Bio 2 Flashcards
Calcium transport mechanisms
Action potentials involve influx of sodium causing depolarization leading to action potential
\sodium and potassium serve the role for electrical impulses and not highly recative with proteins, doesn’t change it, doesn’t act as a chemical signal
AP invades nerve terminal and have calcium channels, which open and enter cytoplasm, interact with protiens with synaptic vesicles, it fuses and releases neurotransmitters
Example in muscle cell- neuromuscular junction
And have ach receptos- sodium chnanels activated by binding of receptor, causing depolorization, muscle cell causes ap and calcium is released and interacts with proteins causing the contratction
Calxcium takes ap and causes movements of proteins etc
cytoplasmic Ca2+
Why cytoplasmic Ca2+ is good?
Binds oxygen atoms (i.e., carboxyl and carbonyl groups on amino acids)
Causes conformational changes in proteins (good for signalling or activating
mechanical processes)
e.g., vesicle exocytosis, muscle contraction, activating other ion channels, changes in gene expression, apoptosis, intracellular signaling
Why is cytoplasmic Ca2+ is bad?
It precipitates phosphates (CaPO4), which can accumulate and become toxic
Can trigger apoptosis
Cannot be chemically altered for neutralization, cant be converted to something else
Thus, cytoplasmic Ca2+ is kept at very low levels
This makes Ca2+ a good transient cytoplasmic signaling molecule
Missing 2 electrons
And calcium is unhappy without ectra electrons
The cells and proteins have oxygen,
Oxygen is highly electronegative, binds with calcium to use its electrons
Oxygen surrounded by electrical acticity
This binding on a protein causes confirmational changes- triggering changes in the cell
Calcium is translatopr of electrical activity
But calcium precipitates phopsphates, which accumulate in cell
Cells use calcium as checkpoint for apoptosis
Lose control of calcium- will kill cells
Remove calcium for cytoplasm
Can tolerate calcium for short time, use ito cause confirmational change then get rif of it
[Ca2+]in «< [Ca2+]out
[Ca2+]in «< [Ca2+]out
10,000 fold difference!
1,500,000-fold less concentrated inside the cytoplasm than K+!
Calcium is extremely low outside the cell
[Ca2+]cyt can increase transiently…
During neuronal excitation & muscle contraction
Highly conserved proteins
[Ca2+]cyt can increase transiently…
After a stroke: positive feedback triggers ↑[Ca2+]cyt Why do neurons die in a stroke
Blood vessel that carries oxygen and glucose cant get to the brain, so the neurons in that region experience glucose and ocygen depletion
Brain consumes most energy of all organs
The sodium potassium pump needs atp, this activity diminishes during stroke, so our concentration gradient decreases, leading to depoloirization, causing ap when shouldn’t be happening
This increases calcium
A lopt of cells use glutamate to cause activation, releases during AP
Now have excess glutamate excretion, and cant reuptake it, casuing constant excitation, calcium is also glutamergic, causing increase in calcium, causing cell death
Would have to remove clot and apply drugs that dec sodium potassium to reduce ap, or block glutamate or calcium channels
How calcium is removed from the cytoplasm quickly
Ca2+ needs to be removed from the cytoplasm quickly
Cytoplasmic chelators/buffers bind free Ca2+ to “remove” it from solution- bind calcium and remove it from soloution
Pumps and exchangers extrude Ca2+ from the cytoplasm to the cell exterior or intracellular compartments (sarco/endoplasmic reticulum, mitochondria)take calcium and push out of cell or bring into the cell(mitochondria
2 types of Ca2+ pumps
