LEC19: Voltage-Clamp and Patch-Clamp

Question 1

why can’t you use a current clamp to figure out an action potential’s conductances?

Answer 1

b/c current-clamp provides poor control over the membrane potential or the currents that flow through the membrane -

when inject current, it bypasses the membrane, triggers conductance changes that cause an action potential

both conductance and resistance are changing in action potential, so cannot solve Ohm’s law and know what happens to currents

Question 2

how does a voltage-clamp work?

Answer 2

voltage-clamp keeps Vm constant, measures Im (command potential)

you set the command voltage, and it compares changes in voltage inside the cell to that command voltage, and converts the voltage difference to a proportional current thats delivered to the cell through an electrode

thus it detects different btwn set command potential and the actual membrane potential; and produces a proportional current to maintain your set command potential in the cell

Question 3

what is the sign of an inward/outward current in a voltage clamp?

Answer 3

inward current on voltage-gated Na+ channels: voltage clamp is **negative **(injects negative charged ions)

outward current on voltage-gated Na+ channels: voltage clamp is positive (injects positive charged ions)

Question 4

action potential of squid giant axon

Answer 4

??

Question 6

what can an I-V plot show?

Answer 6

study ion currents through membranes - which ions mediate the action potential

use I-V plot to detemrine reversal potential

Question 7

how do you know the equilibrium potential, V_Na, from this I-V graph?

Answer 7

flat area

V_Na = +50 mV

Question 8

what is TTX?

Answer 8

tetrodotoxin

a neurotoxin that blocks voltage Na+ channels

eliminates transient inward current

makes your tongue numb when you eat fugu

Question 9

what is TEA?

Answer 9

tetraethylammonium

blocks K+ channels

blocks K+, prevents persistent outward current

Question 10

what is the I-V plot for a membrane containing only non-rectifying K+ Channels?

what is the slope equal to?

Answer 10

slope = all values of Vm b/c conductance doesn’t change w/ voltage

Question 11

what does the patch clamp method allow study of?

Answer 11

single-channel level study

Question 12

how does the patch clamp work?

Answer 12

pipette is applied to outside of cell membrane; creates high resistance seal between glass and membrane; pipette now records the channels that’re present in that patch of membrane

may be 1, or several, channels

thus allows single channel microscopic recording or many channel macroscopic recording

Question 13

what is the cell-attached mode for the patch clamp?

Answer 13

initial patch configuration

intracellular environment remains intact

Question 14

what is the inside-out patch?

Answer 14

pull pipette away from cell; patch remains sealed to pipette, former cytoplasmic face of memrbane is exposed to bathing sol’n, where ion concentratiosn and other conditions can be manipulated

Question 15

what is the whole cell configuration of path clamp configuration?

Answer 15

patch is ruptured while pipette is still attach to the membrane; as result, the pipette, containing much larger volume of solution thatn cell, dialyzes interior of cell; diffusible molecules are lost; cytosol replaced w/ pipette sol’n

here, macroscopic combined activity of channels in intact membrane is recorded