currents at the voltage Amplifier c records it Charges from b are passing through resisitor (known resistance)into measured current c Apply current across resistor will have voltage drop C records voltage drop as current passes through Recording voltage- can trabsfporm into current

Answer 21

Use Vc to hold voltage at -65 mV At the onset of the voltage change pulse, Vc is changed to -9 mV, so the voltage clamp amplifier B quickly injects current into the axon raising Vm to -9 mV Vc holds the Vm at -9 mV for 5 milliseconds (ms) Vc is then changed back to -65 mV, which returns Vm back down to -65 mV Amplifier b detects difference and injects current to change voltage for 5 seconds, then take the voltage back down- voltage steo

Answer 22

Use Vc to hold voltage at -65 mV At the onset of the voltage change pulse, Vc is changed to -9 mV, so the voltage clamp amplifier B quickly injects current into the axon raising Vm to -9 mV Vc holds the Vm at -9 mV for 5 milliseconds (ms) Vc is then changed back to -65 mV, which returns Vm back down to -65 mV Amplifier b detects difference and injects current to change voltage for 5 seconds, then take the voltage back down- voltage steo

Answer 23

A capacitative current An early current- voltage sodium channels A late current- potassium channels Notice how nice and flat Vm stays during the voltage clamp step, compared to the AP Can study GNa and GK at fixed voltages! Convert currents to conductances with Ohm’s Law (more to come) 5 ms

Answer 24

Used the voltage-clamp to record voltage-gated sodium and potassium channel currents at different fixed voltages Mathematically converted recorded currents to conductances Derived equations that mathematically simulate conductances at different voltages Used these equations to define the change in voltage over time over the course of an action potential

Answer 25

H & H separated the Na+ and K+ currents in the squid axon The capacitative current is the current required to change the voltage The early inward current is attributed to Na+ influx The late K+ outward current is attributed to K+ efflux We now know that these currents are caused by NaV and KV channels Needed to separate both sodium and potassium currents, did this by removing sodium See late current- this is potassium If remove potassium- only see the early current

Answer 26

Start at -65(holding voltage)- Amplifier b- charges mebrane Amplifier c- records changes Depolarize cell- insert into command voltage which goes to amplifier b- so it injects current into axon to change the voltage As current goes through resitor amplifier c records it- this is the capacitive current(spike) Amplifier a- tells us voltage of axon Wwhat happens in axon- axon is working with voltage gated sodium channels, they open and rush in to cause a depolorizatiuon - this is a problem for amplifier b- want to stay at injected current, sodium wil try to depolarize more- injects an equal and opposite current to hold constant voltage Depolarize more- current gets larger Every voltage step- dec driving force for sodium but conductance for sodium is larger At -30= activate more sodium channels, have larger conducatance but smaller driving force When reach certain amount- conductance will not change- no more channels can open and have decreased driving force At +54 mv don’t have current, no driving force so don’t see a current Can depolarixe so much but current reverses because driving force has reversed As you depolarize, inc driving force while inc activation of the channels

Answer 27

Recall that the goal was to observe GNa and GK at different fixed voltages, not INa and IK Hodgkin and Huxley thus transformed the recorded Na+ and K+ currents into conductance curves using Ohm’s law Notice that peak Na+ and K+ conductances increase with stronger depolarization, towards a maximal value To get conductance- use ohms law and rearrange for conductance Vm= command voltage Ek= equilibrium Conducatnce= how many channels opening at given voltaghe

Answer 28

The next step was to derive equations that could mathematically simulate the GNa and GK conductance curves at all fixed voltages GK(max) is the maximal conductance The parameter n changes according to both time (t) and the voltage- dependent parameter (τn) Plugging in values for time and voltage into the n equation, and then plugging the resulting n values into the GK(t) equation, simulates the K+ conductance curve Gk(t)- correlates with the potassium conductance = asymptote of potassium conductance Different tn- at different voltage steps and plug in increasing values of time Take sigmodial curve- like potassium conductance- shows many ion channels opening over time Processes that have 4 separate elements all influencing the ion channels independently Throygh this they could demonstrate the structure of the ion channels both time (t) and voltage (τn) Plugging in values for time and voltage into the m and h equations, and then plugging the resulting m and h values into the GNa(t) equation, simulates the Na+ conductance curve This isn’t sigmodial- rises fast and decreases fast One of the factors that contributes to potassium, doesn’t contribute to sodium After sodium conductance reaches max- sodium channels plugf themselves causing the decay H= decay M3= activation/growth

Answer 29

how H&H used this to simulate the action potential: Assumed a mild depolarization of Vm takes place at t=0.01 ms (e.g., an EPSP) Used this new value of Vm to determine new GNa and GK values at t=0.01 ms GNa(t)=GNa(max)m3h GK(t)=GK(max)n4 *Remember m, h and n change according to time and voltage *GNa and GK undergo a slight increase at t=0.01 ms Used the new GNa and GK values to determine new total Na+ and K+ currents acting on the membrane at t=0.01 ms We are ignoring leak currents, although H&H accounted for these in their calculations Calculated the change in Vm that resulted from the increased INa and IK values Gna- cant predict- steady state, doesn’t include time And doesn’t account for size of capacitance- a;;pwa you to measure ap Summed the change in Vm plus the previous Vm value to give Vm at time=0.02 ms Then repeated step 2… 2. Used the new value of Vm to determine new GNa and GK values at t=0.02 ms And repeated step 4… 4. Calculated the change in Vm that resulted from the increased INa and IK values And repeated step 5… 5. Summed the change in Vm and the previous Vm value, giving Vm at time=0.02 ms *By repeating steps 2-5 over and over again, H&H were able to simulate Vm, GNa, and GK curves over the course of an entire action potential!

Answer 30

Why does the inward Na+ current have a funny “U” shape, while the outward K+ current has a sigmoidal shape? It turns out that the conductance equations derived by H&H predicted the molecular properties of voltage-gated Na+ and K+ ion channels Why is sodium a U shape, Transmembrane helices that span the membrane 4 repeating structures M^3- predicts existance of alpha helix and contribution to opening channels- current caused by opening the ion channels- ion channels need 3 specific qualities to open- also have a fourth domain- activation must be complete by 3 particles H= inactivation- single particle causes this- fourth domain Can use flourescnet labeling to show individual activation- fourth domain doesn’t contribute to gating the channels K= inactivation- inbetween domain 3 and 4 Cant physically look at the channels and see what happens Sodium opens causing decrase of current(positive flowing in), open more sodium channels= more likely to be inactivation- doesn’t happen at same time, happens randomly but overtime more will become inactivated, causing current to reach zero\ Potassium doesn’t have inactivation, current stays open Potassium is similar, has transmembrane helices One protein just encodes this instead of four separate domains Sodium channels- the single protein is enough, but potassium need 4 of these proteins- 4 coltage sensing domains to give rise

Answer 31

Nav and Kv channels are part of the P-loop family of ion channels Nam ed after extracellular pore (P)-loops that project into the center of the folded protein P-loop regions are responsible for ion selectivity Need four of these pore structures- in middle= ion permanation pathway First contact site of ions as they leave the pore Amino acids in loop, determines which cations allow it to flow

Answer 32

All P-loop channels need 4 P-loops to be functional: Nav channels consist of four repeat domains (Domains I to IV) encoded by a single gene/protein Kv channels also consist of four domains, but these are encoded by a single-domain gene (i.e., four separate proteins must come together to form a complete channel) Need four p loops for the channel to work In sodium- single protein encodes for the 4 domains- enough to form a working channel Potassium- need 4 proteins to come together and make the domains to have a working channel In centre have a core- made by the four helixes Sodium- has same structure as potassium channel, but all encoded by same gene Each domain/subunit possess 6 transmembrane alpha helices a.k.a. segments 1 to 6 or S1-S6 S5 and S6 helices make up the lining of the ion channel pore These possess the extracellular P-loops Define ion selectivity (i.e., Na+ vs. K+ vs. Ca2+) S1-S4 make up the voltage sensors of the channels Pore loops used to sleect fpr different cations The S4 helices bear positively-charged amino acids Counterbalanced by negatively charged amino acids in S2 and S3 helices At RMP, +ve charges outside and -ve charges inside the membrane (on the capacitor) keep S4 helices within the plane of the membrane Pore loops closest to ion permeation pathway, have amino acids that select for ions In phospholipid bilayer- to have positive energy in ion channel, needs to be stabilized

Answer 33

Upon membrane depolarization, S4 helices protrude outward This causes a conformational change in the channel, opening the pore This process is referred to as channel activation In the squid axon, activation of numerous Nav channels causes the onset of the inward early current The charge distribution becomes diminished, loose the conc gradient Before had enough negative inside to keep it down Because of depolorization- pulls open pulling on s4 helices opening the channel Rise in current= activation

Answer 34

Nav channels have a plug in the cytoplasmic linker between domains III and IV that quickly plugs the pore This process is known as channel inactivation In the squid axon, inactivation of numerous Nav channels causes decay of the inward early current When activated, more likelyt to be plugged Causing the reverse of current

Answer 35

Kv channels undergo activation, but more slowly than Nav channels Also, Kv channels in the squid axon do not inactivate Note: various other Kv channel types, such as A-type Kv channels, do undergo inactivation (i.e., some Kv channels have a plug mechanism) Slwoer to respond Go through same processed S4 tugs on s5,s6 to open the gate Once channel activated don’t get plugged Repolorization- causes normal conc gradient, causing s4 to be pulled down and causing gate to close In dquid they don’t inactivate

Answer 36

The channels must become activated by depolarization This temporarily increases GNa and GK compared to when only leak channels are active There must be driving force for ion flow through open channels To move current- need to activate channels via depolorization, causing more pathways for ions to pass the membrane and need driving force These currents are product of actication of channels and the driving force

Answer 37

In the squid axon, H&H were recording the sum current though thousands of open channels Referred to as “macroscopic currents” How do macroscopic currents – (result of multiple channels )relate to single channel currents? Enter Erwin Neher and Bert Sakmann… Invented the patch clamp technique Permits recording of single ion channel currents! Need to see single channel current Macroscopic currents are the sum single channel currents of all Nav and Kv channels present in a recorded neuron or axon Opening of channel is random- happens shortly after Individual current- unpredictable at macroscopic level- have same shape of current Single Kv channel currents add up to the macroscopic current with slow activation and no inactivation Shortly after activation, more delayed than sodium Depolarization causes single channels to open and close over time Like macroscopic currents, down means inward cation current, up means outward cation current