Plasma membrane calcium ATPase (PMCA)
Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)
PMCA and SERCA are related to the Na+-K+ ATPase
P-type (phosphorylated intermediate)
However, don’t need a β subunit
2 types of Ca2+ pumps
Plasma membrane calcium ATPase (PMCA)
Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)
PMCA and SERCA are related to the Na+-K+ ATPase
P-type (phosphorylated intermediate)
However, don’t need a β subunit
SERCA
SERCA
2 Ca2+ ions pumped into the SR/ER- into the cell per cycle (hydrolysis of a single ATP molecule)
Humans have 3 SERCA α genes
α1: Muscle contraction
α2: Muscle contraction, neurons
α3: Non- skeletal muscle, but expressed in cardiomyocytes (heart)
SERCA
SERCA
2 Ca2+ ions pumped into the SR/ER- into the cell per cycle (hydrolysis of a single ATP molecule)
Humans have 3 SERCA α genes
α1: Muscle contraction
α2: Muscle contraction, neurons
α3: Non- skeletal muscle, but expressed in cardiomyocytes (heart)
SERCA
SERCA
2 Ca2+ ions pumped into the SR/ER- into the cell per cycle (hydrolysis of a single ATP molecule)
Humans have 3 SERCA α genes
α1: Muscle contraction
α2: Muscle contraction, neurons
α3: Non- skeletal muscle, but expressed in cardiomyocytes (heart)
SERCA (2 Ca2+) and PMCA (1 Ca2+) are sluggish at removing Ca2+
SERCA is highly expressed in the SR to ensure efficient removal of cytoplasmic Ca2+ and restoration of SR Ca2+ stores
PMCA is sparsely expressed at the cell membrane, so it is only good at maintaining low cytoplasmic Ca2+ levels when neurons are not highly active
Calcium transport mechanisms: Pumps
SERCA (2 Ca2+) and PMCA (1 Ca2+) are sluggish at removing Ca2+
SERCA is highly expressed in the SR to ensure efficient removal of cytoplasmic Ca2+ and restoration of SR Ca2+ stores
PMCA is sparsely expressed at the cell membrane, so it is only good at maintaining low cytoplasmic Ca2+ levels when neurons are not highly active
Calcium transport mechanisms: Exchangers
Instead, ion exchangers remove Ca2+ much more quickly
Do not hydrolyze ATP as energy source for moving ions against their gradients
Consume energy from existing ion concentration gradients in exchange for moving desired ions “uphill” against their concentration gradients
Referred to as secondary active transport
NCX exchanger
The NCX exchanger uses the Na+ gradient
Na+ Ca2+ exchanger
a.k.a. sodium-calcium antiporter
1 Ca2+ out for 3 Na+ in (can thus depolarize Vm!)
Most widely distributed sodium-calcium exchanger
3 sodium go in with their conc gradient and calcium goes out
Charges not neutral- influenced by VM
Because NCX is not electrically neutral, it can be made to operate in reverse!
Both Na+ and Ca2+ want to get into the cell (gradient)
Whichever ion type experiences the strongest inward pull, wins
Pull is determined by net ion charge x driving force
To understand this, let’s first look at the typical
equilibrium potentials for Na+ and Ca2+:
If membrane voltage it polarized, will allow calcium to come in and sodium leave
Whoever has greater drive force times the net chstge , causes the movement of ion
Driving force calcium exchanger
For Na+ to go in, it’s net charge (=+3) x driving force (Vm-ENa) must have a greater amplitude than Ca2+ (charge=+2; Vm-ECa)
Near RMP, 3 Na+ ions go in and 1 Ca2+ goes out
At depolarized potentials, 1 Ca2+ goes in and 3 Na+ go out
Which ever line is further from the x axis, causes it to move towards
During stroke have depolirizaion, calcium is coming in while sodium is leaving
|3(Vm - ENa)| > |2(Vm - ECa)|
Ca2+ is extruded
|3(Vm - ENa)| < |2(Vm - ECa)|
Na+ is extruded
NCKX
A different Ca2+ exchanger, NCKX, is better at removing cytosolic Ca2+
Na+-Ca2+-K+ exchanger
