Patch Clamp Technique and Applications

Abstract

There are two main modes in patch clamp recordings: voltage clamp and current clamp. In the voltage clamp mode, the membrane voltage is controlled by the amplifier through the recording pipette and the corresponding current through the pipette is measured. In the current clamp mode, the amplifier controls the amount of current passing through the pipette and the corresponding change in voltage is measured. A third less used mode involves applying no clamp (often designated as I = 0 on the amplifier). This mode is similar to intracellular recordings with a sharp electrode. This chapter will focus on the basic setup and concepts of the patch clamp technique, including considerations of noise and voltage errors in the measurement.

Appendices

Problem

The voltage dependent activation of channel X recorded in whole-cell configuration is shown on the following plot. The R _c is 500 MΩ and the R _seal is 10 GΩ. How do recordings from an R _s of 2 and 20 MΩ change this IV relationship?

Solution

The equivalent electrical circuit for whole-cell configuration is shown in Fig. 2.2d in which R _s = R _a + R _p. Because ${R}_{\mathrm{seal}}\gg \gg {R}_{\mathrm{a}}$ and R _c, the portion of current flowing through R _seal is negligible. The resistance R below the pipette is

$$ \frac{1}{R}=\frac{1}{R_{\mathrm{c}}+{R}_{\mathrm{a}}}+\frac{1}{R_{\mathrm{seal}}}. $$

(3.14)

Again, because ${R}_{\mathrm{seal}}\gg \gg {R}_{\mathrm{a}}$ and R _c, R approaches R _c + R _a.

Thus, Fig. 2.2d can be simplified as R _a and R _p in series with R _c, or R _s in series with R _c (the diagram presented in Fig. 2.4) and the main consideration is the voltage error created by R _s. At more hyperpolarized voltages (−100 to ~−60 mV), the currents are small and thus the actual membrane voltage at each voltage step can be estimated as

$$ \Delta {V}_{\mathrm{c}}={V}_{\mathrm{c}\mathrm{om}}\frac{R_{\mathrm{s}}}{R_{\mathrm{c}}+{R}_{\mathrm{s}}}. $$

(3.15)

In this case, the cell’s membrane voltage for a step from −100 to −60 mV (a 40 mV step) is −60.16 and −61.54 mV for an R _s of 2 and 20 MΩ, respectively (a voltage error of 0.16 and 0.54 mV, respectively). At more depolarizing voltages, large currents are generated and the voltage drop across R _s becomes larger. For example, the cell generates ~2.5 nA of current when membrane is depolarized to 0 mV. In this case, the voltage drop across R _s as the current flows through the R _s is

$$ \Delta {V}_{\mathrm{s}} = {I}_{\mathrm{c}}\times {R}_{\mathrm{s}}. $$

(3.4)

Thus, the voltage error is estimated to be 5 mV for a R _s of 2 MΩ and 50 mV for a R _s of 20 MΩ. However, with an R _s of 20 MΩ, because the membrane voltage is expected to be significantly lower than 0 mV due to the voltage error, the actual current generated by the cell membrane will be less than 2.5 nA. Thus, the actual voltage error would be less than 50 mV. Nonetheless, with higher R _s, the slope of the linear portion of the IV relationship becomes “flatter,” as shown as follows.

Additional notes: If the current is measured at a time when membrane capacitance has not been fully charged, the voltage error will be larger than that estimated with Ohm’s law.

Further Study

Sakmann, B., Neher, E.: Single-channel recording. Plenum Press, New York (1995)

Walz, W., Boulton, A.A., Baker, G.B.: Patch-clamp analysis. Humana Press, Totowa (2002)