Answer 38

Microscopic current- one channel Sodium- open in 1-2 ms when activated Depolorized- voltage sensor opens channel and inactivated ater 1 ms, stays plugged after depolorization, opens once return to normal Channels open in 1-2 ms, causing depolorixation, then inactivated Can either pull patch away- inside out Or do whole cell configuration and pull away to do outside out Patch-Clamp Methods: The patch-clamp technique is used to study these tiny currents at the level of individual ion channels. There are two primary configurations relevant here: • Inside-Out Patch: In this configuration, a small piece of the cell membrane is pulled away with the inner surface of the membrane facing outward. This setup allows researchers to manipulate the intracellular environment and observe how it influences the channel. • Outside-Out Patch: In this configuration, a whole-cell configuration is first established, and then the pipette is pulled away to reseal the membrane with the extracellular side facing outward. This setup is ideal for studying the effects of extracellular signals or drugs on channel activity. 4. Whole-Cell Configuration: Sometimes, the entire cell membrane is clamped to measure the total current flowing across all the channels in the membrane. This “whole-cell” configuration gives a broader view of ionic currents but lacks the precision of single-channel measurements. In summary, Na⁺ channels open and inactivate quickly (within milliseconds), playing a central role in action potential initiation and propagation. Patch-clamp techniques allow researchers to study these currents either at the single-channel level (microscopic currents) or across the whole cell, giving insight into the channel’s behavior and response to various conditions.

Answer 39

Stronger voltage/ depolorization= larger the current Depolorize with strong voltage, more sodium channels will open However, with even stronger depolarization, they found that peak INa maxed out, and began to decline Eventually, peak INa reached zero and reversed Once reach peak current, increase voltage anymore, the effect will reverse ] Why- bringing closer to equilibrium potential, decreasing the driving force- eventually when get to ENA, no driving force= no curren6 Why? At first, Na+ currents got bigger because increased depolarization activated more and more Nav channels (i.e., ↑GNa) But note how each voltage step incrementally decreases driving force for the Na+ current This would make currents shrink, not grow However, at these -ve voltages: ↑GNa > ↓DF Once all Nav channels have become activated (GNa maxes out to a constant), peak current will shrink and then reverse as Vm approaches and surpasses ENa (i.e., only DF influences INa: Vm - ENa)

Answer 40

Identified sodium channel mutation causing pain and delayed limb impairement This is expressed in peripherial system 2 children had it, tracked genomes and those who didn’t Had mutation. In sodium channel domain 2- have glycine but in these people have arginine Amplified activity of channel, made current way larger Driving stringer action potentials and more often, send excess information

Answer 41

For the K+ currents, both GK and DF increase with each depolarizing voltage step… Increased depolarization activates more and more Kv channels, increasing GK Ek= negative As you activate potassium channels, inc driving force, causing inc activation For the K+ currents, both GK and DF increase with each depolarizing voltage step: Increased depolarization moves Vm further and further away from EK, increasing DF

Answer 42

Single nucleotide mutation- valince to thymine mutation- causes this mutation Lose of function, current is delayed and diminished

Answer 43

Neher and Sakmann received the Nobel Prize in Physiology or Medicine (1991) for their invention of the patch clamp technique https://www.nobelprize.org/prizes/medicine/1991/press-release/

Answer 44

Nav channels are Pore-loop (P-loop) channels that conduct inward Na+ currents that depolarize Vm during the rising phase of the action potential Kv channels are also P-loop channels that become activated after NaV channels, and conduct outward K+ currents that strongly repolarize Vm Nav channels, Kv channels, and indeed all P-loop channels have their evolutionary origins in bacteria… Action Pore loop channels select for ions- sodium potassium Ena battery drives voltage up to equilibtrium, then potassium channels open causing potassium to drove membrane back down What is it about these channels that let them discriminate? P-loop channels (or P-loop ion channels) are a family of transmembrane proteins that function as ion channels, allowing specific ions to pass through the cell membrane. They are characterized by the presence of a P-loop (or pore loop), a structural motif that forms part of the channel’s ion-conducting pore. The P-loop plays a crucial role in determining ion selectivity and conducting properties of the channel. Key Features of P-loop Channels: 1. Structure: • P-loop channels typically have a repeating motif in their transmembrane domains. • The P-loop is located between two transmembrane helices (usually the fifth and sixth helices in many cases). • The P-loop folds back into the pore to form the selectivity filter, which ensures only specific ions can pass through. 2. Function: • They mediate the selective passage of ions like sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), or chloride (Cl⁻) across the cell membrane. • These channels are critical for processes like electrical signaling in neurons and muscle cells, osmoregulation, and maintaining ionic gradients. 3. Types of P-loop Channels: • Voltage-Gated Ion Channels: Channels like voltage-gated potassium (Kv), sodium (Nav), and calcium (Cav) channels. • Ligand-Gated Ion Channels: Activated by neurotransmitters (e.g., AMPA, NMDA receptors). • Two-Pore Domain Potassium Channels (K2P): Involved in background potassium conductance. • Cyclic Nucleotide-Gated Channels (CNG): Activated by cyclic nucleotides like cAMP and cGMP. 4. Ion Selectivity: • The P-loop contains a specific sequence of amino acids that determines the ion selectivity of the channel (e.g., the GYG motif in potassium channels). 5. Clinical Significance: • Mutations in P-loop channels are associated with diseases such as epilepsy, cardiac arrhythmias, and channelopathies. • They are important drug targets for treating neurological, cardiovascular, and other disorders. Example: Potassium Channel (Kv): • In voltage-gated potassium channels, the P-loop creates a selectivity filter that allows only potassium ions to pass while excluding smaller ions like sodium due to precise geometric and electrostatic constraints.

Answer 45

P-loop channels emerged billions of years ago in bacteria Initial prototype: inward rectifying potassium channel (KIR channels) 2 TMHs separated by an extracellular P-loop that dips back into the membrane Like all P-loop channels, 4 subunits make up a complete channel (i.e., tetrameric) The four P-loops project key amino acids into the pore to define K+ ion selectivity Make up the “ion selectivity filter“ motif/structure- specific amino acids give rise to selectivivity KIR homologues are found in all cellular lifeforms, including humans Between TMH- has pore loops, 4 of them ‘the pore loops have specific amino acids, allow the channels to be selective The channels are ubiquiotous- found in all cellular forms

Answer 46

Evolutionary changes in the original KIR channel structure gave rise to many other P- loop channel types: Tandem duplication of a KIR gene gave rise to 2-pore K+ leak channels (K2P channels) Dimeric → only need 2 proteins to make a channel Major contributors to K+ leak currents Thus, critical for setting RMP (↑ GK) K2P channels are only found in eukaryotes During evoloution the gene that encodes it, duplicated itseld, to give rise to 2 pore demains- connected together Each protein codes 2 pore loops Major channels in cells that give rise to leak potassium conductance- gives to rmp- many of these in channels, main conductors of leak potassium channels Only found in eukaryotes- happened after diversion of bacteria and eukaryotes

Answer 47

Although K2P channels are “leak” channels, they are not as boring as their name implies: They exhibit gating/enhanced current flow in response to numerous stimuli including pH, membrane stretch, and temperature Name can be misleading- leak implies always open but conductance isn’t constant, exhibit gating, currents grow or shrink in response to factots like ph, temp Enhancing or dimninishing conducatnace Modulated by these factors 2 configurations- upstate vs down state- down state is more conductive

Answer 48

Inversion across the membrane, plus addition of a transmembrane helix gave rise ionotropic (ligand-gated) glutamate receptors AMPA, NMDA, and Kainite receptors of vertebrates- 3 main classes Crucial roles in excitatory synaptic transmission i.e., Glutamatergic synapses Like all P-loop channels → tetrameric Ancient proteins that appear to have originate in bacteria- may in bacteria, happened before eukaryotes Coming up: guest lecture by my PhD student Anhadvir Singh Same inward potassium structure flipped itself- became intracellular and added extra helix Created ionotropic glutamate receptords- bind glutamate excitatory glutamtergic signalling- use glutamate as transmitter post synaptic cells have these recpetords Need 4 of these proteins- tetrameric ligand gated chanbnels Chen- found bacterial channel with potassium and glutamate channel- link between the transition

Answer 49

Fusion with a separately-evolved protein in bacteria, the voltage sensor (S1-S4), gave rise to voltage-gated channels i.e., Kv channels, Ca2+-sensitive SK channels Some lost selectivity for K+ to conduct Na+ and/or Ca2+ (mostly in eukaryotes) Hyperpolarization-activated cyclic nucleotide- gated channels (HCN) Activated by hyperpolarization, cAMP, and/or cGMP Transient receptor potential channels (TRP)- discovered in fruit flies sensory research, ubiquiotous in animals- eat spicy food- activates this, many of them have voltage sensor, but lost voltage sensitibvity Still have pore loops and 4 TMH Most lost voltage sensitivity Activated by various chemicals, mechanical stimuli (touch), and temperature/heat Important roles in sensory biology Voltage gated potassium channels have a pore loop- in evoloytion this protein the voltage sensor proteins, fused with the protein of the pore to create voltage gated potassium channels Calcium sensoitive potassium channels- class of voltage potassium channels Once this evoloutionary event occurred- evolved into HCN- HCN- have in hearts and muscle cells- regulate heartbeat- regulated by camp- open or closes channels- modulate conductance and currents to control heart beat

Answer 50

The voltage sensor domain appears to have originated as a separate ion channel... Humans have 2 genes for voltage sensor proteins (S1-S4 helices) without P-loops: Transmembrane Phosphatase with Tensin Homology (TPTE and TPTE2) These conduct voltage-sensitive outward H+ currents gated by pH Idea that evoloution of voltage sensor and the pore structure are involved with sepetayte proteins Red alpha helices- evolved on own as separate ion channels and these channels then fused together and over time lost voltage sensor capability We have two genes which look like voltage sensors but lack pore structure- exisat as own ion channel- (TPTE and TPTE2) S1-s4= homogolous to voltage gated channel Voltage sensor- alpha helices- need s4 to detect voltage - pos charged amino acids detect charges to allow s4 helices to open during depolorizatiuon Alignment between voltage gated potassium channel of s4 helix we have amino acids with repeating pos charge because of amino acids Then have TPE- which have the s4 but are ion channels- they produce outward current that gets bigger when pipette soloution is acidic The currents we are seeing are protons- conduct protons in response to acidic cytoplasm These proteins conjugated to enzyme