Uses sodium and potassium gradients to remove Ca2+
4 Na+ in and 1K+ out in exchange for 1 Ca2+ out
|4(Vm-ENa) - 1(Vm- EK)| v.s. |2(Vm- ECa)|
This one never reverses
NCX:
NCX:
9 transmembrane segments
Mammals have 3 genes: NCX1-3 (1 in muscle, 2 & 3 in brain)
See the human protein atlas (SLC8A1 to 3)
Why do we have the calcium sodium exchanger ? Helps replace the calcium potassium echanger, incase have no atp
NCKX:
NCKX:
11 transmembrane segments
N-terminus is cleaved
Mammals have 5: NCKX1-5
NCKX1: retina
NCKX2: retina, brain
NCKX3: brain & smooth muscle
NCKX4: brain & smooth muscle
NCKX5: not expressed at the membrane; polymorphism is associated with white skin in individuals from Europe and Asia; might regulate Ca2+ in melanosomes
Human atlas SLC24A1 to 5
Chloride transport mechanisms
Neurons are strange when it comes to Cl- gradients
For immature neurons and almost all other cells…
[Cl-]in ~ [Cl-]out
Some mature neurons actively extrude Cl- from the cytoplasm such that…
[Cl-]out»_space; [Cl-]in
Sodium and potassium gradients are kept pretty constant
In immature neuron- works with sodium
Cl gradient is roughly equal- very similar
But as neurons mature- they create a different gradient, where cl in is much less than outside
Mature neruons- work with potassium
Immature neuron
Chloride transport
Membrane voltage is -75, cl and sodium want it to be depolarized from this
So cl and sodium are pulling the resting membrane away from -75
Mature neuron
Chloride transport
Changes driving force so Cl is now working with potassium to reach the equilibrium
𝑬 = −𝟐𝟓 𝒎𝑽 𝒍𝒏 [𝟏𝟎𝟎 𝒎𝑴]𝒐𝒖𝒕/ 𝟓 𝒎𝑴
𝑬𝑪𝒍 = −𝟕𝟒. 𝟗 𝒎𝑽
Glycine (neurotransmitter) activates post-synaptic Cl- channels (glycine receptors
Kakazu et al. saw exactly this in developing superior olive neurons(brain stem neurons important for hearing) of mice
Glycine (neurotransmitter) activates post-synaptic Cl- channels (glycine receptors):
In postnatal day 0 (P0) mice, glycine caused depolarization of Vm
In P15 mice, glycine caused pronounced hyperpolarization of Vm Knew they neurons were activated by glycine- inhibitory neurotransmitter- binds to glycine receptors which are cl channels, opens cl channels
P0 mice- recording intracellular electrode and recorded membrane voltage over time
Had -60 rp, applied glycine while recording and saw depolorixation and ap in immature mice, glycine caused a depolarization
In mature mice- the neurons are constantly firing ap- tonically active, when applied glycine, saw a hyperpolarization and decrease of ap
Mice changed their intrinsic activity, not active vs tonically active and change in response to glycine
How do you end up with high [Cl-]in?
How do you end up with high [Cl-]in?
Co-transporters use the Na+ gradient to move Cl- into the cell
Na+/Cl- Cotransporter (NCC): transports 1 Na+ and 1 Cl- into the cell
Na+/K+/Cl- Cotransporter (NKCC): transports 1 Na+, 1 K+ and 2 Cl- into the cell
Note that these co-transporters, unlike NCX and
NCKX, are electrically neutral
If you want to make a neuron excitable via Cl- channel activation, you need NCC or NKCC to produce [Cl-]in ~ [Cl-]out
This happens because mature neurons chanage their conc
Immature-
Use cotransporters, that use energy from the na/k gradient and move cl into the cell
Sodium potassium exchanges are influenced by the voltage
But these transporters aren’t affected by voltage
Want to make conc gradient=, have to use these cotransporters, which borrow energy and push cl into the cell
Chloride transport mechanisms
Nomenclature in mammals:
1 NCC gene, a.k.a. solute carrier 12 A3 (SLC12A3)
2 NKCC genes in humans:
NKCC1 (SLC12A2)
NKCC2 (SLC12A1)
How do you end up with less [Cl-]in?
