Topic 3 Flashcards
Primary protein structure
sequence of amino acids
Secondary structure
alpha and beta sheets as a result of the nature of the amino acid sequences
- Polar side chains will face the water
- Non-polar will wrap themselves inside of the protein structure
Tertiary structure
full length of the protein all folded up together in a 3D structure
Quaternary structure
subunits of proteins are bound together with other proteins to form a functional units
Domain
a certain region of a protein which can be structurally and/or functionally different
- Alpha helix structures like to be embedded in membranes which is good for ion channels
replication
DNA synthesis
Transcription
RNA synthesis
Translation
protein synthesis
what drives the folding of proteins?
the nature of the amino acids
alpha helix
ribbon like structure forming a helical structure
- Individuals proteins can have multiple helices and other types of secondary structures
- the charges are lined up and there are 0.54 nm between twists
Beta sheets
flat folds of amino acids that stack on top of one another and have 2-3 bridging H-bonds
- form a twisted, pleated sheet
what type of protein folding makes good transmembrane domains?
alpha helices
- the external region is hydrophobic which allows it to integrate into the membrane and the inside is hydrophilic which allows aqueous solution to flow through
- When they all come together they make a functional transmembrane protein used as a channel ⇒ ion flow
what are the ways proteins interact with the lipid bilayer? (4)
- transmembrane
- mono layered
- lipid linked
- protein attached (to another protein)
what did early voltage clamp experiments tell us?
how current changes over time at a particular voltage
- Required large axons to accommodate the relatively large electrodes
- These were whole cell currents ⇒ for the whole population of channels on the cell
what is different about the modern voltage clamp?
has much smaller/thinner electrodes
- Can record whole cell currents from single mammalian neurons
- Can record current through a single channel
Microscopic current
any current that flows through an individual ion channel
Patch clamp
glass needle microelectrode pulled to a fine and sharp point attached to an amplifier ⇒ touches wherever you want to record in the cell
- The electrode has an internal core thats hollow that we can fill up with saline solution and when we apply a mild suction it will pull the membrane to make a tight seal against the glass
- Current cannot flow in our out of the electrode ⇒ you can get a single ion channel in the tip of the electrode
how are the patch clamp and voltage clamp similar?
the patch clamp is a subtle version of voltage clamp
T/F It is possible to clamp voltage and current
False
- not both at the same time
Whole cell
give it a strong transient suction which pops a hole in the membrane so the cytoplasm is continuous with your recording electrode
Single channel inside out recording
move the electrode away from the cell out of the aqueous environment into air and then back in so now you have an individual ion channel that you can record for current
how do patch clamps work in frog eggs?
- Get a frog egg and inject DNA
- Overexpression of the protein will be expressed and you get lots of channels in the membrane of the egg
- You can use a patch clamp recording device to use whole cell or inside out recording
how does using a patch clamp with TEA show data?
Blocks the K+ channels and has a transient depolarization causing inward current that disappears right away
- the K+ channels inactivate and stay inactivated during the rest of the pulse
- some Na+ channels chatter on and off but have a delayed start
- on a macroscopic scale the cell has an inward current which dips the graph down before it rises back to current 0 and stays there
how does using a patch clamp with TTX show data?
Depolarization pulse causing an outward current that stays longer than the Na+ inward current before dropping back down
- the K+ channels will open a little bit later but mostly stay on causing an increase in the microscopic measurements and macroscopic measurements
- once a channel opens it will stay open
T/F the probability of Na+ and K+ channels opening increases with increased membrane potential
True
what is the relationship between the probability of a voltage gated channel opening and the whole cell conductance for the ion?
conductance increases for sodium as membrane potential becomes depolarized so this correlates with the whole cell conductance and probability of channels opening
- The same thing happens with potassium which coincides with its conductance
- they are different measurements with different units and scales but the most important thing is that they increase conductance with increased Vm
what differs across neuron action potentials? (3)
- Thresholds for opening
- Kinetics for opening and closing
- Inactivation and de-inactivation properties
- differences in these properties will change the shape of the AP ⇒ changes with neurons and their concentrations of ions inside and outside the cell
what can the refractory period affect?
the frequency of different action potentials
types of voltage gated channels?
