Calcium-dependent chloride currents in isolated cells from rat lacrimal glands

Abstract

Isolated cells from rat lacrimal glands were studied with the tight-seal whole-cell recording technique. Cells were dialysed with K-free solutions containing a high concentration of Ca2+ buffer in order to record Ca-dependent Cl- currents at a Ca2+ level fixed between 0.1 and 10 microM. HEDTA was preferable to EGTA as a Ca2+ buffer, even under conditions of equivalent equilibrium buffering power. After replacement of all internal K+ with Na+, the cells displayed a small conductance component which could be abolished by removal of external K+ or by external application of 2 mM-tetraethylammonium. It is suggested that this conductance is due to Ca-dependent K+ channels. The main part of the cell current was Cl selective. The Cl- conductance was negligible at 0.5 microM-Ca2+, and fully activated at 2 microM-Ca2+. The dose-response curve relating Cl- currents to the internal Ca2+ concentration, [Ca]i, was steeper than predicted by a simple binding isotherm reaction. Relaxations observed in response to voltage jumps could, in most cases, be fitted with single exponentials. At [Ca]i 0.5 microM, the curve relating the relaxation time constant, tau, to the membrane potential, displayed a maximum near +20 mV and 250 ms. At hyperpolarized potentials, tau varied by an e-fold factor in 130 mV. At [Ca]i 1 microM, tau decreased from 100 ms at -120 mV to 60 ms at +60 mV. Relaxation analysis gave an estimate of the variation of the channel open state probability, Po, with potential. At [Ca]i 0.5 microM, Po varied by an e-fold factor in 50-70 mV at hyperpolarized potentials, and saturated above +60 mV. At [Ca]i 1 microM, Po varied e-fold in 100 to 110 mV at hyperpolarized potentials, and saturated near +20 mV. External Cl- was substituted with various anions. From reversal potential measurements, the following permeability sequence was obtained: I- greater than NO3- greater than Br- greater than Cl- greater than F- greater than isethionate, methanesulphonate greater than glutamate. The corresponding normalized permeability coefficients were 2.7, 2.4, 1.6, 1, 0.2, 0.1, 0.1, 0.05. Replacement of external Cl- with Br-, isethionate, methanesulphonate or glutamate did not alter current kinetics as obtained during or after a depolarizing voltage jump.(ABSTRACT TRUNCATED AT 400 WORDS)