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ZOOL 342 > Electrophysiological Principles 2 > Flashcards

Electrophysiological Principles 2 Flashcards

1
Q

Why is the squid giant axon used in H and H experiemnts?

A

large enough to control the membrane potential

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2
Q

What was H and H main research?

A

determine which ion moves when during an AP

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3
Q

What is the effect of decreasing extracellular Na on the AP?

A
  • lower AP peak
  • takes longer to repolarize
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4
Q

At what voltage does the peak of the AP occur?

A

sodium ion equilibrium potential

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5
Q

Why does the membrane potential change during an AP?

A

during an AP, multiple SETS of channels open/close (Na+, K+)

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6
Q

What is the equilibrium potential of Na+?

A

+60 mV

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7
Q

What is the equilibrium potential of K+?

A

-90 mV

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8
Q

The effect on the Na conductance is _______________.

A

regenerative

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9
Q

For Na+, the depolarization is ____________ feedback.

A

positive

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10
Q

For K+, the depolarization is ____________ feedback.

A

negative

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11
Q

How did H and H investigate the activity of Na+ and K+ voltage-gated ion channels and the conductances of Na+ and K+?

A

voltage clamp:

  1. maintains voltage
  2. measure current
  3. calculate R
  4. calculate g
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12
Q

How does the voltage clamp work? Draw the setup.

A
  1. inject charge –> channels open, ions move across the membrane
  2. measure how much Na+ goes in
  3. 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
  4. record current
  5. calculate conductance

(slide 7)

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13
Q

What is an inward current by convention? Example?

A
  • inward flow of positive charge
  • negative
  • Na+ influx
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14
Q

What is an outward current by convention? Example?

A
  • outward flow of positive ions
  • positive
  • K+ efflux
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15
Q

When negative ions flow out of cells, what that be an inward or outward current? Example?

A
  • inward
  • Cl- efflux
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16
Q

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?

A
  • Vm - Eion
  • I = g(Vm - Eion)
  • -/+ sign tells us the direction of the driving force (see current conventions)
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17
Q

What are the 3 currents that occur in a voltage clamp experiment, in order? Draw and label them on a graph.

A
  1. capacitative current
  2. early current (inward)
  3. late current (outward)

(slide 11)

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18
Q

what does the capacitative current represent?

A
  • redistribution of charge across membrane (injection of current/charges)
  • outward, positive current
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19
Q

What does the early current represent?

A
  • inward, negative current
  • Na+ influx
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20
Q

What does the late current represent?

A
  • outward, positive current
  • K+ efflux
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21
Q

TRUE or FALSE: the Na+ current deactivates whereas the K+ current does not deactivate.

How can you tell on a graph?

A

TRUE

early inward current becomes outward current, whereas late outward current continues outward

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22
Q

How did H and H find out that the inward current was carried by Na+?

A
  • 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
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23
Q

TRUE or FALSE: when isolated, the late current begins sooner than the early current

A

FALSE: early current starts sooner than late current

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24
Q

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?

A
  • Na+ flows in faster
  • K+ channels open slower
  • Na+ channels remain INACTIVATED until depolarization ends
  • K+ channels remain ACTIVATED until depolarization ends
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25
Q

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)?

A
  • physically blocks voltage-gated Na+ channels
  • early inward current disappears (slide 15)
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26
Q

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)?

A
  • physically blocks K+ voltage-gated channels
  • late outward current disappears (Slide 15)
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27
Q

TRUE or FALSE: as conductance increases, the driving force decreases.

A

TRUE

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28
Q

When does the early inward current go back to zero?

A

after 3-4 ms

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29
Q

When does the late outward current go back to zero?

A

never

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30
Q

What is the sequence of events of a current?

A
  1. early current activates (Na+ flows in)
  2. early current inactivates (Na+ stops flowing in)
  3. late current activates (K+ flows out)
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31
Q

How did H&H study inactivation of the early current?

A

giving prepulses to the cell before voltage clamping the membrane at a predetermined voltage

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32
Q

How does a hyperopolarizing prepulse affect sodium current at depolarization? Explain. Draw 2 graphs with membrane potential and current to explain.

A
  • 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
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33
Q

What percentage of V-gated Na+ channels are inactivated at the resting membrane potential? Draw the inactivation curve.

A

40% (slide 20)

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34
Q

TRUE or FALSE: Na+ and K+ conductances have the same voltage dependance.