- Na+ channels
- Ca2+ channels
- K+ channel
- Cl- channels
properties of VG channels (6)
- # of proteins required to form a functional channel
- # membrane spanning helices
- Pore-loop
- Voltage dependence
- Ion selectivity ⇒ only let certain ions through
- Regulatory subunit?
what is the structure of a typical VG K+ channel? (6)
- alpha and beta subunits
- beta subunits modify
- usually 4 of each subunit
- linker regions in the cytoplasm between alpha and beta subunits
- pore loops on the external side
- voltage sensor connected to each alpha subunit attracted or repelled by the charge on the inside/outside of the cell => closest ions signal
subunit properties of KV2.1 channels
- 4 alpha and 4 beta
- 6 membrane stannic helices per subunit
- 24 transmembrane helices per channel
Non subunit properties of KV2.1(3)
- Pore loops in each alpha subunit ⇒ form channel
- Voltage sensor ⇒ each alpha subunit
- Ion selectivity = K+ only
What does the beta subunit do in KV2.1?
its regulatory of the alpha subunit
what is the direction of potassium flow for KV2.1 channels?
direction depends on the DF
- Threshold -40 mV
what is the speed of KV2.1 channels?
slow to open and close
when does KV2.1 close?
sustained current above threshold
- does not inactivate
what happens to KV2.1 during depolarization and hyperpolarization
- when we hyperpolarize the cell there is no current flow ⇒ nothing through the channel
- when we depolarize the cell there is a strong outward current ⇒ more depolarization means more conductance
subunit properties of KV4.1 channels
- 4 alpha subunits and 4 beta
- 6 membrane spanning helices/subunit
- 24 transmembrane helices per functional channel
Non subunit properties of KV4.1 (3)
- Pore loops
- Voltage sensory
- Ion selectivity = K+ only
What does the beta subunit do in KV4.1?
beta subunit is regulatory
what is the direction of potassium flow for KV4.1 channels?
direction depends on the DF
- Threshold -40 mV
what is the speed of KV4.1 channels?
fast opening and close
when does KV4.1 close?
Inactivates shortly after opening
- Transient current above threshold
- Doesn’t function very well when repolarizing the membrane after an action potential
what happens to KV4.1 during depolarization and hyperpolarization
- When hyperpolarized there is no current
- When depolarized there is instantaneous activation of the channel and inactivates immediately => different from KV2.1
- Conductance vs Vm looks same as for KV2.1
subunit properties of hERG channels
- 4 alpha and beta subunits
- 6 membrane spanning helices/subunit
- 24 transmembrane helices per functional channel
Non subunit properties of hERG (3)
- Pore loops
- Voltage sensory
- Ion selectivity = K+ only
What does the beta subunit do in hERG?
subunit is regulatory to alpha
what is the direction of potassium flow for hERG channels?
Direction of current depends on DF
Threshold -40 mV
what is the speed of hERG channels?
fast but delayed
when does hERG close?
Inactivates immediately at the depolarizing step until it comes back to rest
- Transient outward current at the end of the pulse
what happens to hERG during depolarization and hyperpolarization
- nothing when hyperpolarized
- when depolarized nothing happens until you stop depolarizing the cell and remove the voltage clamp
Subunit properties of KIR channels
- 4 alpha subunit, no beta
- 2 membrane spanning helices/subunit ⇒ per alpha unit
- 8 transmembrane helices per functional channel
Non subunit properties of KIR (3)
- Pore loops
- Not voltage sensitive
- Ion selectivity = K+ only
what is the direction of potassium flow for KIR channels?
only allows potassium current inward due to electrical gradient
- DF will never be outward
rectifying
allows flow in one direction (KIR channels)
T/F KIR is voltage dependent?
False
- The channel is not voltage dependent and thus does not have a voltage threshold
when is a cell hyperpolarized?
- Not during an AP
- When there are open Cl- channels
when do you get gK and IK with KIR channels
when Vm < EK
- Because the channel is only able to pass IK inward
T/F KIR channels are always open?
the channel is always open but current only flows during hyperpolarization
Subunit properties of Ca2+ gated K+ channels
- 4 alpha and beta subunits
- 7 membrane spanning helices/subunit
- 28 transmembrane helices per functional channel
- Intracellular domain binds Ca2+ (regulatory)
Non subunit properties of Ca2+ gated K+ (3)
- Pore loops
- 1 type is voltage sensitive ( type 1)=> others are not
- Ion selectivity = K+ only
what is the direction of potassium flow for Ca2+ gated K+ channels?
