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Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Physiology Lecture 6

Voltage Clamp

Objectives

1. Understand the use of Ag-AgCl electrodes for measurements of

2. Understand the divider circuit and how it can be used to describe the consequences of leak pathways in membrane potential measurements.

3. Understand the basic principles of two electrode voltage clamp electrophysiology.

4. Understand the types of configurations and how they can be used to characterize channel function.

5. Understand how various voltage clamp protocols can be used to characterize the gating properties of ion channels.

6. Understand how ion selectivity can be determined for voltage-activated ion channels.

Readings

1. Spunger, L.K., Stewig, N.J. and S.M. O’Grady, Effects of charybdotoxin on K channel (Kv1.2) deactivation and inactivation kinetics. European J. Pharmacol. 314:357-64, 1996.

2. Boland, L.M., M. Jiang, S. Y. Lee, S.C. Fahrenkrug, M.C. Harnett, and S.M. O’Grady, Functional properties of a brain-specific N-terminally spliced modulator of Kv4 channels. Am. J. Physiol. [Cell Physiol.] 285:C161-C171, 2003

3. The Guide: A Guide to Electrophysiology & Biophysics Laboratory Techniques. Rivka Sherman-Gold, Editor. Molecular Devices/MDS Analytical technologies. 2007.

1 Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 6

A. Measuring membrane potentials The goal of this discussion is to provide some background information about the principles and techniques that are used to investigate the functional properties of ion channels and electrogenic transporters. The most widely used approach involves voltage clamping the cell or membrane using a amplifier to control the injection of current needed to maintain a constant voltage across the plasma membrane.

Background concepts:

1. Measuring membrane potentials and ionic currents using AgCl electrodes.

Electrodes at a liquid interface must smoothly transform the flow of electrons in a copper wire to the flow of in solution. For each ion, current flow in the bulk solution is proportional to the potential difference. Several types of electrodes can be used in electrophysiological measurements, but the most common is the Ag/AgCl electrode, which is simply a silver wire coated with silver chloride. For this electrode, electrons flowing from the copper wire convert the AgCl to Ag atoms and Cl- ions which become hydrated and enter the solution. If electrons flow in the opposite direction, then Ag atoms in the wire give up their electrons and combine with Cl- ions in solution to form a AgCl precipitate on the surface of the wire. Figure 1: Ag/AgCl electrode reactions in solution Some characteristics of Ag/AgCl electrodes:  These electrodes require Cl- in the recording solution in order to perform properly.  In order to complete the circuit, two electrodes are required.  If the two electrodes interface with solutions with different Cl- concentrations (e.g. 3M KCl vs. 120 mM Cl-), a potential difference between the solution and the electrode will result. This potential difference is the liquid junction potential and must be subtracted from any voltage measurement made using the electrodes.  If AgCl becomes depleted because of excessive current flow, Ag will leak from the wire into the solution and could poison the cells. Additionally, the half cell potentials will become unstable as a result of unpredictable, poorly reversible surface reactions due to other anions in solution and impurities in the silver wire leading to electrode polarization.

2. The voltage divider and the issue of leak pathways

Figure 2 shows a typical voltage divider circuit where a battery is connected to two in series. In this circuit the total voltage (Erp= ) is equal to the sum of the voltage drops across each . V1 + V2 = Erp

2 Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 6

To determine the magnitude of these , simply multiply Erp by the fractional resistance contributed by R each of the resistors. R 1 1 V1  Erp  R1  R2 V + V = E R1 1 2 rp Thus: V1  Erp  R1  R2 R R V  E  2 R 2 2 rp 2 R1  R2 V2  Erp  R1  R2

Figure 2: Voltage divider circuit

An application of the voltage divider is presented in Figure 3.