A

TRUE

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35
Q

At what voltage does maximum conductance for both Na+ and K+ occur? Draw the conductance curve for these ions.

A

+10-20 mV (slide 21)

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36
Q

Compare AP voltage with Na+ and K+ conductances on a graph.

A

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
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37
Q

What is the threshold by definition?

A

voltage at which inward and outward currents are equal and exactly balance each other

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38
Q

Draw a graph to compare the membrane potential curve when excess K+ flows out vs Na+ flows in at threshold

A

slide 23

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39
Q

What is the refractory period by definition?

A

period of time during and immediately following an AP in which another AP is either difficult (RRP) or impossible (ARP) to initiate

40
Q

What are the 2 types of refractory periods?

A
  1. absolute refractory period
  2. relative refractory period
41
Q

Why can an AP not occur during an absolute refractory period (ARP)?

A
  1. Na+ channels have inactivated
  2. large K+ conductance forcing Vm to negative potentials (see graph comparing Ap voltage and Ion conductances, slide 22)
42
Q

Why can an AP occur during the relative refractory period (RRP)?

A
  1. Na+ channels are moving back into activated state
  2. K+ conductance is smaller than in the ARP
43
Q

TRUE or FALSE: the inactivation gate is regulated by voltage.

A

FALSE: regulated by diffusion and binding

44
Q

Draw the Na+ channel with its gates at rest, immediately after depolarization, and 5 ms after depolarization. (slide 27)

A
  • 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
45
Q

Draw the K+ channel with its gate at rest, immediately after depolarization, and 5 ms after depolarization. (Slide 28)

A
  • at rest: n gate closed
  • imm. after depl.: n gate closed
  • 5ms after depol.: n gate open
46
Q

What is the sequence of ion channel gating events during an AP?

A
  1. Na+ channels open (activate)
  2. Na+ channels inactivate
  3. K+ channels open (activate)
  4. K+ channels close (NOT INACTIVATE)
  5. Na+ channels move back to rest
47
Q

How is the h gate indirectly dependent on voltage? what is it directly dependent on?

A
  • m gate closing forces h gate to open (m-gate dependent on voltage)
  • h gate directly dependent on m gate configuration
48
Q

What happens to the activation gate for Na+ and K+ channels when Vm goes back down?

A

activation gates close and reset (resting state)

49
Q

what are the 4 modes of the patch-clamp technique?

A
  1. cell attached
  2. whole cell
  3. inside out
  4. outside out
50
Q

What is the patch clamp technique used for?

A

looking at individual channels

51
Q

Which modes of the patch-clamp technique record single channel activity, and which record APs, macro currents, and miniature currents?

A
  • single channel: cell attached, inside out, outside out
  • APs, etc: whole cell
52
Q

Which mode of patch clamp technique allows access to channels on the inner membrane?

A

inside out

53
Q

Which mode of patch clamp technique allows access to channels on the outer membrane? what kind of channels are these?

A

outside-out; ionotropic receptors (GABA, glutamate, nicotinic, etc.)

54
Q

How do we classify channels?

A

based on conductance (because it is the only constant in I = gV)

55
Q

How do you identify conductance on an I-V graph?

A

slope

56
Q

Current flow through ion channels depends upon _______________.

A

membrane potential (e.g. see I-V graph)

57
Q

How many domains does the Na+ channel have? How many and which subunits? How many transmembrane units does each domain have?

A

4 domains - 1 alpha subunit; 6 units in each domain (S1-S6)

58
Q

How man domains does the Ca2+ channel have? How many and which subunits? How many transmembrane units does each domain have?

A

4 domains - 1 alpha subunit; 6 units S1-S6 (almost identical to Na+ channel)

59
Q

How many domains does the K+ channel have? How many and which subunits? How many transmembrane units does each domain have?

A

4 domains - 4 alpha subunits and 4 beta subunits; 6 units S1-S6

60
Q

TRUE or FALSE: K+ channels are larger than Na+ channels and Ca2+ channels.

A

FALSE: when assembled, they are all roughly the same size

61
Q

TRUE or FALSE: H&H treated potassium channels as if they had an inactivation gate

A

FALSE: H&H treated them as if NO inactivation gate

62
Q

Which region of ion channel domains is involved in activation?

A

S4

63
Q

How does saxitonin affect Na+ channels? Where can this substance be found?