Answer 51

Digression on the VSD... Key point: mutations can “re-awaken” voltage sensor pores Mutations in the voltage sensor of P-loop channels can result in leak cation currents that are toxic to cells… e.g., Arginine→Histidine mutation in the domain II S4 helix of Nav1.4 creates a leak proton current Abberant voltage sensor currents can also conduct Ca2+, which is toxic to cells Voltage sensor once ion channel- Have mutations that reopen conductance of ion channels Mutations occur in pos charged amino acid WT- have sustained current after fast step R1H- secondary current going to voltage sensor- causes it to be conductance pathway, causing pathology Mutations on voltage gated channels in voltage sensors that open conductance pathways

Answer 52

Addition of a transmembrane helix to the N-terminus of the KV/SK/TRP channel structure gave rise to BK channels Like SK channels, BK channel gating is regulated by rises in cytoplasmic Ca2+ This structure makes diverse ion channels Adding additional helix- creating BK channels- calcium sensitive potassium channels- become activated by inc calcium- during excitation- calcium controls open and closinh

Answer 53

Tandem duplication of a Kv-like channel gave rise to Two Pore Channels (TPC) Found in plants and animals Dimeric channels (two separate proteins make a functional channel) Cationic (Ca2+, Na+)- conduct cations Release Ca2+ into the cytoplasm from acidic organelles called endo-lysosomes(involved in breaking down bacteria) present here to conduct calcium from here to cytoplasm TRP- underwent duplictation- single gene encodes 2 proteins- need 2 proteins now- formed TPC channels

Answer 54

6. Tandem duplication of a TPC-like channel gave rise to 4-domain channels One protein makes a channel Voltage gated Na+ channels (Nav) Depolarization during action potential Voltage-gated Ca2+ channels (Cav) Synaptic transmission, muscle contraction, gene expression- drive exocytosis, contraction of muscles NALCN → Na+ leak channel Contributes leak Na+ current that helps set RMP in many neurons (remember Hodgkin and Horowicz?)- berstaein thought main contributor to action potential is voltage gated sodium channels, this channel also at play Yeast Ca2+ channel 1 (CCH1) Found in fungi Considered homologous to NALCN- has 3 proteins presnt in fungi 4-domain channels have only been documented in eukaryotes There are vast lineages of undescribed 4-domain channels in eukaryotes Underwnet anothore duplication to form 4 domain channels One protein makes channel one makes pore loops Related to atp and voltage gated sodium channels NALCN- discovered in 80s- cloned calcium and sodium channels, found genes that appeared similar, couldn’t record current through it until 2020 Cch1- counterpart in yeast to NALCN- associated with virulence of bacteria evoloutionary related to NLCN

Answer 55

The NALCN “channeolosome” consists of 4 subunits NALCN is also not “boring”; its activity is highly modulated to affect RMP and cellular excitability NALCN- structure of the channel through crytoecrome Has 3 subunits - fungi have these subunits How it operates neurons Like KTP- not just static regulated by other factors NALCN regulated to inc or dec activity inc sodium leak- causes depoloriaztion- intracellular paths activate NALCN to cause depolorization- can change to quiescent neuron tonically active neuron- fires AP a lot Can inc firing for a while

Answer 56

Mutations in the pore region: Congenital contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD) Mutations in the voltage sensor regions: Infantile hypotonia, with psychomotor retardation and characteristic facies 1 (IHPRF1) 16 Mutations in NALCN affect diseases The blue mutations- clustered around- ion conductance pathway, associated with CLIFAHDD- causes developemental condition- bent hands irregular brain developemen- cause excess depolorization IHPRF1 mutation around voltage sensor, causing dampening of activity- not depolorizing as much as should Have mutations that make it hyperactive or mutations that cause dampening

Answer 57

The X-ray crystal structure of a KIR-like channel revealed the mechanism for K+ selectivity Specific arrangement of carbonyl oxygen atoms in the selectivity filter mimics the hydration shell of K+ ions Makes it energetically favorable for K+ ions to strip their H2O molecules and enter the pore Conditions are unfavorable for other cations like Na+ or Ca2+ Crystal structure should us how conduct ion channels- how selectivity works\ Have k ion in soloution- put charged ion in polar water- oxygens of water rearrange around atom creating a hysration shell- specific to diameter- k has larger ionic radius- positioning of oxygen is different K used this during evoloytuon to select for k instead of na Positioned in same distance as hydration channels- k selectivity mimics hydration shell of k- can enter pore without lots of energy- strip water and enter pore Sodium has different diameter- doesn’t match hydrsation channel not favourvale to lose water Goes from hydrated state, dehydrates and interacts with oxygen to enter ion Enter vestibule become hydrated again and enters gate Key insight: K+ ions are selectively (and temporarily) stripped of their water molecules as they pass through the K+ channel selectivity filter Allow through hydration but don’t let water in- just k

Answer 58

Roderick MacKinnon received the Nobel Prize in Chemistry (2003) for discovering the mechanisms for K+ channel ion selectivity Discovered this The carbonyl oxygen on amino acid backbone- positioned in same orientation as water molecule- select for potassium

Answer 59

Although structures of NaV channels are now available, the exact mechanisms for ion selectivity are still unclear… Unlike KV channels, Na+ ions likely pass through the pore in a semi-hydrated state Like KV channels, amino acids in the selectivity filter select for Na+ (see next slide) Altogether, pore dimensions and spatial positioning of selectivity filter amino acids are optimized for interactions with Na+ ions Sodium channels are more complicated- have abstract notion of selectivity Have voltage gated sodium channel- Smaller proteins crystal technique works but hardder for larger proteins CRY_EM- has enhanced over the years and continues to improve Potassium channels easy to visualize In sodium- only have partial dehydration- don’t know how much dehydrated Have 3d structure and charged aspect in pore to cause ion selectivity

Answer 60

Nav channel selectivity filter in humans and most invertebrates: DEKA Glutamate (E) and aspartate (D) residues have negatively charged side groups that attract cations The positively-charged lysine (K) seems to be key for Na+ selectivity These channels are extremely selective for Na+ Structure of sodium channel Purple- space filled bubble showing pore Selectivity filter has 4 amino acids- each domain has different amino acids- form narrowest part of conduction pathway, here is where dehydration occurs- why only sodium and not cations- D and E- have neg charged carbon and oxygen- attracting cations Lysine in d3- is critical for Na Calcium has 3, not lysine Lysine kicks calcium out making selective to na only Asparate and glutuamte- have carboxyl group- coorfdinate cations- projecting into pore to cause selectivity 4 amino acids projected into core causing selectivity Purple= pore space

Answer 61

Nav channel selectivity filters in early-diverging invertebrates (e.g., jellyfish) are different: DKEA channels are Na+ selective DEEA channels are more not selective for Na+, and are sometimes highly selective for Ca2+ Why? Their selectivity filters resemble those of voltage-gated Ca2+ (CaV) channels… Priginally sodium channels didn’t have this filter- had dkea- has neg charge highly calcium permeable First evolved as calcium channels then changed amino acids to cause true sodium channel- happened in our group of organisms- with bilateral symmetry Glutumate to lysine In jellyfish- independently evolved na channels- altered second domain Before we didn’t have dkea- hughly calcium permeable Sodium channels were originally calcium channels- they got a lysine in d3 to form sodium channels Carboxyl from D and E- create binding affibity for calcium Connect ocgen to protein- hogs electrons has a negative charge- attracting cations- esp calcium l

Answer 62

Bilaterians evolved Na V1 and Na V2 type channels, while non -bilaterians only possess ancestral Na V type channels. • True Na + -selective sodium channels evolved via glutamate (E) to lysine (K) mutations in bilaterian Na V1 channels and some (but not all) cnidarian Na V channels. Bilaterians- bilateral symettery Protosomes- non vertebraes Non-bilaterians- primitive animals Choanoglfagellates- closet to animals 2 groups of sodium channels- in bilaterians nv1 vs nv2 evolve from mutant We lost nv2 Non bilaterians- ancestral sodium channek Nv2 have deaa They eviolve from glutamate to lysine channel mutation- happened in nv1 - medusozoan- have mutation in nv2 channels to become sodium chanbels

Answer 63

Ca2+ selectivity (Cav channels) involves a high-affinity Ca2+ binding site made up of key acidic amino acids from the selectivity filter Cav1 and CaV2 channels (will learn more about these later): Four glutamates (selectivity filter motif = EEEE) Cav3 channels (also, more later): Two glutamates and two aspartates (selectivity filter motif = EEDD) Position carbonlys to be in pore- to select for calcium- calcium has high affinity here to bond w oxygen, calcium repels other cations E and D carboxyl oxygens create high-affinity binding sites for Ca2+ (recall: Ca2+ ions have a high affinity for oxygen atoms) Bound Ca2+ repels other cations away from the pore, thus preventing Na+ and K+ permeation (i.e., Ca2+ blocks Na+ and K+ permeation)

Answer 64

UCSF ChimeraX demonstration: visualizing an NaV channel protein structure Next-generation molecular visualization program Free to use for academia https://www.cgl.ucsf.edu/chimerax/ Can search for and download structure files from the Protein Data Bank website: https://www.rcsb.org/ e.g., NaV1.5 structure: PDB number 7FBS Using ChimeraX, you can visualize, analyze, manipulate, and create images of protein structures Mechanisms for ion selectivity of P-loop channels • UCSF ChimeraX demonstration: visualizing an NaV channel protein structure • Next-generation molecular visualization program • Free to use for academia • https://www.cgl.ucsf.edu/chimerax/ • Can search for and download structure files from the Protein Data Bank website: https://www.rcsb.org/ • e.g., NaV1.5 structure: PDB number 7FBS • Using ChimeraX, you can visualize, analyze, manipulate, and create images of protein structures