This figure shows the measurement of membrane potential using a conventional voltage sensing microelectrode. Note that in the inset figure cell impalement produces a pathway for current to leak across the membrane. The magnitude of this leak depends on the ability of the membrane to reseal around the electrode. The consequences of this leak on measurement of membrane potential can be analyzed using a voltage divider circuit described above. Since the internal resistance of Intracellular the voltmeter is extremely high relative to R K E (resistance of K selective channels in the K RS membrane) and RS (seal resistance) essentially no Rs V V  EK  RS  RK current flows to the voltmeter so that RK and RS RK are in series. Analysis of the circuit shows that as RS increases relative to RK, V approaches EK. Thus the measurement becomes more accurate Extracellular when RS is maximized. Figure 3: Measurement of Vm

B. Voltage clamp circuits 1. axon In the 1940s, Kenneth Cole invented the voltage clamp method that was used in the pioneering experiments of Hodgkin and Huxley to determine the ionic basis of the in the . For these Differential experiments, microelectrodes amplifier V were not necessary. The axon cmd A was simply removed from the 2  V squid and perfused with A intracellular solution and 1 bathed in physiological saline Figure 4: electrode configuration for axon VC

3 Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 6 solution. In order to eliminate the space clamp problem, a wire constituting the current passing electrode was coiled around shaft of a glass capillary. This configuration ensured that the membrane along the entire length of the axon could be maintained at the same voltage.

Figure 5: Setup for voltage clamping squid giant Vcmd + A2 2. Two-electrode voltage (TEV) clamping A1 -  Vm Two electrode voltage clamping involves two microelectrodes, one that senses voltage and a second that passes current into the E1 E2 cell to maintain a constant voltage specified by the experimenter. Membrane potential is recorded through a unity gain buffer RE1 RE2 amplifier (A1) that is connected to the voltage sensing microelectrode. Output from A1 is fed into the negative input of a high gain differential amplifier (A2;  = gain) that compares the measured membrane potential with the command potential Rm Cm (Vcmd). The output of A2 is proportional to the difference () between Vm and Vcmd. The voltage at the output of A2 forces current to flow through the current passing microelectrode (E2) A into the cell, setting the membrane at Vcmd. Changes in Rm resulting from opening or closing of electrogenic transport pathways (e.g. ion channels) will require changes in the amount RE 2C of current injected into the cell to hold the voltage constant. The V   m  current flowing across the membrane is recorded by the m ammeter (A) shown in Figure 6. 

m Note that unlike the ideal case where Vm would change to Vcmd Figure 6: TEV clamp instantaneously following the voltage step, a finite time is setup in Xenopus oocytes required to charge the membrane capacitance. The (t) is the product of the E2 resistance (RE2) times the membrane capacitance (Cm) divided by the gain of A2. Thus, if the gain is infinite, or RE2 is zero, the voltage and current responses would approach the ideal condition.

4 Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 6

3. Patch clamp electrophysiology Patch clamp recording is a technique well suited for c small spherical cells where instead of impaling the cell with a sharp microelectrode (as in TEV clamping) a suction pipette is used to form a b gigaohm seal between the plasma membrane and the electrode glass. Different configurations can be used to study currents with this technique. a Examples shown below include i) whole cell recording, where the membrane patch is disrupted by a strong suction or voltage pulse so that the entire can be voltage clamped: ii) inside- Intracellular out configuration for measuring single channel space currents, iii) outside-out configuration also for single channel measurements or possibly whole cell recording if the membrane is large enough and contains enough channels. Other variations also exist including perforated patch where the whole cell configuration is achieved by using a pore forming antibiotic instead of a voltage or suction pulse or single channel recording using “on cell’ mode where the patch is allowed to stay attached to the cell and not ripped off as in the case for the inside-out or outside-out configurations.

R Patch clamp circuitry: f - The technique uses a single A1 - microelectrode and a current-to-voltage + A2 converting amplifier (A1) with either + resistor or feedback headstages. Capacitance feedback amplifiers are Vcmd desirable for single channel recording R or R because the noise characteristics of series a are better behaved and lower than resistors. The basic design involves transmitting the command voltage (Vcmd) Rm Cm into the positive input of A1 and the negative input of the differential amplifier (A2). The output voltage of A1 is then fed into the pipette and the negative terminal of A1 so that the voltage matches Vcmd. The output of A1 is then transmitted to the negative terminal of A2 so that Vcmd can be subtracted off of the signal coming from the electrode. Unlike TEV however, the actual membrane potential is not measured. The voltage is assumed to be that set by the bias current that flows into the negative input of A1. Note that a

5 Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 6

series resistance (or sometimes called the access resistance: Ra) exists between A1 and the electrode tip. If this resistance is high, then a significant voltage drop will occur across the series resistance and Vm will not equal Vcmd. For this reason patch clamp amplifiers have series resistance compensation circuitry that can be used to partially correct for this problem.

