Electrophysiological Principles 2 Flashcards
Why is the squid giant axon used in H and H experiemnts?
large enough to control the membrane potential
What was H and H main research?
determine which ion moves when during an AP
What is the effect of decreasing extracellular Na on the AP?
- lower AP peak
- takes longer to repolarize
At what voltage does the peak of the AP occur?
sodium ion equilibrium potential
Why does the membrane potential change during an AP?
during an AP, multiple SETS of channels open/close (Na+, K+)
What is the equilibrium potential of Na+?
+60 mV
What is the equilibrium potential of K+?
-90 mV
The effect on the Na conductance is _______________.
regenerative
For Na+, the depolarization is ____________ feedback.
positive
For K+, the depolarization is ____________ feedback.
negative
How did H and H investigate the activity of Na+ and K+ voltage-gated ion channels and the conductances of Na+ and K+?
voltage clamp:
- maintains voltage
- measure current
- calculate R
- calculate g
How does the voltage clamp work? Draw the setup.
- inject charge –> channels open, ions move across the membrane
- measure how much Na+ goes in
- when Vm is different from the command potential, clamp amplifier injects current into the axon through a second electrode (inject equal and opposite charge to maintain voltage) –> Vm becomes same as command potential
- record current
- calculate conductance
(slide 7)
What is an inward current by convention? Example?
- inward flow of positive charge
- negative
- Na+ influx
What is an outward current by convention? Example?
- outward flow of positive ions
- positive
- K+ efflux
When negative ions flow out of cells, what that be an inward or outward current? Example?
- inward
- Cl- efflux
What is the driving force for an ion? How does it relate to V=IR? How do we signify the direction of the driving force?
- Vm - Eion
- I = g(Vm - Eion)
- -/+ sign tells us the direction of the driving force (see current conventions)
What are the 3 currents that occur in a voltage clamp experiment, in order? Draw and label them on a graph.
- capacitative current
- early current (inward)
- late current (outward)
(slide 11)
what does the capacitative current represent?
- redistribution of charge across membrane (injection of current/charges)
- outward, positive current
What does the early current represent?
- inward, negative current
- Na+ influx
What does the late current represent?
- outward, positive current
- K+ efflux
TRUE or FALSE: the Na+ current deactivates whereas the K+ current does not deactivate.
How can you tell on a graph?
TRUE
early inward current becomes outward current, whereas late outward current continues outward
How did H and H find out that the inward current was carried by Na+?
- replace Na+ in the bath with equimolar choline
- this abolished the early inward current, and left only the outward current
- subtracted outward current from original/complete current trace
- left with inward current
TRUE or FALSE: when isolated, the late current begins sooner than the early current
FALSE: early current starts sooner than late current
Na+ and K+ channels both open in response to depolarization. However, why does Na+ influx begin sooner than K+ efflux, and why do they not end at the same time?
- Na+ flows in faster
- K+ channels open slower
- Na+ channels remain INACTIVATED until depolarization ends
- K+ channels remain ACTIVATED until depolarization ends
What is the effect of TTX (tetrodotoxin) on ion channels? which ion channels does it affect? How does this affect the entire current trace (draw)?
- physically blocks voltage-gated Na+ channels
- early inward current disappears (slide 15)
What is the effect of TEA (tetra ethyl ammonium) on ion channels? which ion channels does it affect? How does this affect the entire current trace (draw)?
- physically blocks K+ voltage-gated channels
- late outward current disappears (Slide 15)
TRUE or FALSE: as conductance increases, the driving force decreases.
TRUE
When does the early inward current go back to zero?
after 3-4 ms
When does the late outward current go back to zero?
never
What is the sequence of events of a current?
- early current activates (Na+ flows in)
- early current inactivates (Na+ stops flowing in)
- late current activates (K+ flows out)
How did H&H study inactivation of the early current?
giving prepulses to the cell before voltage clamping the membrane at a predetermined voltage
How does a hyperopolarizing prepulse affect sodium current at depolarization? Explain. Draw 2 graphs with membrane potential and current to explain.
- larger sodium current (slide 19)
- hyperpolarization resets the sodium channels and opens the inactivation gates –> more channels are available to open at depolarization –> larger sodium current
What percentage of V-gated Na+ channels are inactivated at the resting membrane potential? Draw the inactivation curve.
40% (slide 20)
TRUE or FALSE: Na+ and K+ conductances have the same voltage dependance.
TRUE
At what voltage does maximum conductance for both Na+ and K+ occur? Draw the conductance curve for these ions.