Answer 65

Highly selective for K+ ions: • K2P, IR, KV, SK, and BK • Highly selective for Na+ ions: • NaV1 (DEKA), some cnidarian NaVs (DKEA) • Highly selective for Ca2+: • CaV (EEEE, EEDD), NaV2 and ancestral NaVs (DEEA) • Non-selective cationic (Na+, K+, Ca2+): • iGluRs • HCN • TRP • TPC • NALCN

Answer 66

H&H's pivotal studies provided avenues for understanding other aspects of the action potential How much depolarization is required for an action potential to occur? i.e., What is the action potential voltage threshold? Graded- computes the strength of stimulation- help discern what is important

Answer 67

Let's look more closely at action potential threshold: A synapse releases NT to evoke an excitatory synaptic potential (EPSP) By the time the graded EPSP reaches the axon, it is below threshold See strong pulse in area, local electrical event, but it needs to head across dendrites and neuron to the axon to reach voltage gated sodium channels Put two electrodes- charges diffuse and absorbed by membrane A second synapse is much closer… When this EPSP reaches the axon, it depolarizes Vm above the action potential threshold, so we get an action potential Closer= easier to create ap, depolarization doesn’t travel as far Note: The region of the axon where APs initiate is called the axon initial segment (AIS)- far into axon, space where AP starts, propogates from here, The AIS can be variable in its location, even in the same neuron where its location can be modulated by neurohormones, not static, modulation by neurons moves site, moves through the axon

Answer 68

In the axon, Nav and Kv channels are "waiting" for a depolarization Threshold voltage is a tipping point, need to surpase the voltage where enough axonal Nav channels become activated such that they can overcome the outward K+ leak currents (and currents though any Kv channels that might be open as well) AP- sodium activated opens more na channels and is explosive- all channels get activated, driving rising phase, to do this they have to overcome the k current, k channels are trying to prevent that and generate response to counteract Spodium channels start to close down while the potassium channels start to wake up Once over the tipping point, the depolarizing Nav currents activate even more Nav channels, creating an all or none response This causes the rising phase of the action potential Threshold voltage is a tipping point, where enough axonal Nav channels become activated such that they can overcome the outward K+ leak currents (and currents though any Kv channels that might be open as well)

Answer 69

However, Kv channels are like sleeping giants- contribute to falling phase- rise of k conductance They are slower to respond to depolarization from EPSPs and Nav channels Once activated, they conduct massive outward K+ currents that quickly repolarize the membrane (i.e., cause the falling phase of the action potential) NaV channel inactivation also (passively) contributes to the falling phase- not pulling it down but inactivating and passively allowing the k conductance to hyperpolarize Stay open after repolarization- causing undershoot, large k conductance and current However, Kv channels are like sleeping giants Furthermore, activated Kv channels temporarily drive Vm below RMP to generate the AP undershoot, a.k.a. the afterhyperpolarization Rising phase- sodium conductance but then undergo inactivation- coinciding with falling phase, as this happens k conductance is high At end of AP, sodium conductance turned off but still residual potassium conductance- undershoot

Answer 70

There is an important point to address with respect to AP threshold: The voltage of AP threshold is not static, not fixed, can have different threshold under conditions It can change significantly depending on the RMP of the neuron, expression levels and gating properties of Nav, Kv and other channels in the axonal membrane- can change activity, changing threshold, the degree of inactivation of NaV channels at RMP, amplitude and duration of conductance and even temperature- proteins move more freely at high temps, faster at gating at higher temps

Answer 71

To understand how threshold voltage works, we need to revisit the activation and inactivation properties of NaV and KV channels If we plot the peak conductance of NaV and KV channels at different depolarizing voltages, we get corresponding “conductance curves” Sodium conductance- peak at top of AP, k max conductance= at end of AP Plot this data on a graph to understand conductance changes as a product of the AP Causes s shaped curve- conductance curves

Answer 72

Q: What gives rise to Na+ and K+ conductances in a squid axon or neuron?- opening of the na and k voltage channels Populations of Nav and Kv channels present in the membrane and how they respond to voltage These conductance curves thus reflect the activation properties of pupolations of NaV and KV channels present in the membrane- tells us how population responds to depolarixation

Answer 73

Q: Why do the peak Na+ and K+ conductances plateau at voltage steps greater than +20 mV?- the inc conductance stops, why, there are a fixed number of channels, cant get any more conductance, any inc voltage will not open more channels The membrane bears a finite number of Nav and Kv channels We max out the # of channels that can be activated at voltages >20 mV

Answer 74

Exercises for interpreting the conductance curves: Say RMP or the holding voltage (Vm) is at -80 mV Here, a depolarizing voltage step to -60 mV does not open (activate) Nav or Kv channels (no change in conductance) Interpreting the curves: Say RMP or the holding voltage (Vm) is at -80 mV What voltage step is required to activate/open ~50% of the Nav and Kv channels?- find where along x axis, conductance is halfway i.e., Turn on 50% of the respective conductances ~-10 mV Interpreting the curves: RMP or the holding voltage (Vm) is at -80 mV What voltage step is required to activate/open ~75% of the Nav and Kv channels? ~0 mV- again look at graph Interpreting the curves: RMP or the holding voltage (Vm) is at -80 mV Do we get more NaV channels if we depolarize to +50 mV compared to +30 mV? No Why?- have same conductance, at 100% activation, cant open more

Answer 75

Key point: Conductance curves can be interpreted as the percentage of channels from the population that open when we quickly depolarize Vm from a negative voltage to different depolarized voltages This is how you need to interpret the X axis in the plot See a graph like this- at top is 100% activation- the x axis is a step voltage- start at zero and step up to that voltage- step to -60 what happens etc This graph not over time- steady state voltage 20mv- 100% channels activated for both- but what channels open first- the sodium channels- first responderfs to depolarization letting them cause ap, however voltage responses are causing similar conductance patterns This makes sense of you think about how the Nav and Kv voltage sensors work Stronger depolarizations make S4 helices slide out of the membrane more efficiently, opening channel- this is probalistic- depolarize further- activate more, get highe rprob of opening all channels

Answer 76

By holding Vm through a series of voltages (RMPs), and then stepping to a fixed depolarizing voltage to open Nav channels (Step Vm), H&H were able to characterize the Nav channel steady state inactivation properties Important points: the holding (steady state) voltage is considered to emulate the RMP potential of a cell, hold for extended period of time. If RMP is very negative (-100 mV), few/no NaV channels are inactivated and hence most/all are available for activation If RMP is depolarized (-50 mV), only 1/2 of the NaV channels- less available bc inactivated are available for activation Held MV for extended periods of time and took single voltage step to cause inward sodium current Holding at -100, hard to get sodium activation and do depolarization- none are inactivated will get max amount of channels at largest amp of current bc so negative As inc voltage- sodium channels undergo inactivation, more depolarized voltage, more inactivation, less channels open At 0- all inactivated cant opne So, now we have the voltage properties of Nav and Kv channelactivation, and Nav channel inactivation % available- how rmp affects ability for ap x= starting voltage

Answer 77

So, now we have the voltage properties of Nav and Kv channelactivation, and Nav channel inactivation % available- how rmp affects ability for ap x= starting voltage Important notes: The % available curve (left) shows us how RMP influences the number of Nav channels available should a depolarization take place Thus, the x axis reflects “starting” voltages Depolarization will activate Nav and Kv channels according to the GNa and GK conductance curves on the right Here, the x axis reflects step voltages

Answer 78

Notice how the conductance curves for NaV and KV channels are highly similar However, when you depolarize to a given voltage, NaV channels will respond faster than KV channels because they have faster activation kinetics

Answer 79

Scenario 1: A neuron's RMP is at -80 mV Resting voltage- use graph on the left 90% of Nav channels are available If a +30 mV EPSP reaches the axon Vm reaches -50 mV- use percent activated now Very few Nav channels become activated (say roughly 2% of the 90% available) In our "simulation", not enough to overcome the K+ leak The EPSP fades as a graded potential

Answer 80

Scenario 2: A neuron's RMP is at -80 mV 90% of Nav channels are available If a +40 mV EPSP reaches the axon Vm reaches -40 mV More Nav channels activate (~5% of the 90% available) Now, we get enough activation to overcome K+ leak Activated NaV channels depolarize the voltage further, causing an explosive, positive feedback activation of more Nav channels leading to an AP

Answer 81

Scenario 3: A neuron's RMP is at -60 mV 60% of Nav channels are available Note how some K channels are activated (~2% of 100%)- don’t ever fatigue If a +20 mV EPSP reaches the axon Vm reaches -40 mV Some Nav channels activated (~5% of 60% available) However: we started with less Nav channels (60%), so we get less actual NaV channel current for overcoming K+ leak and the few open Kv channels Notice how before, Vm = -40 mV generated an AP, but this time it didn’t Hence the threshold voltage has changed- its higher At same depolarizing voltage have different activation- less channels available the same depolorization voltage will cause less channels to be open

Answer 82

Scenario 6: A neuron's RMP is at -30 mV Some K channels are activated Only 10% of Nav channels are available If a -40 mV IPSP reaches the axon Vm drops to -70 mV Some Nav channels lose their inactivation (i.e., recover from inactivation) After the IPSP ends, the Na+ leak channels depolarize Vm again to -30 mV This activates Nav channels, leading to an AP This process is inhibit neuron resulting in AP known as post-inhibitory rebound (PIR) excitation in cells with depolarized RMPs

Answer 83

The % available curve shows us how RMP influences the number of Nav channels available for an AP Depolarization from RMP will activate Na and K channels according to the GNa and GK conductance curves Approximates the percent of channels that get activated

Answer 84

NaV channels become inactivated during an action potential During the afterhyperpolarization/undershoot (driven by KV channels), the negative membrane voltage promotes recovery from inactivation of Nav channels The time required for this recovery, such that we can get another AP, is referred to as the refractory period

Answer 85

There are two types of refractory periods... Absolute refractory period: Period of time where no amount of stimulation can generate a second AP Due to excessive Nav channel inactivation Relative refractory period: Period where we can generate another AP…strength But, it depends on the strength of stimulation after the first AP At first, we need a strong stimulus to get an AP since we have few available Nav channels As more Nav channels recover, less depolarization is required for a second AP Called relative bc need larger current

Answer 86

Why are refractory periods important? In the nervous system, information is often encoded by AP frequency Stronger signal = higher frequency e.g., retinal ganglion cells e.g., nociceptive neurons Sensory coding We also control our muscle this way Higher frequency = stronger contraction

