Characterization of membrane currents in dissociated adult rat pineal cells.

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Abstract
Membrane currents, particularly the outward components, were studied in pineal cells acutely dissociated from adult rats using the whole-cell variant of the patch-clamp technique. In current clamp, outward constant current elicited a transient graded depolarizing response. A sustained membrane rectification developed within 20 ms; this phenomenon was reduced in cells internally dialysed with 120 mM-CsCl. Study of the membrane current revealed the existence of a transient and a delayed outward current. These currents were virtually eliminated when the cell was internally dialysed with CsCl. The delayed outward current, isolated from a holding potential of -50 mV, activated at potentials near -20 mV, reached a steady-state current amplitude within 60 ms and had little or no decay during steps up to 400 ms in duration. This component was reduced by 80% or more with the addition of 5 mM-TEA. From -100 mV, the treatment outward current reached a peak within 15 ms and decayed with a single-exponential time course. The mean decay time constant was 66 .+-. 10 ms (at -33 mV) and it showed little voltage sensitivity. This current, which activated at potentials positive to -60 mV and displayed half-inactivation at -76 .+-. 8 mV, was reduced by 50% with the addition of 5 mM-4-AP (4-amino-pyridine). In the presence of external Ca2+, the current-voltage relationship for the delayed current did not display a region of negative-slope conductance (N-shape). Increasing the intracellular ionized Ca2+ concentration by varying the Ca-EGTA buffer ratio did not alter the dependence of the current on the membrane potential. Block of outward currents with internal Cs+ revealed a small (< 90 pA) inward Ca2+ current when the external Ca concentration was increased to 10 mM. From a holding potential of -50 mV, it had a threshold at -30 mV and peaked at +5 mV. Evidence for an inward Na+ current was not obtained. We conclude that acutely dissociated pineal cells display two distinct K+ current: (i) a slowly activating, sustained current similar to the delayed rectifier (IK); and (ii) a transient A-current (IA). At normal Ca2+ concentrations, no macroscopic Ca2+-activated outward current was observed.