+10-20 mV (slide 21)
Compare AP voltage with Na+ and K+ conductances on a graph.
slide 22
- Na+ and K+ conductances change during and AP
- AP voltage already decreasing by the time K+ starts flowing out (i.e. membrane potential drives conductance)
- K+ conductance ends before the AP ends
What is the threshold by definition?
voltage at which inward and outward currents are equal and exactly balance each other
Draw a graph to compare the membrane potential curve when excess K+ flows out vs Na+ flows in at threshold
slide 23
What is the refractory period by definition?
period of time during and immediately following an AP in which another AP is either difficult (RRP) or impossible (ARP) to initiate
What are the 2 types of refractory periods?
- absolute refractory period
- relative refractory period
Why can an AP not occur during an absolute refractory period (ARP)?
- Na+ channels have inactivated
- large K+ conductance forcing Vm to negative potentials (see graph comparing Ap voltage and Ion conductances, slide 22)
Why can an AP occur during the relative refractory period (RRP)?
- Na+ channels are moving back into activated state
- K+ conductance is smaller than in the ARP
TRUE or FALSE: the inactivation gate is regulated by voltage.
FALSE: regulated by diffusion and binding
Draw the Na+ channel with its gates at rest, immediately after depolarization, and 5 ms after depolarization. (slide 27)
- at rest: m gate closed, h gate open
- imm. after depol.: m gate open, h gate open
- 5ms after depol.: m gate open, h gate closed
Draw the K+ channel with its gate at rest, immediately after depolarization, and 5 ms after depolarization. (Slide 28)
- at rest: n gate closed
- imm. after depl.: n gate closed
- 5ms after depol.: n gate open
What is the sequence of ion channel gating events during an AP?
- Na+ channels open (activate)
- Na+ channels inactivate
- K+ channels open (activate)
- K+ channels close (NOT INACTIVATE)
- Na+ channels move back to rest
How is the h gate indirectly dependent on voltage? what is it directly dependent on?
- m gate closing forces h gate to open (m-gate dependent on voltage)
- h gate directly dependent on m gate configuration
What happens to the activation gate for Na+ and K+ channels when Vm goes back down?
activation gates close and reset (resting state)
what are the 4 modes of the patch-clamp technique?
- cell attached
- whole cell
- inside out
- outside out
What is the patch clamp technique used for?
looking at individual channels
Which modes of the patch-clamp technique record single channel activity, and which record APs, macro currents, and miniature currents?
- single channel: cell attached, inside out, outside out
- APs, etc: whole cell
Which mode of patch clamp technique allows access to channels on the inner membrane?
inside out
Which mode of patch clamp technique allows access to channels on the outer membrane? what kind of channels are these?
outside-out; ionotropic receptors (GABA, glutamate, nicotinic, etc.)
How do we classify channels?
based on conductance (because it is the only constant in I = gV)
How do you identify conductance on an I-V graph?
slope
Current flow through ion channels depends upon _______________.
membrane potential (e.g. see I-V graph)
How many domains does the Na+ channel have? How many and which subunits? How many transmembrane units does each domain have?
4 domains - 1 alpha subunit; 6 units in each domain (S1-S6)
How man domains does the Ca2+ channel have? How many and which subunits? How many transmembrane units does each domain have?
4 domains - 1 alpha subunit; 6 units S1-S6 (almost identical to Na+ channel)
How many domains does the K+ channel have? How many and which subunits? How many transmembrane units does each domain have?
4 domains - 4 alpha subunits and 4 beta subunits; 6 units S1-S6
TRUE or FALSE: K+ channels are larger than Na+ channels and Ca2+ channels.
FALSE: when assembled, they are all roughly the same size
TRUE or FALSE: H&H treated potassium channels as if they had an inactivation gate
FALSE: H&H treated them as if NO inactivation gate
Which region of ion channel domains is involved in activation?
S4
How does saxitonin affect Na+ channels? Where can this substance be found?
- binds to AAs in the S5-S6 region and physically blocks the channel (like TTX)
- found in shellfish
Na+ currents have the same general shape and overall characteristics. What does this imply about different organisms?
during evolution, Na+ channels developed for fast activation for APs
on average, how long does it take Na+ channels to open once the cell is depolarized?
within the first few milliseconds of the depolarization
TRUE or FALSE: Na+ channels are mostly closed at very negative potentials.
TRUE
TRUE or FALSE: Na+ channels spend less time in the open state at depolarized potentials.