Answer 87

The absolute refractory period determines the maximum AP frequency of a neuron Given the strongest stimulus possible, how fast can APs fire in succession? e.g., for a 1 ms absolute refractory period, max. freq. = 1000 APs/second or 1000 Hz The relative refractory period determines the relationship between stimulation strength and AP frequency A neuron with a faster (shorter) relative refractory period will have a higher AP frequency for a given stimulus than one with a slower relative refractory period

Answer 88

As always, the in vivo situation is much more complex… There are many types of Nav and Kv channels (especially in vertebrates) Humans have 9 Nav channel genes And 40 Kv channel genes! DRG- sense pain project from periphery into spinal cord- na v 1.7-1.9 Different sodium channels have different properties, different responses to stimulation As always, the in vivo situation is much more complex… There are many types of Nav and Kv channels (especially in vertebrates) Humans have 9 Nav channel genes And 40 Kv channel genes! Can have very different activation and inactivation properties Functional differences would influence AP threshold and refractory period As always, the in vivo situation is much more complex… Different neurons can express different combinations of Nav and Kv channels! There are also many other types of ion channels… the electrical properties of a given neuron depends on the numbers, types and localization of all of the different ion channels that are expressed at the cell membrane

Answer 89

Many neurons respond to changes in RMP by altering AP frequency In retinal ganglion cells, increasing injected depolarizing current increases AP frequency Thus, the frequency of action potentials can be modulated by ion channels that alter RMP Injecting current, depolarizes the cell

Answer 90

Na+ leak channels can modulate RMP: ↑ Na+ leak conductance depolarizes RMP Increases AP frequency in a "tonically" firing neuron- default is to fire ap ↓ Na+ leak conductance hyperpolarizes RMP K+ leak channels can also modulate RMP: ↑ K+ conductance hyperpolarizes RMP Decreases AP frequency ↓ K+ conductance depolarizes RMP Think of RMP as a dial that can be controlled by “turning” leak channel conductances up or down http://www.metaneuron.org/Windows?sid=27664 -set sweep duration to 200 ms -Change holding current from +10 to +200 μA (like a RMP dial) If have sodium leak and inc can inc rate of ap

Answer 91

As noted, 2-pore K+ (K2P) channels provide the major K+ leak current at rest (GK) 15 K2P genes in mammals Highly modulated by intracellular signaling pathways such as G proteins potential frequency The channel behind Na+ currents remained elusive for quite some time… Recent discovery: NALCN is the major Na+ leak channel

Answer 92

Previously: NALCN is an evolutionarily conserved channel that requires the ancillary subunits UNC-79, UNC-80, and FAM155 to function. CryoEM structure:

Answer 93

Another mechanism for Na+ leak currents: Nav channel “window currents” occur in Vm ranges where not all channels are inactivated, and some can become activated

Answer 94

Quick digression: relevance of the window current: A single amino acid mutation in the channel NaV1.9 enhances the window current, leading to depolarization of DRG nociceptive neuron RMP This mutation also causes higher frequency firing which causes stronger pain signaling to the brain (i.e., this mutation is a channelopathy) Valine to A- causes more firing, conducts more na leak current

Answer 95

Voltage-gated Ca2+ (Cav) channels are important modulators of AP shape (and frequency) P-loop channels- four domains Similar structure to Nav & NALCN channels

Answer 96

Humans have 10 Cav channel genes Referred to as Cavα subunits → make up the core channel structure: 4 Cav1 α subunit genes: Cav1.1, Cav1.2, Cav1.3 and Cav1.4 3 Cav2 α subunit genes: Cav2.1, Cav2.2 and Cav2.3 3 Cav3 α subunit genes: Cav3.1, Cav3.2 and Cav3.3

Answer 97

Cav1 and Cav2 also associate with Cavβ and Cavα2δ subunits: Cavβ is a cytoplasmic protein that binds to the alpha interaction domain (AID) within the domain I-II intracellular linker of the channel α subunit Cavα2δ is an extracellular protein that binds to the outside of the α subunit Cav3 channels do not generally associate with Cavβ or Cavα2δ

Answer 98

Cav channels are also classified based on their activation properties: “Low voltage activated” (LVA) Cav3 channels require only slight depolarization for activation Activated by graded potentials and other subthreshold depolarizing events Start activating before Nav channels, so they can help determine when APs take place Think of them as regulators or "decision makers" for AP generation

Answer 99

“High voltage activated” (HVA) Cav1 and Cav2 channels require strong depolarization for activation Activate only after Vm is considerably depolarized, hence, after an AP has been initiated Thus, they tend to play important roles in coupling APs with cellular events… Pre-synaptic Cav2 channels drive NT exocytosis Somatic neuronal Cav1 channels regulate gene expression for activity-induced learning and memory Muscle Cav1 channels drive contraction Think of them as the executors of the AP In the dendirtes- where decisions are made Cav1- drive muscle contraction, postsynaptically at muscles

Answer 100

In the heart, both LVA and HVA channels play important roles… LVA channels- decide to fire ap (Cav3.1 and Cav3.2) help set heart rate in the human embryo (not in adults) Prominent roles in heart of small adult mammals and invertebrates In the adult heart, high numbers of HVA (Cav1.2) channels widen the cardiac action potential for more Ca2+ influx and stronger contraction i.e., drive afterdepolarizations (ADPs)

Answer 101

How exactly do Cav channels modulate the AP??? LVA conductances active at subthreshold voltages contribute to membrane depolarization towards AP threshold, and towards Nav channel activation

Answer 102

Not surprisingly, Cav3 (aka T-type) channels are associated with numerous pathologies involving excessive excitability

Answer 103

How exactly do Cav channels modulate the AP??? At high enough levels, HVA conductances promote ADPs by countering Kv channel conductances that are trying to repolarize Vm after the rise of the AP Important notes: ADPs require lots of HVA Cav channels Not all cells expressing Cav channels exhibit ADPs (i.e., those with less HVA Cav channel expression)

Answer 104

Generally, Cav channels conduct depolarizing Ca2+ currents into neurons and muscles to promote membrane depolarization and cellular excitation Ca2+ ions also bind cytoplasmic proteins to promote intracellular processes such as vesicular exocytosis In some neurons/cells, Cav channels can actually inhibit excitation! They do this by activating Ca2+-sensitive K+ channels that hyperpolarize Vm in response to ↑ intracellular [Ca2+] We’ll learn more about this next!

Answer 105

SK and BK channels are P-loop channels Evolutionarily-related to Kv and Nav channels Tetrameric (need four proteins to make a channel) BK channels acquired an additional N-terminal alpha helix

Answer 106

Structure and Ca2+ sensitive mechanisms… BK channel subunits bear C-terminal Ca2+ bowls that bind Ca2+ BK subunits associate with accessory β subunits (4 per channel) SK channel C-termini associate with Ca2+ sensor protein calmodulin- binds calcium (CaM) Remember CaM from GCaMP? CaM

Answer 107

BK channels are activated by the combined action of depolarization (due to voltage sensors) and cytoplasmic [Ca2+] increases (through the calcium bowl), unlocked by cytoplasm calcium e.g., BK channel inside out whole cell currents… [Ca2+]cytoplasm Voltage clamp steps

Answer 108

Instead, SK channels lost voltage sensitivity when they evolved from a KV channel Activated strictly by Ca2+/CaM Calcium binds commoldiun and interacts with the channel to opens the gate Don’t use voltage sensors

Answer 109

Often, BK and SK channels are positioned close to Cav channels at the cell membrane Interestingly, this coupling can lead to excitation-induced neural inhibition Excitation opens Cav channels, which normally provide depolarizing Ca2+ currents Ca2+ activates BK/SK channels, which produce strong outward K+ currents that hyperpolarize Vm

Answer 110

In neurons, Cav channel activation of BK and SK channels prolongs action potential afterhyperpolarization (AHP) BK channels activate very quickly and contribute to fast AHP SK channels are slower and stay open longer, leading to slow AHP More APs → more Ca2+ influx → stronger SK activation → stronger slow AHP Calcium comes in potassium leaves

Answer 111

Cav-SK channel coupling can also drive spike frequency adaptation (SFA)… Sequential APs cause accumulating Ca2+ influx through HVA Cav channels This activates more and more SK channels, leading to ever-increasing AHPs Increased AHPs cause AP frequency to slow down and eventually stop Like turning up the GK “dial” we discussed before Proof of SFA: Cav channel blocker Cd2+- binds to calcium channels prevents spike frequency adaptation and AHP Every ap that occurs activates sk activation, next ap has to deal w the k influx, make it harder for next ap because of inc k current Eventually causes it to stop firing

Answer 112

Cav channel-SK/BK channel coupling can be used to counter-balance depolarizing Na+ leak currents that generate afterdepolarizations (ADPs) In rat hippocampal neurons, excitation turns on a Na+ leak channel that causes a prolonged ADP Experimentally removing external Ca2+, which reduces Cav channel Ca2+ currents, and hence BK/SK channel activation, increases the amplitude of the ADP Thus, under normal circumstances, Cav channel activation of SK/BK channels counters the Na+ leak and its ADP Couple it with na leak ap Reduce the calcium, the neuron fires 2 ap, we diminished neg feedback

Answer 113

Action potential threshold is not static The refractory period between APs is determined by the recovery of inactivated Nav channels Absolute refractory period sets the maximum AP frequency of a neuron Relative refractory period determines the frequency of action potentials for a given fixed amplitude stimulus Cav channels can promote and regulate APs and neuronal excitability LVA channels regulate the formation/frequency of APs HVA channels can promote ADPs Cav channels can couple with SK/BK channels to inhibit APs and excitability Generate fast and slow AHPs (BK = fast, SK = slow) Spike frequency adaptation Counter depolarizing Na+ leaks that generate ADPs

Answer 114

Members of the P-loop channel super family. • Ligand gated cation channels. • Homo- or Hetero-tetramers. • Each subunit: • Amino-terminal domain • Ligand binding domain (LBD) • Transmembrane Domain (TMD) • Intracellular carboxyl terminal Major mediators of excitatory synaptic transmission in the CNS. • Classically of 3 main types: • NMDA • AMPA • Kainate

Answer 115

Summary of Part 1 • The iGluR LBD has switched ligand selectivity from glutamate to glycine many times. • More early diverging iGluR subunits are predicted to be glycine sensitive. • Possibly iGluR played a role in environment sensing in early diverging animals. (As opposed to cell-to-cell communication)

Answer 116

Most iGluRs are non-selective for monovalent cations. • NMDA and some AMPA receptors conduct Ca2+ ions which contribute to synaptic plasticity. • NQR site in the M2 helix controls Ca2+ permeability

Answer 117

Canonically iGluR NQR site has N ( in NMDA receptors), Q (in AMPA/Kainate receptors), or R (in AMPA receptors) residues. • Some placozoan epsilon receptor have an atypical S residue in this position Nonselective for monovalent cations (Na+ vs. K+) • Highly permeable to Ca2+ Monovalent selectivity? (Na+ vs. K+) • Ca2+ permeability?

