BB Lecture 19: Analysis of membrane behavior (voltage and patch clamps) Flashcards
What is the current-clamp method?
using an intracellular electrode to manipulate the membrane potential and seeing how Vm responds (in accordance with Ohm’s law)
can be used to change the voltage-dependent conductance
When are we more interested in measuring conductance of a membrane?
when we are trying to analyze how channels respond to changes in membrane potential, second messenger drugs, and other factors
How does the voltage-clamp method solve the technical problems with using Ohm’s law in the current clamp method?
keeps Vm constant and measures Im (even though the gm might change due to the behavior of voltage-dependent channels)
with Vm constant, any time dependent variation in Im must reflect changes in underlying membrane conductance
Describe the set-up of the voltage-clamp method.
two microelectrodes are inserted into the cell
one measures Vm (voltage recording electrode) and one passes current into the cell (current passing electrode)
between these two there is a differential amplifier that compares the voltage read by the recording electrode and regulates it to the (user-set) command voltage to be passed into the cell by the pass electrode (thus keeping Vm constant via negative feedback)
What does the drug tetrodotoxin do?
TTX
blocks voltage-gated Na+ channels
(blocks inward current- shows persistent outward current)
What does the drug tetraethylammonium do?
TEA
blocks most K+ channels
(blocks outward current- shows persistent inward current)
What is an I-V Plot?
a graph of the relationship between current and voltage where slope=g
What is the I-V Plot used for?
analytical tool for the study of ion currents through membranes
What is the equilibrium potential for the ions carrying a current and how can it be identified on an I-V Plot?
the voltage at which permeant ions are equally likely to move inward or outward through open channels (ie there is no driving force)
the value of Vm where Im measures zero
In voltage clamp recordings outward currents have ____ values, and inward currents have ____ values. Why?
positive values
negative values
because the direction of the current that the amplifier injects into the cell offsets currents flowing through membrane channels
What would the I-V Plot look like if you were to add or take away K+ leak channels?
add: slope is steeper, but Vm (the x-intercept) remains constant at Vk
subtract: slope is less steep and Vm (the x-intercept) remains the same at Vk
How does the shape of an I-V Plot differ between a membrane with only leak channels vs a membrane with only Kir channels vs a membrane with only voltage-gated K+ channels?
leak: linear
Kir: logarithmic
voltage-gated: sigmoidal
Describe the parts of the I-V Plot for a membrane with only voltage-gated K+ channels.
starts with all channels closed (slope=0)
second half is when progressively more channels are open (slope is constantly increasing)
last section when max channels are open (slope is constant)
Describe the shape of the I-V Plot for a membrane with only voltage-gated Na+ (or Ca+) channels.
upside-down parabolic (flat until Vm=-50 mV, then steep negative slope due to relatively small inward current until Vm=-30mV)
then positive linear slope until maximal g (x-intercept at VNa)
What is the patch-clamp method?
an electrolyte-filled glass pipette forms a tight, high electrical resistance seal with the outside of a cell’s bilayer
ion concentration inside the pipette resembles extracellular fluid, but can be manipulated for experimental conditions
area of the patch normally has one or a small number of channels so you can track only those channels reactions to your stimuli