FALSE: spend more time open at depolarized potentials
TRUE or FALSE: the activation curve for voltage gated Na+ channels shifts right in the presence of batrachotoxin.
FALSE: shift left (i.e. activated at more negative potentials in the presence of batrachotoxin)
What are the effects of batrachotoxin on Na+ channels?
- opens channels at more negative potentials
- prevents inactivation (may lead to cell death)
Draw the activation curve of Na+ channels in the presence of batrachotoxin.
slide 41
which animal do batrochotoxin and pumiliotoxin come from?
poison arrow frogs
Which region of the ion channel acts as the voltage sensor?
S4
TRUE or FALSE: every 2nd AA in the S4 region is positively charged.
FALSE: every 3rd
every 3rd AA in the S4 region is positively charged. what does this imply?
- repel positive charges
- when the inside of the cell is negative, the S4 region keeps the m/n (activation) gate closed
activation vs inactivation vs deactivation
- activation: opening of voltage-gate channels caused by depolarization, or a change in voltage
- inactivation: closing of voltage-gated channels through movement of an inactivation gate that is separate and distinct from the activation gate
- deactivation: closing of voltage-gated channels through movement of the opening/activation gate back into closed position (inactivation gate does not close the channel)
Describe activation, inactivation, and deactivation in terms of m, n, and h gates.
- activation: m and n gate open
- inactivation: h gate close
- deactivation: m and n gate close
How does the inactivation gate close an ion channel?
by diffusion and binding (i.e. conformation change)
TRUE or FALSE: inactivation is voltage-dependent
FALSE: At rest, when m-gate closed, channel is in conformation such that h gate cannot bind to pore and block channel. At depol, when m-gate open, channel is in conformation such that h gate is able to bind to pore and block channel.
(i.e. dependent on conformation change)
What acts as the inactivation gate for most K+ channels?
amino terminus (also beta unit)
When do K+ channels open after depolarization?
some time after depol, not immediately following
TRUE or FALSE: K+ channels stay open for the duration of the depolarizing pulse.
TRUE
What is rectification?
change in resistance/conductance that is dependent on the direction of ion flow
On an I-V graph, how can we tell if a current has experienced rectification?
there is a change in the slope
What is an ion channel analogous to in a circuit?
resistor
What kind of rectifier was considered by H&H in K+ channels?
delayed outward rectifier (above axis = high g, below axis = low g)
(see H&H K+ curve on slide 49)
How can you tell if an ion channel is an inward or outward rectifier based on an I-V graph?
- the greater slope = greater conductance = greater current (I = g(Vm - Eion))
- if greater current is negative = greater current flowing in –> INWARD RECTIFIER
- if greater current is positive = greater current flowing out –> OUTWARD RECTIFIER
(slide 49)
Arrange the following K+ channels from experiencing the most inactivation to the least inactivation:
Shaw, Shaker, Shal, Shab
Shaker > Shal > Shab > Shaw
(note: Shaw experiences no inactivation)
Which enzyme removes inactivation of K+ shaker channels?
proteinase (trypsin)
How did they know that the inactivation gate on K+ shaker channels was found on the alpha unit?
when trypsin was administered, part of alpha subunit was the only thing that was reconstituted (while inactivation did not occur)
which part of the amino terminus is important for inactivation?
first 20 AA
how many inactivation gates do K+ channels have? explain how you know this.
4 inactivation gates bc 4 alpha units
TRUE or FALSE: as the number of inactivation gates gets smaller, the rate of inactivation got smaller.
TRUE
How do we know that K+ channels inactivate by diffusion and not voltage?
if only one of the 4 alpha units in a K+ channel have an inactivation gate, it takes 4x as long to inactivate the channel
TRUE or FALSE: Na+ and Ca2+ channels are composed of single domains, but 4 of these domains come together to form a channel.
FALSE: K+ channels are composed of 4 single domains; Na+ and Ca2+ are composed of 4 domains LINKED together
Which region of the domain forms part of the pore of the channel?
S5-S6
What kind of charges is the S4 region lined with?
positive
How do selectivity filters ensure that only Na+ ions move through Na+ channels? K+ through K+ channels? Draw a diagram to explain
- ions are surrounded by hydration shell
- Na+ hydration shell is very large, Na+ is small
- K+ hydration shell is very small, K+ is very large
- Na+ channel strips away H2O –> only Na+ is small enough to fit (K+ without hydration shell too large)
- K+ channel does not have the energy to strip away H2O –> only K+ with its hydration shell can fit (Na+ with hydration shell too large)