Answer 118

Mammals have Ca2+ permeable and Ca2+ impermeable AMPA receptors. • Presence/absence of Q in NQR site determines this. • Trichoplax has evolved separate iGluR subunits with S or Q the NQR site to control Ca2+ permeability without affecting monovalent selectivity

Answer 119

Mammals have Ca2+ permeable and Ca2+ impermeable AMPA receptors. • Presence/absence of Q in NQR site determines this. • Trichoplax has evolved separate iGluR subunits with S or Q the NQR site to control Ca2+ permeability without affecting monovalent selectivity

Answer 120

Electrical signals have to work their way through neurons EPSPs IPSPs APs Devopped protein that acts as voltage sensor indicator- flourescneces at differenent voltage, can see electrical events throughout neuron AIS- starts To roght- to depolarization, propogating, also propogating back up yhe axon to cell body and dendrites

Answer 121

Electrical signals have to work their way through neurons EPSPs IPSPs APs Devopped protein that acts as voltage sensor indicator- flourescneces at differenent voltage, can see electrical events throughout neuron AIS- starts To roght- to depolarization, propogating, also propogating back up yhe axon to cell body and dendrites

Answer 122

We can simplify axons/dendrites to cylinders Have a radius r and length l Say we injected a depolarizing current into the middle of an axon... Where would the injected ions go? Some would go to the membrane capacitor to depolarize membrane voltage Rate of flow would be dependent on the size of the membrane (capacitance or Cm) Some would move out of the axon through leak channels Cylinder of length and radius, is on two directions Size of structure impacts how capicatance flowx Down te axon- propogating event have to moce through the conductances of the cell membrane Dependent on membrane resistance or Rm Some would travel down the axon Dependent on axial resistance or R

Answer 123

Current that goes through Ra subsequently will either travel... Further down the axon (i.e., through Ra again) To Cm or through Rm located farther away from the site of current injection It is evident that injected current available to depolarize an axon gets consumed as you move farther and farther away from the site of injection l l r Current that goes through Ra subsequently will either travel... Further down the axon (i.e., through Ra again) To Cm or through Rm located farther away from the site of current injection It is evident that injected current available to depolarize an axon gets consumed as you move farther and farther away from the site of injection l l r

Answer 124

Current that goes through Ra subsequently will either travel... Further down the axon (i.e., through Ra again) To Cm or through Rm located farther away from the site of current injection It is evident that injected current available to depolarize an axon gets consumed as you move farther and farther away from the site of injection l l r Current that goes through Ra subsequently will either travel... Further down the axon (i.e., through Ra again) To Cm or through Rm located farther away from the site of current injection It is evident that injected current available to depolarize an axon gets consumed as you move farther and farther away from the site of injection l l r

Answer 125

We can model the movement of current through an axon with the following circuit diagram: The same circuit can be considered for Nav channels that "inject" depolarizing current into the axon during an action potential Inject current and paths are on membrane capacitorm resistors, or Ra

Answer 126

What we will seek to understand next is how the radius of an axon influences the flow of current to/through Cm, Rm, and Ra The flow of current dictates how fast voltage signals, such as action and graded potentials, can propagate through tubular nerve structures How radius influences charge of axon, determines how fast electrical signals can travel through Speed of axon potential propogation- 120 m/s

Answer 127

There are two important considerations... Resistance for current flow is inversely proportional to the surface area through which the ions are flowing More surface area means more avenues for ions to flow This applies to both the axial (Ra) and membrane (Rm) resistance A wider "tunnel" presents a lower axial resistance (Ra) A wider "tunnel" also has more membrane, and hence presents a lower membrane resistance (Rm) Resistance through any media- inversely proportional to surface area Why- cytoplasm in axon trying to move through it is surface area is big, can have lots of ways u can move with smaller area- less options Bigger radius= greater surface area so it reduces resitstance, same applies to membrane Bigger surface area smaller resitsnace, more leak channels Applys to axon and membrane There are two important considerations... The capacitance of the membrane is directly proportional to the membrane surface area Wider axons = more membrane = higher membrane capacitance (Cm) A wider "tunnel" presents a higher membrane capacitance (Cm) Bigger axon with more membrane has more capacitance Wider membrane- more capacitance

Answer 128

Let's start by look at membrane resistance (Rm) in more detail: Rm depends on the number of leak channels (holes) in the membrane A membrane with lots of holes is bad at resisting ion flow (low Rm) A membrane with few holes is good at resisting ion flow (high Rm) Rm also depends on the surface area (SA) of the membrane More SA means more channels which provide more routes for ion flow, hence lowering resistance Lots of holes, bad at resisting membrane- has low resistance If not, has high resistance Surface area affects this, holes per surface area the same, larger tube has lower resitsnace

Answer 129

Rsm is the specific membrane resistance This is a property of different cell membranes determined by the number of leak channels present per unit surface area (density), more dense spots, more leak channels Typical values range from 1,000 Ωcm2 (lots of leak channels) – low resistance to 50,000 Ωcm2 (few leak channels)- high resistance 2πrl is the SA of the outside of our cylinder- formula r is the radius of the cylinder l is the length of the cylinder Resistance inversely proportional to surface area, inc r inc SA R = Rsm/ 2π𝑟𝑙

Answer 130

Let's now look at axial resistance (Ra) in more detail: Ra depends on the width of the tunnel, radius A wider tunnel provides more routes for ion flow (lower Ra) Increased surface area for axial current flow Travelling further down the axon increases axial resistance (Ra), inc length inc resistance i.e., current has to travel further through the resistive cytoplasm

Answer 131

The axial resistance (Ra) through a cylindrical cellular structure can be determined as follows: Rsa is the specific axial resistance- resistance of charges mothing through cytoplasm Reflects the conductive properties of the cytoplasm/axoplasm Squid (high [salt], lower resistance) ~ 30 Ωcm at 20oC, low salt bald at conducting charger Mammals (low [salt], higher resistance) ~ 125 Ωcm at 37oC Frogs (even lower [salt]) ~ 250 Ωcm at 20o Aqeuous- think of a copper wire πr2 is the surface area of a cross section of the axon- moving through a cross section Increasing r therefore decreases Ra, gives you more SA The further the currents need to travel, the higher the resistance it will encounter Increasing l therefore increases Ra Ra = Rsa 𝑙 / π𝑟2 I’m

Answer 132

And finally let's look at membrane capacitance: c is directly proportionate to SA Capacitance increases with increasing surface area of the cylinder More membrane SA makes for a larger capacitor and hence more places for charges to go to A wider axon has more membrane (higher Cm) A thinner axon has less membrane (lower Cm)

Answer 133

cellular structure can be determined as follows: 𝐶𝑠𝑚 is the specific membrane capacitance per unit surface area Physical property of the cell membrane (nothing to do with ion channels) Typically 1 micoFarad/cm2 2πrl is the SA of the outside of a cylinder with radius r and length l Based on the equation, increasing r or l increases Cm C = 𝐶 2π𝑟𝑙 These don’t change from obe cellular system to the other

Answer 134

cellular structure can be determined as follows: 𝐶𝑠𝑚 is the specific membrane capacitance per unit surface area Physical property of the cell membrane (nothing to do with ion channels) Typically 1 micoFarad/cm2 2πrl is the SA of the outside of a cylinder with radius r and length l Based on the equation, increasing r or l increases Cm C = 𝐶 2π𝑟𝑙 These don’t change from obe cellular system to the other

Answer 135

Rm decreases for larger radius axons This means that wider axons loose more injected current through membrane leak channels Ra decreases for larger radius axons This means that currents travel more easily through wider axons Cm increases for larger radius axons This means that more current/charges are used up charging the membrane Capacitance All depend on radius Inc radius, dec resistance

Answer 136

What we're going to look at next: How these properties explain why "giant" radius axons produce faster propagating action potentials How axon myelination, which reduces membrane capacitance, can also produce faster action potentials Rm decreases for larger diameter axons This means that wider axons lose more injected current through leak channels Ra decreases for larger diameter axons This means that currents travel more easily along wider axons Cm = 𝐶𝑠𝑚 2π𝑟𝑙 Cm increases for larger diameter axons This means that more charges are used up charging the membrane capacitance Travels axon from brain to muscles directly – occurs via synaptic actovation

Answer 137

Recall: when you inject current into an axon, where will it go? To the membrane capacitance (Cm) Through membrane leak channels (Rm) Along the axon (Ra) Injected charges are used up as you move away from the site of injection... Less ions become available to charge membrane capacitance at distal sites for changing Vm Thus, the degree of Vm depolarization decays as you move away from the site of depolarizing current injection . Voltage response weakons along axon- closet to current injection- strongest voltage response

Answer 138

Recall: when you inject current into an axon, where will it go? To the membrane capacitance (Cm) Through membrane leak channels (Rm) Along the axon (Ra) Injected charges are used up as you move away from the site of injection... Less ions become available to charge membrane capacitance at distal sites for changing Vm Thus, the degree of Vm depolarization decays as you move away from the site of depolarizing current injection . Voltage response weakons along axon- closet to current injection- strongest voltage response

Answer 139

ΔV0 is the maximal depolarization at the site of injection ΔVx reflects the decay in ΔV0 as distance from site of injection (x) is increased λ is the length constant of an axon Distance (x) where ∆Vx has decayed by 63% from its initial value of ΔV0 Axons with larger λ can spread depolarization further than ones with lower λ (e.g., compare λ1 to λ2) Equation to describe decay- change in voltage= to initial current times the constant X- is length for site, change values for x and creates decaying voltage graph Lamda- unique factor for length of axon Larger axon- less decay So larger axons can send info further- can move further down axon

Answer 140

Simplifying: increasing radius has the net effect of increasing λ; length has no bearing! Bigger radius- dec lamda – in membrane In axon- bigger radius bigger lamda Length cancels out for lamda- proportional to radius So inc radius inc lamda, inc depolarization across axon

Answer 141

How does increasing λ speed up the action potential? First, let's look at how APs move along axons... At any given point along the axon, membrane voltage is either at rest, reaching threshold, reaching peak depolarization, or becoming repolarized/hyperpolarized Depolarization at one point in the axon, where Nav channels conduct inward Na+ currents, spreads along the axon (like injected current) Behind the depolarizing wave, is a refractory region of the axon, where Nav channels are inactivated and Kv channels are active As action potential moves through- voltage depolarizes then see hyperpolarization Refractory regain- potassium channels activated, no more sodium open- as wave propogates through causes these regions How does increasing λ speed up the action potential? ↑λ means that the wave of depolarization spreads further down the axon, activating Nav channels located farther away The blue line represents the spread of depolarization along an axon with a low λ The red line represents the spread of depolarization along an axon with a high λ Notice how the red line reaches threshold voltage further down the axon than the blue line Thus, high λ means larger steps in depolarization along the length of the axon = faster action potential propagation!

