Lecture 12: Patch Clamp Electrophysiology and Single-Channel Recordings

Describe the voltage-clamp process.

clamp the membrane potential at a particular voltage, and change the V_m as desired for a certain experiment/measurement

What is the voltage-clamp used to reveal?

used to reveal ionic currents flowing across the cell membrane

True or False: In a good voltage-clamp recording, you have a great degree of control of the cell you’re recording from.

true

What does the current clamp reveal?

reveals changes in voltage (including action potentials)

True or False: In the current clamp, some current can be injected, or no current can be injected.

true

True or False: In a good current clamp recording, you’re not really controlling the cell.

true

Describe how to use the voltage-clamp to measure ionic current.

• one electrode injects current to set V_m to particular value

• second electrode detects current in the membrane required to maintain V_m at designated holding potential or move it to a new V_m

True or False: Most modern patch clamp equipment uses one electrode to both inject and detect changes in current/voltage simultaneously.

true

What is I_m?

total membrane current; sum of individual currents through parallel branches of circuit

What is the equation for I_m?

• I_m = I_C + I_X

• I_m = I_C + G_X(V_m - E_X)

True or False: In cell-attached configuration, tight contact is created between the pipette and the plasma membrane.

true

True or False: In whole-cell configuration, the pipette has direct access to the cytoplasm.

true

What is cell-attached configuration?

tight contact is created between the pipette and the plasma membrane

What is inside-out configuration?

the pulled membrane breaks away from the cell and allows access to the cytoplasmic face of the ion channel

What is whole-cell configuration?

the pipette has direct access to the cytoplasm

What is outside-out configuration?

the extracellular face of the ion channel is now accessible, and the broken ends of the membrane anneal

What is unitary current?

current generated by a single ion channel

True or False: Unitary currents can summate (add together) to generate larger amplitude (larger size) currents.

true

What is macroscopic current?

the current activity of a population of channels sampled

What can macroscopic current be?

either large (across whole cell) or few (outside-out or inside-out patch)

What type of gating does macroscopic current represent?

represents nonsynchronous gating (opening/closing) of single channels

What is macroscopic current obtained by?

obtained by averaging several microscopic current recordings

True or False: Single channels fluctuate between open and closed states (as long as voltage and/or ligand-binding requirements for channel opening are met).

true

True or False: Channels are probabilistic (stochastic) and occurs randomly.

true

What is the probability of channel opening (P_O)?

fraction of time channel is in open state (open time / total time)

What is the slope of a single channel I-V relationship?

the unitary conductance (gamma) of that channel

What is the slope of the I-V/unitary conductance typically?

1-500 pS

How do you generate macroscopic current (I_X)?

averaging and summating individual single channel records

What is the equation for I_X?

I_X = n x i x P_O where n is number of channels, i is unitary current, and P_o is probability of opening

What determines the shape of an I-V curve?

• the number of ion channels in the membrane

• unitary current

• probability of opening

What are the 2 characteristics of unitary current?

• ion channel intrinsic properties

• [X_O] / [X_i] ratio

True or False: P_O can be voltage-independent or voltage-dependent.

true