Various and complementary approaches are used to study exo-/endocytosis and the trafficking of plasma membrane proteins. Herein, membrane capacitance (C_m) is a good indicator of membrane surface area. C_m can be measured with unmatched precision and time resolution in small cells using the patch-clamp technique. C_m measurements by means of the two-electrode voltage-clamp (TEVC) technique, e.g. in Xenopus (X.) laevis oocytes, may provide equally important insights. We sought to improve ease of use, accuracy, speed and robustness of C_m measurements in X. oocytes.

To this end, we designed asymmetrical triangular voltage stimuli with two different absolute slopes |dV/dt |. These stimuli elicit current (I_m) responses that allow the calculation of membrane resistance (R_m) and C_m from the I_m integrals, as opposed to conventional symmetrical ramps which need to rely on the amplitude of a current jump for this. Integrals, however, are much more robust under non-ideal clamp conditions and are easier to measure than theoretically instantaneous jumps that are necessarily distorted in real TEVC experiments. Continuous application of these stimuli, acquisition of I_m data, calculations and on-line display of C_m were automated via simple macros in ‘Pulse’ and ‘X-Chart’ (HEKA). We tested our approach systematically in a custom-built model circuit (NPI) with tunable C_m, R_m, and series resistance (R_s), and in X. oocytes.

In the model circuit, inaccuracy of the C_m estimates was < 0.5 % under widely varying conditions (100 k¢ < R_m < 2 M¢, 0 ¢ < R_s < 1 k¢, 60 nF < C_m < 1000 nF). Imprecision of C_m estimates was small, independent of R_s, and inversely related to C_m (< 1.5 % at 50 nF, < 0.4 % at 200 nF). Using signal averaging, C_m deviations from 200 nF as little as 0.1 nF, i.e. 0.05 %, could be detected reliably. In X. oocytes, C_m could be recorded fast (~15 Hz) and in parallel with the standard parameters I_m, V_m and R_m. Our method could resolve C_m changes that were small (e.g. upon 100 µM 8-Br-cAMP, ▓Dgr│C_m ~2 nF) or fast (e.g. upon 1 µM PMA, ▓Dgr│C_m/▓Dgr│t = 20 nF (30 s)^-1), or occurred over extended periods in complex patterns (see Fig. 1).

Taken together, our modified ramp approach affords C_m measurements in X. oocytes with high temporal resolution, accuracy and low noise. It can be implemented easily on standard electrophysiology software and does not require extra hardware. Thus it provides a straightforward approach to continuous surface area monitoring for cell biologists who study plasma membrane dynamics. For electrophysiologists who study regulation of ion channels or transporters by hormones or drugs, this approach facilitates the distinction between effects on insertion/retrieval vs. effects on protein function.