Voltage errors imposed by uncompensated series resistances

The series resistance limits the flow of current required to bring Vm = Vcmd. To determine Vm, a fractional resistance term must be multiplied by Vcmd.

Rm Vm  Vcmd  Rm  Ra Remember that at steady state, no net current is flowing onto or off of the capacitor, thus Rm and Ra are in series with each other, thus Rm + Ra = total resistance (Rt). In contrast, in the pre- steady-state condition when the membrane is charging, the two resistances add in parallel so that:

Ra Rm Rt  . Thus the time constant is:  RtCm Rm  Ra

Voltage error: Verror = ImRa: For example, If Im = 2 nA, and Ra is 10 M then the voltage error -9 7 -2 would be (2 x 10 A) (1 x 10 ) = 2 x 10 V or 20 mV! In other words, Vm would underestimate Vcmd by 20 mV.

How would a high series resistance affect the kinetics of the measured currents?

C. Examples of whole cell voltage clamp data

Measuring activation of Kv1.2 channels expressed in Xenopus oocytes:

1. Assessing voltage dependence of channel activation: Figure 9: Kv1.2 activation

A standard activation voltage protocol and resulting current responses are shown in Figure 9. In this experiment the membrane potential is held at -60 mV, a voltage where the channel is -60 mV in the closed state. In fig. 9A, oocytes are bathed in standard physiological saline solution. Note that current doesn’t flow through the channel until the voltage is stepped to ~ -30 mV and increases steadily with depolarization. Thus the activation threshold is -30 mV. In part B, oocytes are bathed in a symmetric K solution where intracellular and extracellular [K+] are equal. Note that the initial set of current responses are negative at voltages less than zero mV, which makes sense since K+ ions are moving into the cell in response to these voltage steps. Remember, by convention, negative current results from either anions moving out of

6 Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 6 the cell or cations entering the cell. Since Kv1.2 is selective for K+ ions, the inward current represents K+ entry into the cell. Why don’t you see this in part A? Figure 10: Conductance-voltage plot. Figure 10 shows a conductance-voltage plot where the oocyte membrane conductance as a function of voltage has 1.0 been normalized to the maximum conductance. Of course K the conductance at each voltage can be calculated from the 0.8

I 0.6

current voltage relationship using Ohm’s law ( G  ). max Vcmd 0.4 Typically, the data points are fit using the Boltzmann G/G 0.2 G (Gmin  (Gmax  Gmin ) function [  ]. Note that two V G (V V ) 50 max (1 exp 50 ) 0.0 K -60 -40 -20 0 20 40 Voltage (mV) factors (V50 and the slope factor, K), often used to describe voltage-dependency of the channel, can be derived from the fit. The V50 value is the voltage at which the conductance is 50% of its maximum. The slope factor gives an indication of how sensitive channel activation is to changes in voltage. A higher slope indicates greater voltage sensitivity. It is important to remember that in the case of voltage- activated ion channels, the activation plot shown in Figure 9A does not provide any information about the ion selectivity of the channel since the channel is closed over most of the negative voltage range where one would expect that a K+ selective ion channel would exhibit inward (negative) currents when bathed in physiological saline solution (high intracellular [K+], low + extracellular [K ], so EK should be approximately -60 mV in oocytes).

Figure 11 Kv1.2 inactivation 2. Inactivation Kv1.2 channels also undergo slow inactivation sometimes referred to as C-type inactivation. This process is voltage independent as shown in the traces from Figure 11. Note that time constants for inactivation are essentially identical for each of the three traces. So no matter what voltage is used to activate Kv1.2, inactivation, proceeds at the same rate. The plot below the traces shows both activation and inactivation curves for the channel. The inactivation curve was generated by holding the cell at different voltages (-60 mV, -50 mV, - 40 mV, etc) and waiting a couple of minutes to allow a certain fraction of the channels to undergo inactivation. The voltage was then stepped to a maximum activating voltage and the resulting peak current was measured. As the channels inactivate at more depolarized holding potentials, less current can be evoked in the subsequent depolarization step. Note that there is overlap between the activation and inactivation curves for this channel. The area inscribed by the intersection of these curves is known as the “window current”. This region defines the range of voltages where the channel will exhibit a measureable open probability so that if the membrane potential of the cell is within this range, the channel will be open. Thus at -20 mV, approximately 50% of the channels will be open.

