Voltage-clamp analysis of a Ca2+- and voltage-dependent chloride conductance in cultured mouse spinal neurons

Abstract

Current and voltage-clamp recordings were made at room temperature from cultured mouse spinal neurons using conventional two-electrode voltage-clamp techniques and electrodes filled with either 3 M KCl, 3 M CsCl, or 3 M Cs2SO4. In the presence of tetraethylammonium and tetrodotoxin, "fast" (rapidly rising and falling) action potentials (FAP) of variable duration were recorded in most neurons. "Slow" (slowly rising and falling) depolarizing potentials (SDP) occurred in 23% of the cells, when using KCl-filled electrodes, and in 82% of the cells with CsCl-filled electrodes. The SDP was frequently preceded by an FAP, although in some cells activation of the SDP occurred before the FAP threshold was reached and in a graded fashion. Both the FAP and SDP were abolished by Cd2+ and other Ca2+ antagonists. In cells exhibiting SDPs, voltage-clamp analysis revealed a sustained (noninactivating) inward current (Isin) during depolarizing steps to potentials more positive than -45 mV. Repolarizing steps resulted in slowly decaying inward tail currents (Itail). Both Isin and Itail were abolished in solutions nominally free of Cao2+, or containing Ca2+-channel antagonists. Bao2+ did not support Isin. The data indicated a U-shaped activation curve for Isin, peaking at about -10 mV. Activation of Isin occurred exponentially with a time constant of approximately 140 ms at -23 mV, becoming faster at more depolarized potentials (ca. 50 ms at -2 mV). Deactivation was slow, giving rise to tail currents lasting seconds. In some cases deactivation could be described by a single exponential process, although frequently the kinetics were more complex. Deactivation was faster at hyperpolarized potentials and sensitive to extracellular ([Ca2+]o), duration of activating voltage steps, and the degree of activation of Isin. Using CsCl-filled electrodes, the reversal potential (Erev) for Isin was -1.7 mV (SEM 3.5 mV, n = 20). Erev always corresponded to the reversal potential for gamma-aminobutyric acid-evoked currents in the same cell. In experiments in which Cs2SO4-filled electrodes were used, Erev was estimated to be -44 mV (SEM 2.3 mV, n = 9). Neither complete substitution of Nao+ with choline ions nor elevation of [K+]o 10-fold significantly affected the estimated Erev. However, substitution of Cl0- with isethionate or methanesulphonate increased the amplitude of inward currents (recorded with CsCl-filled electrodes) and shifted Erev to more depolarized potentials. The results indicate that Cl- are the primary charge carriers for this current and that Cai2+ is required for its activation, leading us to identify it as ICl(Ca).(ABSTRACT TRUNCATED AT 400 WORDS)