An improved method for real-time monitoring of membrane capacitance in Xenopus laevis oocytes.
Measurements of membrane capacitance (C(m)) in Xenopus laevis oocytes offer unique experimental possibilities but are difficult to perform with current methods. To improve C(m) measurements in the two-electrode voltage clamp (TEVC) mode, we developed a paired-ramp protocol and tested its performance in a model circuit (with tunable C(m), membrane resistance R(m), and series resistance R(s)) and in Xenopus oocytes. In the cell model and with R(s) = 0 Omega, inaccuracy of C(m) estimates was <1% under widely varying conditions (R(m) ranging from 100 to 2000 kOmega, and C(m) from 50 to 1000 nF). With R(s) > 0 Omega, C(m) was underestimated by a relative error epsilon closely approximated as epsilon approximate 2 x R(s)/(R(s) + R(m)), in keeping with the theoretical prediction. Thus, epsilon may be neglected under standard conditions or, under extreme conditions, corrected for if R(s) is known. Relative 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). Averaging allowed reliable detection of C(m) deviations from 200 nF of 0.1 nF, i.e., 0.05%. In Xenopus oocytes, we could resolve C(m) changes that were small (e.g., DeltaC(m) approximate 2 nF upon 100 muM 8-Br-cAMP), fast (e.g., DeltaC(m)/Deltat approximate 20nF/30s upon 1 muM phorbol myristate acetate (PMA)) or extended and complex (e.g., fast increase, followed by prolonged C(m) decrease upon 1 muM PMA). Rapidly alternating between paired ramps and a second, step protocol allowed quasi-simultaneous monitoring of additional electrical parameters such as R(m), slope conductance g(m), and reversal potential E(rev). Taken together, our method is suited to monitor C(m) in Xenopus oocytes conveniently, with high temporal resolution, accuracy and precision, and in parallel with other electrical parameters. Thus, it may be useful for the study of endo- and exocytosis and of membrane protein regulation and for electrophysiological high-throughput screening.
Schmitt
Bernhard M
BM
Department of Anatomy & Cell Biology, University of Würzburg, 97070 Würzburg, Germany. bernhard.schmitt@mail.uni-wuerzburg.de
Koepsell
Hermann
H
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004594:Electrophysiology / Q000295:instrumentation* / Q000379:methods*; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D012984:Software; D013997:Time Factors; D014982:Xenopus laevis