Membrane voltage, resistance, and channel switching in isolated mouse fibroblasts (L cells): a patch‐electrode analysis.

Abstract

The whole‐cell patch‐electrode technique of Fenwick, Marty & Neher (1982) has been applied to single suspension‐cultured mouse fibroblasts. Seals in the range of 10‐50 G omega were obtained without special cleaning of the cell membranes. Rupture of the membrane patch inside the electrode was accompanied by a shift of measured potential into the range ‐10 to ‐25 mV, but in most cases with little change in the recorded resistance. The latter fact implied that the absolute resistance of the cell membrane must be in the same range as the seal resistance and the recorded potential is a poor measure of actual cell membrane potential. Steady‐state current‐voltage curves (range ‐160 mV to +80 mV) were generated before and after rupture of the membrane patch, and the difference between these gave (zero‐current) membrane potentials of ‐50 to ‐75 mV, which represents a leak‐corrected estimate of the true cell‐membrane potential. The associated slope conductivity of the cell membrane was 5‐15 microS/cm2 (assumed smooth‐sphere geometry, cells 13‐15 microns in diameter) and was K+‐dominated. With 0.1 mM (or more) free Ca2+ filling the patch electrode, membrane potentials in the range ‐60 to ‐85 mV were observed following patch rupture, with associated slope conductivities of 200‐400 microS/cm2, also K+‐dominated. Similar voltages and conductivities were observed at the peak of pulse‐induced 'hyperpolarizing activation' (Nelson, Peacock, & Minna, 1972), and the two phenomena probably reflect the behaviour of Ca2+‐activated K+ channels. Both the pulse‐induced conductance and the Ca2+‐activated conductance spontaneously decayed, the latter over periods of 5‐15 min following patch rupture. Sr2+, Ba2+, and Co2+ could also activate the putative K+ channels, but only Sr2+ really mimicked Ca2+. Co2+ and Ba2+ activated with a delay of several minutes following patch rupture, and deactivated quickly with a small decrease of conductance and a large decrease of membrane potential. Evidently, Co2+ and Ba2+ affect channel specificity as well as channel opening and closing kinetics.