7 Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 6

In contrast to Kv1.2, the Kv4.2 channel rapidly inactivates (Figure 12). These channels are expressed in ventricular myocytes and are responsible for the initial hyperpolarization response occurring at the peak of the action potential. The inactivation exhibited by these channels is referred to as N-type inactivation and is known to be modulated by a K channel auxiliary subunit, specifically KChIP 1B. The inset figure shows that coexpression of KChIP 1B significantly slows the rate of Kv4.2 inactivation. Figure 12: Kv4.2 inactivation 3. Deactivation To determine the kinetics of the deactivation (channel closing) process a tail current protocol is used to produce maximum channel opening followed by incremented hyperpolarization steps to induce channel closure. Figure 13A shows the voltage step protocol for activation of the Kv1.2 channel which involves an initial depolarization to +40 mV to completely activate the channels and then a series of hyperpolarization steps to drive current through the open channels. Figure 13B shows data from Kv1.2 channels in oocytes bathed in symmetric [K] solution. You can see from this tracing that at strong hyperpolarized voltages, the deactivation is relatively rapid, but then slows down with less hyperpolarization until the channels A B do not close at all (at voltages +30 mV above -10 mV). This result demonstrates that the deactivation rate is voltage-dependent. Figure hp = -80 mV 13C shows the effects of the K channel toxin charybdotoxin (CTX) -100 mV on deactivation. Note the sharp inward currents relative to the C D traces in part B. Figure 13D shows the voltage dependence of deactivation along with the effects of increasing CTX concentrations on the time constants at the different voltage steps (upper curve is the control without CTX). These results demonstrate that CTX promotes transition of Kv1.2 from the open to the closed state. Figure 13: Kv1.2 deactivation

4. Selectivity In order to determine the ion selectivity for a voltage-activated channel it is necessary to first activate the channels so that a current-voltage relationship can be determined over a range of voltages that allows for resolution of the reversal potential (or zero current potential) of the channel. The reversal potential provides critical information regarding the ion selectivity. Thus the tail current protocol described above is well suited for this purpose. Figure 14 shows the I-V

8 Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 6 relationship taken at the peak of the inward tail currents shown at the arrow on the current traces. Currents measured at this point in time represent the near-instantaneous I-V relation which shows the current at each voltage step flowing through the open channels. This is also called the open channel I-V relation and since the oocyte is bathed in symmetric [K] solution, the reversal potential should be zero mV. If the oocyte were bathed in physiological saline solution and the channel was perfectly selective for K and no other ion within the solution then the reversal potential should equal the equilibrium potential for K.

Figure 14: The near-instantaneous I-V relation +20 mV 5. Recovery from inactivation

To determine the time required for an inactivated channel to hp = -80 mV t return to the closed state, a recovery from inactivation protocol is necessary. In this experiment, cells are stepped from a holding potential where the channels are in their closed state to a voltage where the channels are open and maintained at this voltage until inactivation is complete. The membrane is then stepped back to the holding potential for varying lengths of time when a test depolarization step is delivered to the membrane. The resulting current traces form an envelope of peak currents that can be fit with an exponential function in order to determine the time constant(s) for inactivation. Often these can be fit with a single exponential function. An example of a recovery protocol is shown in Figure 15. Note the voltage protocol at the top and the resulting current traces shown in the middle panel. Peak currents are plotted at different holding potentials to illustrate the voltage- dependence of recovery from inactivation with the most hyperpolarized voltage (-80 mV) at the top. Figure 15: Kv1.2 recovery protocol

Figure 16 shows the effects of KChIP 1b on the recovery from inactivation of the Kv4.2 channel. Note that co- expression of KChIP 1a or 1b speed up the recovery rate compared to control oocytes that only express the Kv4.2 subunit.

Figure 16: Kv4.2 recovery from inactivation

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