Characterization of a chloride conductance activated by hyperpolarization in Aplysia neurones

A voltage-clamp study was made of some properties of the non-synaptic hyperpolarization-activated Cl- conductance recently described in Aplysia neurones loaded with Cl- ions (Chesnoy-Marchais, 1982). The experiments were performed on an identified family of neurones, which present cholinergic responses allowing an easy measurement of the equilibrium potentials of Cl- (ECl) and K+ ions (EK). The Cl- selectivity of the hyperpolarization-activated conductance was deduced from four observations: (1) the extrapolated reversal potential of the hyperpolarization-activated current, Er, was close to the reversal potential of the cholinergic Cl- response, which is the equilibrium potential for Cl- ions, ECl. (2) Modifications of the intracellular or extracellular Cl- concentration induced changes of the reversal potential Er. (3) A prolonged and intense activation of the current lowered the intracellular Cl- concentration. (4) The current persisted after complete substitution of intracellular and extracellular cations by CS+ ions, as well as after replacement of extracellular Na+ ions by Tris. The steady-state Cl- conductance (gss) increases steeply with hyperpolarization. The kinetics of activation and deactivation are exponential and are characterized by the same voltage-dependent time constant (tau), of the order of a few seconds or fractions of seconds. The curves gss(V) and tau (V) can both be fitted by a two-state model in which the rate constants are exponential functions of the membrane potential (e-fold change for 12-16 mV). The Cl- current is much more affected by changes of the intracellular Cl- concentration than predicted simply from the change in Cl- driving force. Both the conductance and the time constant of activation are strongly modified. Modifications of the extracellular Cl- concentration do not always alter the amplitude of the hyperpolarization-activated Cl- current, but systematically affect its kinetics. The hyperpolarization-activated current is abolished after prolonged exposure of the cell to an artificial sea water where NO3- ions replace Cl- ions, as well as after intracellular injections of NO3- ions. Increasing the external pH shifts the gss(V) and tau (V) curves to the left. Lowering the external pH has reverse but less pronounced effects. In cells which were not loaded with Cl- ions and did not present the hyperpolarization-activated Cl- current, this current could be detected if the hyperpolarizing jump was preceded by short depolarizing pulses. In cells which were loaded with Cl- ions, the Cl- current became larger after a short depolarizing pulse. In the presence of extracellular Co2+ ions, depolarizing pulses no longer increased the Cl- current.(ABSTRACT TRUNCATED AT 400 WORDS)