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 «_space; [Cl-]out, change expression of transporters, start expressing kcc, inhibit neuron
Mature neruons
Cotransporters that use k gradient to move cl out of cell
4 KCC genes
SLC12A4 (KCC1)
SLC12A5 (KCC2)
SLC12A6 (KCC3)
SLC12A7 (KCC4)
Model for the developmental switch in Cl- gradient
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
Cl- co-transport also plays an important role in pH regulation
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
H+ and Na+ gradients drive membrane transport of neurotransmitters
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
Vesicular transporters
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
structure of the human V-ATPase was solved via cryo-electron microscopy
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
. H+ and Na+ gradients drive membrane transport of neurotransmitters
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
Revisiting the circuit diagram of the excitable cell
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»_space; GNa)…
The cell membrane is packed with leak K+ channels (called 2-pore K+ channels)
simplified cell circuit diagram…
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)
RC circuit
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
GHK equation can be used to conceptualize what happens during cellular excitation
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
graded potentials:
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)
action potentials?
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
The voltage clamp technique and the squid giant axon
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
Alan Hodgkin and Andrew Huxle
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
How Cole’s voltage clamp let’s you record channel currents at fixed voltages…
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
A second voltage amplifier (B)
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)
A third voltage amplifier (C)
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
A third voltage amplifier (C)
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
voltage clamp experiment:
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
voltage clamp experiment:
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
is what Hodgkin and Huxley saw in their first voltage-clamp recordings
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
Hodgkin and Huxley mathematical description of the action potential
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
H & H separated the Na+ and K+ currents in the squid axon
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
Used the voltage-clamp to record voltage- gated sodium and potassium channel currents
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
Mathematically converted recorded currents to conductances
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
Derived equations that mathematically simulate conductances at different voltages
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
how H&H used this to simulate the action potential:
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!
Why does the inward Na+ current have a funny “U” shape, while the
outward K+ current has a sigmoidal shape?
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
P-loop family
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
All P-loop channels need 4 P-loops to be functional:
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
Upon membrane depolarization, S4 helices protrude outward
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
channel inactivation
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
Kv channels undergo activation
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
2 things are required for Na+ and K+ currents through Nav/Kv channels:
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
macroscopic currents
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
Various configurations of the patch clamp
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.
H noted in their voltage clamp experiments that at first, depolarization caused an increase in peak macroscopic INa amplitude
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)
Voltage gated sodium Chanel mutation
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
For the K+ currents, both GK and DF increase
with each depolarizing voltage step…
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
Single nucleotide mutation
Single nucleotide mutation- valince to thymine mutation- causes this mutation
Lose of function, current is delayed and diminished
Neher and Sakmann
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/
P-loop channels
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.
P-loop channels emerged billions of years ago i
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
Evolutionary changes in the original KIR channel structure gave rise to many other P- loop channel types:
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
Although K2P channels are “leak” channels,
they are not as boring as their name implies:
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
Inversion across the membrane, plus addition of a transmembrane helix gave rise
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
Fusion with a separately-evolved protein in bacteria, gave rise
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
Digression on the VSD…
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
Digression on the VSD…
Key point: mutations can “re-awaken” voltage sensor pores
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
- P-loop channels
Addition of a transmembrane helix to the N-terminus of the KV/SK/TRP channel structure gave rise to
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
P-loop channels Tandem duplication of a Kv-like channel gave rise to
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
Tandem duplication of a TPC-like channel gave rise to
- 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
NALCN
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
Mutations in NALCN affect diseases
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
Mechanisms for ion selectivity of P-loop channels: K+ and KV channels
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