Answer 142

Why do giant axons exist anyway?-escape behaviour Large λ values allow APs to propagate faster Giant axons are used when speed is imperative, incapable of mylenation In the squid, the giant axon permits rapid action potentials to stimulate the mantle muscles for efficient escape swimming

Answer 143

A digression on where these ideas came from... In the 1850s, transatlantic cables were a tantalizing prospect for connecting the new world with Europe- wire to connect the continets Ships took 11 days to carry messages over the Atlantic The Atlantic Telegraph Company and its derivatives (backed by investors from England, US and Canada) sought to achieve this goal

Answer 144

William Thompson (later knighted Lord Kelvin) came up with a mathematical model to explain how electrical signals flow though underwater cables, and helped design a cable that finally worked! More insulation (lower Cm and increase Rm) and lower axial resistance (thicker metal wires that decreases Ra) Put wire through ocean tp transmit the info Wire needed low capacitance, by inc insulation, allowing it to move trhough and dec axonial resistance- decreases resitsnace and get faster A digression on where these ideas came from... Hodgking and Huxley later adopted Lord Kelvin's mathematical model to describe the movement of electrical signals through the squid giant axon

Answer 145

Large axons are rather imposing... you can only bundle so many of these into a nerve (bundled axons and some blood vessels) Clearly, there must be another way to speed up action potentials... Limitation to amount of giant axons

Answer 146

One challenge for AP speed is that membrane capacitance introduces a time delay for membrane depolarization... Initially, injected current is consumed by closest capacitor As that capacitor reaches its maximal voltage, more and more current is diverted to more distal capacitors, each also initially consuming most incoming current, accumulate until reach fixed voltage, takes time, and then move the charges diwn the axon, accumulating delay of charges Thus, charging a tube-shaped capacitor takes longer and longer as you move farther and farther from the site of current injection This significantly delays AP propagation

Answer 147

Instead, imagine if we could prevent charges from accumulating on the membrane capacitor, not being used up and not losing charges Charges would not be lost to charge the membrane capacitor locally, and would instead be directed along the axoplasm more efficiently Effectively, we would be changing the specific membrane capacitance (Csm): Cm = 𝐶𝑠𝑚 2π𝑟𝑙 Shwann- PNS and glial cells wrap around axon creating insulation, dec capacitance

Answer 148

Evolution has found a way to solve the capacitance problem... Glial/Schwann cells wrap themselves around axons to insulate them from the extracellular saline This decreases Cm while increasing Rm, both of which promote faster signal propagation Seperated by node of navier Dec capacitance, block leak channels and inc resistance and inc lamda

Answer 149

Myelin sheaths are interrupted with bare regions called nodes of Ranvier Packed with Nav (green) and Kv (red) channels Immunolabeling of Nav and Kv channels in a rat Signals essentially “jump” from node to node (i.e., salutatory conduction) The nodes boost the AP as it travels along the neuron Nav channels respond to incoming depolarization and amplify it to boost depolarization further down the axon Sodium channels highly expressed here Allows for saltory conduction- jump along axon

Answer 150

In a nerve containing numerous axons, conduction velocities differ, even between myelinated fibers Likely due to variability in the degree of myelination, and axon radius e.g., rat sciatic nerve: α → 80-120 meters per second β → 30-80 meters per second, slower δ → 5-30 meters per second, much slower In a nerve with different axons, they project at different speeds a\lpha- very fast Larger diameter- alpha

Answer 151

Multiple sclerosis is an autoimmune disorder that results in the demyelination of neurons Vision problems, difficulty controlling muscles, weakness, tingling. Mylenation around axons becomes degenerated Axon is compressed, can hace complete degeneration

Answer 152

Multiple sclerosis is an autoimmune disorder that results in the demyelination of neurons Vision problems, difficulty controlling muscles, weakness, tingling. Mylenation around axons becomes degenerated Axon is compressed, can hace complete degeneration

Answer 153

Members of the P-loop channel super family. • Ligand gated cation channels. • Homo- or Hetero-tetramers. • Each subunit: • Amino-terminal domain • Ligand binding domain (LBD) • Transmembrane Domain (TMD) • Intracellular carboxyl terminal Ionotropic glutamate receptors (iGluRs) are p-loop channels (same family as inward rectifying K, NaV, CaV, TPC, SK, BK channels, etc) • These are ligand gated channels. Need neurotransmitter like glutamate to bind for opening of the channel. • They are tetramers (4 subunits form 1 channel) • Each subunit has a very conserved structure consisting of extracellular amino-terminal domain, extracellular ligand binding domain, transmembrane domain with 4 alpha-helixes and intracellular carboxyl termin

Answer 154

iGluRs are the main mediators of excitatory synaptic transmission in mammalian CNS. • In mammals iGluRs are of 3 main types: NMDA, AMPA and Kainate iGluRs from early diverging animals do not fit into the categories of NMDA, AMPA and Kainate. • New classification of iGluRs is proposed recently based on phylogenetics: o Epsilon family is the earliest diverging iGluR family and is present in all major animal lineages. o Lambda family is only present in sponges (Porifera) o AKDF family is present in all phyla except Ctenophora. AMPA and Kainate receptors are a part of AKDF family. o NMDA receptors are present in Cnidaria and bilaterians.

Answer 155

A receptor of the epsilon sub-family from the animal Trichoplax adhaerens (Placozoan) is characterized by the Senatore lab. This receptor is activated by Glycine, Alanine, Serine and Valine but not by Glutamat Some NMDA subunits in mammals are also glycine sensitive. • Glycine sensitive iGluRs are present in all iGluR sub-families (epsilon, lambda, AKDF and NMDA).

Answer 156

Ligand sensitivity can be predicted by looking at 5 key residues of the ligand binding domain (LBD). • These residues interact with the side chain of the amino acid ligand. Slide 8: • The ligand sensitivity of the placozoan epsilon receptor was switched from glycine to glutamate by making just 3 mutations of key residues in the LBD. • This was the first experimental confirmation of the hypothesis that those 5 key residues indeed dictate ligand sensitivity of iGluRs. Slide 9 and 10: • These mutations in the LBD also change the response to pharmacological agonists and antagonists of iGluRs. • The m3 variant exhibits nascent sensitivity to the namesake AMPA receptor agonist AMPA. • The m2 and m3 variants also exhibit higher sensitivity to the drug CNQX, which is known for blocking AMPA and kainite receptors. The iGluR LBD has switched ligand selectivity from glutamate to glycine many times, as there are glycine and glutamate sensitive iGluRs in all animal lineages. • More early diverging iGluR subunits are predicted to be glycine sensitive. • Possibly iGluRs played a role in environment sensing in early diverging animals. (As opposed to cell-to-cell communicatio

Answer 157

iGluRs are roughly equally permeable to Na+ and K+ . • NMDA receptors and some AMPA receptors can also conduct Ca2+ . • Ion selectivity is controlled by the NQR site (selectivity filter), which is the narrowest part of the pore formed by the p-loop (M2 helix) of the transmembrane domain. Slide 13: • In mammalian iGluR the NQR site has asparagine/N (in NMDA receptors), glutamine/Q (in AMPA and kainate receptors), or arginine/R (in AMPA and kainite receptors). • The placozoan epsilon receptor has an atypical serine/S residue at this site. • The consequences of S at the NQR site were not known. Does the S change monovalent selectivity or Ca2+ selectivity? Slide 14: • S at the NQR site does not change monovalent vs. monovalent selectivity (i.e., the channels are non-selective for one cation over the other, and mutating the pore from S to Q or N did not affect the monovalent selectivity in a significant way. Slide 15: • S at the NQR site considerably diminishes Ca2+ vs. monovalent permeability (i.e., the channel is less permeable to Ca2+ relative to monovalent cations). • Mutation of the pore S to Q or N considerably increased Ca2+ permeability.

Answer 158

• Mammals have Ca2+ permeable and Ca2+ impermeable AMPA receptors. o Presence/absence of Q in NQR site determines this. o Ca2+ permeable iGluRs play a key role in learning and memory and in synaptic plasticity. • Trichoplax has evolved separate iGluR subunits with S or Q the NQR site, perhaps to control Ca2+ permeability without affecting monovalent selectivity.