A
  • binds to AAs in the S5-S6 region and physically blocks the channel (like TTX)
  • found in shellfish
64
Q

Na+ currents have the same general shape and overall characteristics. What does this imply about different organisms?

A

during evolution, Na+ channels developed for fast activation for APs

65
Q

on average, how long does it take Na+ channels to open once the cell is depolarized?

A

within the first few milliseconds of the depolarization

66
Q

TRUE or FALSE: Na+ channels are mostly closed at very negative potentials.

A

TRUE

67
Q

TRUE or FALSE: Na+ channels spend less time in the open state at depolarized potentials.

A

FALSE: spend more time open at depolarized potentials

68
Q

TRUE or FALSE: the activation curve for voltage gated Na+ channels shifts right in the presence of batrachotoxin.

A

FALSE: shift left (i.e. activated at more negative potentials in the presence of batrachotoxin)

69
Q

What are the effects of batrachotoxin on Na+ channels?

A
  1. opens channels at more negative potentials
  2. prevents inactivation (may lead to cell death)
70
Q

Draw the activation curve of Na+ channels in the presence of batrachotoxin.

A

slide 41

71
Q

which animal do batrochotoxin and pumiliotoxin come from?

A

poison arrow frogs

72
Q

Which region of the ion channel acts as the voltage sensor?

A

S4

73
Q

TRUE or FALSE: every 2nd AA in the S4 region is positively charged.

A

FALSE: every 3rd

74
Q

every 3rd AA in the S4 region is positively charged. what does this imply?

A
  • repel positive charges
  • when the inside of the cell is negative, the S4 region keeps the m/n (activation) gate closed
75
Q

activation vs inactivation vs deactivation

A
  • 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)
76
Q

Describe activation, inactivation, and deactivation in terms of m, n, and h gates.

A
  • activation: m and n gate open
  • inactivation: h gate close
  • deactivation: m and n gate close
77
Q

How does the inactivation gate close an ion channel?

A

by diffusion and binding (i.e. conformation change)

78
Q

TRUE or FALSE: inactivation is voltage-dependent

A

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)

79
Q

What acts as the inactivation gate for most K+ channels?

A

amino terminus (also beta unit)

80
Q

When do K+ channels open after depolarization?

A

some time after depol, not immediately following

81
Q

TRUE or FALSE: K+ channels stay open for the duration of the depolarizing pulse.

A

TRUE

82
Q

What is rectification?

A

change in resistance/conductance that is dependent on the direction of ion flow

83
Q

On an I-V graph, how can we tell if a current has experienced rectification?

A

there is a change in the slope

84
Q

What is an ion channel analogous to in a circuit?

A

resistor

85
Q

What kind of rectifier was considered by H&H in K+ channels?

A

delayed outward rectifier (above axis = high g, below axis = low g)

(see H&H K+ curve on slide 49)

86
Q

How can you tell if an ion channel is an inward or outward rectifier based on an I-V graph?

A
  • 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)

87
Q

Arrange the following K+ channels from experiencing the most inactivation to the least inactivation:

Shaw, Shaker, Shal, Shab

A

Shaker > Shal > Shab > Shaw

(note: Shaw experiences no inactivation)

88
Q

Which enzyme removes inactivation of K+ shaker channels?

A

proteinase (trypsin)

89
Q

How did they know that the inactivation gate on K+ shaker channels was found on the alpha unit?

A

when trypsin was administered, part of alpha subunit was the only thing that was reconstituted (while inactivation did not occur)

90
Q

which part of the amino terminus is important for inactivation?

A

first 20 AA

91
Q

how many inactivation gates do K+ channels have? explain how you know this.

A

4 inactivation gates bc 4 alpha units

92
Q

TRUE or FALSE: as the number of inactivation gates gets smaller, the rate of inactivation got smaller.

A

TRUE

93
Q

How do we know that K+ channels inactivate by diffusion and not voltage?

A

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

94
Q

TRUE or FALSE: Na+ and Ca2+ channels are composed of single domains, but 4 of these domains come together to form a channel.

A

FALSE: K+ channels are composed of 4 single domains; Na+ and Ca2+ are composed of 4 domains LINKED together

95
Q

Which region of the domain forms part of the pore of the channel?

A

S5-S6

96
Q

What kind of charges is the S4 region lined with?

A

positive

97
Q

How do selectivity filters ensure that only Na+ ions move through Na+ channels? K+ through K+ channels? Draw a diagram to explain

A
  • 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)

ZOOL 342 (21 decks)

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