Direction of current depends on DF (likely outward)
- High Ca2+ will let the channel open at the same threshold as KV2.1 (-40 mV)
- Can cause outward current without and AP
what is the speed of Ca2+ gated K+ channels?
fast opening and delayed close like KV2.1 but calcium induced
- when an action potential is fired calcium will flow inward so this channel activates an outward current that helps repolarize the cell
- If this channel is mutated then you will have seizures ⇒ the cell will stay depolarized from the calcium
when does Ca2+ gated K+ open?
when calcium binds to the channel it opens and potassium leaves
- When there is low internal calcium there isn’t much activation so amplitude is small
- When there is high internal calcium we get a larger current
what happens to Ca2+ gated K+ channels during depolarization and hyperpolarization
when hyperpolarized there is no current ⇒ we can control the internal calcium concentrations in the cell
- Reduce calcium by injecting a calcium buffer and reduce readily available calcium or we can increase it
- same effect to depolarization as KV2.1
what is the calcium sensor for Ca2+ gated K+ channels?
C terminal region is the calcium sensor ⇒ when Ca2+ levels are high enough it binds and activates the channel
subunit properties of 2P K+ leak channels
- 2 alpha subunits and 4 beta
- 4 membrane spanning helices for each alpha subunit
- 8 transmembrane helices per functional channel
Non subunit properties of 2P K+ leak channels (4)
- Pore loops
- Not voltage sensitive ⇒ potassium flows regardless of membrane potential
- Sensitive to pH and mechanical stretch
- Ion selectivity = K+ only
T/F K+ leak channels have a regulatory sub unit?
False no regulatory subunit
what is the direction of potassium flow for 2P K+ leak channels?
Direction of current depends on DF
- This channel is not voltage dependent and thus does not have a voltage threshold
what factors affect 2P K+ leak channel conductance?
Max g at basic pH 8.0 but also open at biological pH 7.4-7.8
at Vm > EK which direction does K+ flow through leak channels?
outward current
at Vm < EK which direction does K+ flow through leak channels?
inward current
T/F 2P K+ leak channels will leak K+ current without an action potential?
True
how does pH affect leak channels from acidic to basic?
the change in pH from acidic where there is no current to a basic pH creates a large outward current
- Conductance are not voltage dependent but are pH dependent
what is true of both Na+ and Ca2+ channels?
you only need 1 protein to form a functional channel but it has multiple domains similar to the subunits in VG K+ channels
- 1 gene encodes the whole channel in comparison to the 4 genes in K+ channels
subunit and domain components of Na+ channels?
- 1 protein forms a channel (single alpha subunit )
- 4 domains (1-4), each of which is similar in structure to a single alpha subunit of the VG K+ channel
- Total of 24 membrane spanning helices
non subunit components of Na+ channels (3)
- 1 pore loop per domain confers Na+ selectivity (4 total)
- Voltage dependence ⇒ 1 voltage sensor per domain (4 total)
- Intracellular inactivation loop (regulatory) ⇒ inactivate soon after activation
what does the beta subunit of Na+ do?
it is regulatory and a different gene
subunit and domain components of Ca2+ channels?
- 1 protein forms a channel (1 alpha subunit)
- Has 4 domains (1-4), each of which is similar in structure to a single alpha subunit of the VGK+ channel
- Total of 24 membrane spanning helices
non subunit components of Ca2+ channels (2)
- 1 pore loop per domain confers Ca2+ selectivity
- Voltage dependence ⇒ 1 voltage sensor per domain
what does the beta subunit of Ca2+ do?
it is regulatory and a different intracellular gene
what do scorpion alpha toxins do?
prolongs Na+ currents
how do scorpion alpha toxins change Na+ currents?
they affect the action potential by changing toxicity ⇒ membrane potential does not rise as high with the alpha toxin
- Still get a somewhat early inward current but this doesn’t turn off
- There is a small inward sodium current throughout the whole voltage step
what do scorpion alpha toxins do to the AP?
In real life this makes the action potential longer in duration by widening the curve
- The channel is more slowly and not completely inactivating
what do scorpion beta toxins do?
make Na+ currents occur at a lower Vm
- shifts the voltage dependance (threshold) to a more hyperpolarized state
what do scorpion beta toxins do to the AP?
- the action potentials will occur at a more negative hyperpolarized state making them hyperactive
- Scrambles the network they are in which makes the animal less able to respond to stimuli or threat
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