Answer 159

Previously we discussed excitatory post-synaptic potentials… • Acetylcholine (ACh) secreted by vertebrate pre-synaptic motor neurons activates ion channels called nicotinic ACh receptors (nAChRs) in post-synaptic muscle cells • nAChRs conduct mostly Na+ to depolarize cells, leading to: action potentials → Cav channel activation → Ca2+ influx → muscle contraction

Answer 160

post-synaptic potentials mediated by Cl- channels… • Kakazu et al. documented a shift in post-synaptic responses of mouse superior olive neurons to the neurotransmitter glycine • Glycine (Gly) activates post-synaptic Cl- ion channels called glycine receptors • A developmental shift in [Cl-]in vs. [Cl-]out causes glycine (and GABA) receptors to change from excitatory to inhibitory

Answer 161

nACh, glycine, and GABA receptors belong to a large family of ligand-gated ion channels called Cys-loop channels • Pentameric (5 separate protein subunits make a complete channel) • Each subunit contains an extracellular loop bearing 2 cysteine amino acids • These form a disulfide bond that stabilizes the loop • Hence the name: “Cys-loop” • Cys-loop channels can be cationic or anionic • e.g., nAChRs are cationic (Na+ and K+) • e.g., Glycine and GABA receptors are anionic (Cl-)

Answer 162

Here’s the general strategy: 1. Biochemical isolation → purify and identify desired channel proteins/subunits 2. Sequence the isolated channel proteins, and use that to sequence mRNA 3. Clone channel mRNA (cDNA) → express in cell lines → characterize ion conducting properties via electrophysiology in vitro 4. Mutate channel coding sequences → test hypotheses about how amino acids and channel structures contribute to channel function 5. Determine/predict the structure of a channel protein

Answer 163

Biochemical isolation → identify channel proteins/subunits • Nicotinic acetylcholine receptors (nAChRs) were first isolated from the electric organs of the ray Torpedo • Torpedo bear modified muscle cells in electric organs packed with cholinergic synapses and nAChRs • The snake α-bungarotoxin was known to bind nAChRs very strongly (peptide toxin) • Researchers used α-bungarotoxin as bait to isolate nAChRs from protein extracts of Torpedo electric organ • Like “fishing” for a desired protein • Analysis of the isolated proteins revealed that nAChRs in the ray each consisted of 5 separate proteins: • 2α subunits • 1β subunit • 1γ subunit • 1δ subunit

Answer 164

Sequence purified channel proteins, identify corresponding mRNAs, and sequence them also • This is a very laborious type of research! • The 5 nAChR subunit proteins were found to be similar to each other • Each contained: a. 4 predicted transmembrane helices (TMHs), dubbed M1 to M4 b. A large extracellular N-terminal loop with 2 cysteines c. An extended intracellular loop between M3 and M4 helices d. An extracellular C-terminus

Answer 165

Ion channel properties arise from the physical properties of their constituent amino acids… • Different amino acids arranged in a sequence will promote different secondary structures • e.g., alpha helices, beta strands, disordered loops • Side groups of amino acids have different polarities and hence hydrophilicity/hydrophobicity • Some amino acids have charges • D & E are negatively charged • H,K & R are positively charged you would expect, TMHs contain mostly hydrophobic amino acids physical properties of their constituent amino acids… • Secondary structures assemble into tertiary structures • This is the final "shape" of the folded subunit/protein • Subunits assemble into quaternary structures to make up functional channels • These are the protein complexes that make up a functional channel unit • e.g., The nAChR quaternary structure consists of 5 subunits

Answer 166

Remember the voltage sensor TMHs of Kv, Nav and Cav channels? • S4 helices contain: • Hydrophobic amino acids • Positively-charged lysine (K) and arginine (R) amino acids crucial for voltage sensing Nav and Cav channels? • S2-S3 helices contain: • Hydrophobic amino acids • Negatively-charged aspartate (D) and glutamate (E) residues that act as "counterions" to S4 charges Using the deciphered protein sequences, researchers were then able to sequence the corresponding mRNAs • This is a crucial step since it allows you to identify which gene gives rise to the ion channel/receptor in question

Answer 167

Further cloning and gene sequencing efforts identified a large "superfamily" of Cys-loop channels • Some are cationic, some are anionic… • Gamma aminobutyric acid (GABA) receptors • Anionic (conduct Cl-), found at inhibitory synapses (vertebrates and invertebrates) • Glycine receptors • Anionic, inhibitory synapses (vertebrates/chordates) • Serotonin receptors • Cationic (Na+ and Ca2+), excitatory synapses (vertebrates) • Invertebrate MOD-1 (serotonin) receptors • Anionic (invertebrates) • Glutamate-gated receptor • Anionic (invertebrates) • EXP-1 (GABA) receptors • Cationic (invertebrates

Answer 168

Clone channel mRNA (cDNA) → expression in cell lines → characterize ion conducting properties via in vitro electrophysiology • Clone the channel coding sequence (complementary DNA or cDNA) into a DNA plasmid vector that will permit expression in desired cell line • e.g., Human Embryonic Kidney-293T (HEK-293T) cells • e.g., Chinese Hamster Ovary (CHO) cells • e.g., Xenopus (frog) oocytes • Perform electrophysiology on the in vitro expressed channels Human embryonic kidney (HEK)-293T cells are one common system: • Gene cDNA is cloned into a plasmid DNA vector bearing a strong mammalian promoter • e.g., cytomegalovirus (CMV) • Introducing the vector into HEK-293T cell nuclei (via transfection) will lead to transcription, translation and channel membrane expression • Very easy to record molecular biology to insert your channel's cDNA into a mammalian expression vector23 HEK-293T cells…Frog oocytes are also commonly used for ion channel research • e.g., from the species Xenopus laevis • Clone channel cDNA into a plasmid DNA vector that permits synthesis of synthetic mRNA (in vitro) • Inject the mRNA into isolated Xenopus oocytes (will express your channel protein) • Perform patch clamp/sharp electrode electrophysiology

Answer 169

Mutate channel coding sequences → test hypotheses about how amino acids and channel structures contribute to channel function Mutation studies • Use molecular biology to alter/mutate amino acids so that you can study... • Channel folding, structure, function, ion selectivity • Channel gating (e.g., voltage-gating, ligand binding, inactivation) • Mutations associated with diseases • Binding by drugs that can alter/correct channel function (pharmacology)

Answer 170

ray crystallography: • Make a crystal of the protein of interest (can be difficult) • Shine an X-ray beam through the crystal • Use the resulting diffraction pattern to infer the protein's structure ray crystallography: • Very high resolution possible, given good quality crystals • Caveats: • Difficult to generate crystals for membrane-spanning proteins that have hydrophobic TMHs • Can use tricks to crystalize membrane proteins (e.g., use antibodies), but these can create artefacts in the structure

Answer 171

High-resolution electron microscopy • Embed proteins of interest in isolated membrane preparations (e.g., cylindrical vesicles) • Take transmission electron microscope images • Average images of single proteins to get a consensus structure • Caveats: • More representative of channel structure but requires fixation • lower resolution than X-ray crystallography

Answer 172

Cryo-electron microscopy • A newer iteration of high-resolution electron microscopy • Cryogenically freeze the sample before imaging with an electron beam (reduces damage and hence has better resolution) • Most “realistic” structural analysis due to lack of chemical fixation • Recent advances have permitted resolution similar to that of X-ray crystallography • Now a leading method to determine structures of ion channels and large protein complexes that are not amenable to X-ray crystallography

Answer 173

Predict structures de-novo with AlphaFold • Developed by DeepMind (also known for AlphaGo) • Artificial intelligence system capable of accurately predicting protein structure directly from user- inputted protein sequences!

Answer 174

Cys- loop receptor structure and function: • Each receptors is made up of 5 subunits, each with large extracellular domains and 4 TMHs • Extracellular loops contain 2 cysteine amino acids linked by a disulfide bond, hence the name Cys-loop Key insights into Cys-loop receptor structure and function - Ion selectivity: • M2 helices line the aqueous pore and interact with permeating ions • Different M2 sequences are used to create cation vs. anion selectivity: • e.g., cationic nACh receptors vs. anionic Gly receptors Cys-loop receptor ligand binding and channel activation: • The outside domain contains 2 sets of beta strands arranged in sheets (red and blue) • Ligand binding causes a conformational change in the sheets loop receptor ligand binding and channel activation: • Upon ligand binding, conformational changes are transmitted to the pore region • Pore-forming M2 helices move outward, opening the ion permeation pathway Like ionotropic glutamate receptors (P-loop channels), Cys-loop channels evolved in bacteria • Cationic and anionic channels appear to have evolved separately prior to animals • There are many Cys-less channels, including in animals Cys-loop and Cys-less channels share a conserved phenylalanine/tyrosine-proline-X-aspartate (F/YPxD) motif located between the two cysteine residues • This proline is the only amino acid conserved in all receptors

Answer 175

Gap junctions are aqueous conduits between juxtaposed cells • When enough gap junctions are clustered together, we get an electrical synapse • Potentials (action and graded) can travel through gap junctions/electrical synapses • Gap junctions are formed by the bringing together of two “hemi-channels” from adjacent cells

Answer 176

• Three types of hemi-channel genes/subunits are known about • Vertebrates have 20 connexins and 3 pannexins • Invertebrates have > 25 innexins • Pannexins and innexins are evolutionarily related • Connexins vs. pannexins/innexins have similar structures, but there is no evidence for homology • Independent evolution or extreme divergence in protein sequence?

Answer 177

Protein complexing and nomenclature: • 6 connexin proteins → 1 connexon hemi- channel • 6 x pannexin proteins → 1 pannexon hemi- channel • 6 x innexin proteins → 1 innexon hemi- channel

Answer 178

Connexins, pannexins and invertebrate innexins • Connexons: • Can couple to form gap-junctions in vertebrates • Innexons: • Can couple to form gap-junctions in invertebrates • Pannexons: • Do not form gap-junctions • Have extracellular glycosylation moieties that prevent gap-junction formation

Answer 179

General features of hemi-channels and gap junctions • Outwardly permeable to ATP • i.e., leak ATP out of the cell • Inwardly permeable to dyes including Lucifer yellow and 6 carboxyfluorescein • i.e., cells can readily take up external dyes • Pore opening is induced by membrane depolarization • Other factors can induce opening for different types, such as ischemia (inadequate blood supply), mechanical stress, osmotic shock, and cytokines • Closure can be induced by several drugs, low cytoplasmic pH, and cytoplasmic kinases General functions of gap-junctions: • Mediate electrical coupling between cells • Permit sharing of resources (soluble, membrane impermeable molecules) • General functions of hemi-channels: • Mediate release of signaling molecules such as ATP and glutamate • Activate ATP-gated cation channels (Purinergic receptors) for cell-cell (paracrine) and self (autocrine) signaling

Answer 180

Several structures of gap junction forming proteins have been solved • e.g., A recent Cryo-EM structure of the homohexameric connexin-43 This recent structure revealed putative mechanism for pore opening • An N-terminal domain (NTD) helix obstructs the pore • TM2 helices move inward, allowing the NTD helix to move outward and open the pore