Characterization of a chloride conductance activated by hyperpolarization in Aplysia neurones.

Abstract

SUMMARY1. A voltage-clamp study was made of some properties of the non-synaptic hyperpolarization-activated C1-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-(EC1) and K+ ions (EK).2. The Cl-selectivity ofthe 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 ofthe cholinergic Cl-response, which is the equilibrium potential for Cl-ions, EC1. 3. The steady-state Cl-conductance (ass) increases steeply with hyperpolarization.The kinetics of activation and deactivation are exponential and are characterized by the same voltage-dependent time constant (r), of the order of a few seconds or fractions of seconds. The curves g.8(V) and r( 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).4. The Cl-current is much more affected by changes of the intracellular Clconcentration 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.5. The hyperpolarization-activated current is abolished after prolonged exposure of the cell to an artificial sea water where N03-ions replace Cl-ions, as well as after intracellular injections of N03-ions.6. Increasing the external pH shifts the g.(V) and r(V) curves to the left.Lowering the external pH has reverse but less pronounced effects. 7. 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 D. CHESNO Y-MARCHAIS 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 C02+ ions, depolarizing pulses no longer increased the Cl-current.8. The Cl-current is not affected by extracellularly applied DIDS (4,4'-diisothiocyano-2,2'-disulphonic acid stilbene), but is markedly reduced by intracellular injection of DIDS.9. Extracellular Cs+ ions, which have been reported to block some cationic hyperpolarization-activated inward currents, do not reduce the hyperpolarizationactivated Cl-current. High concentrations of Cs+ produce complex effects which are probably due to an increased synaptic activity, but the hyperpolarization-activatedCl-current persists after complete substitution of the extracellular and intracellular monovalent cations by Cs+.

Chloride dependence of hyperpolarization‐activated chloride channel gates

“…More detailed studies of ClC_2, including measurements of ClC_2 expressed in smaller cells, are definitely necessary for a more profound understanding of its complex gating mechanisms. In several cell types, native hyperpolarization-activated Cl¦ currents that resemble ClC_2 strongly depend on [Cl¦]é (Chesnoy-Marchais, 1983;Dinudom et al 1993;Staley, 1994;Fritsch & Edelman, 1996;Ferroni et al 1997). Our findings support the hypothesis that these currents are indeed mediated by ClC_2.…”

ClC-2 Voltage-Gated Channels Constitute Part of the Background Conductance and Assist Chloride Extrusion

“…Our data clearly prove that ClC-2 mediates a previously described chloride conductance (Chesnoy-Marchais, 1983;Madison et al, 1986;Smith et al, 1995) in CA1 pyramidal cells and a subset of interneurons. In addition to the absence of this conductance in Clcn2 Ϫ /Ϫ neurons, we observed the same biophysical properties Figure 6.…”

Kinetic properties and selectivity of calcium‐permeable single channels in Aplysia neurones.

“…In the case of the channel studied here, the mean open-time sequence is Ca > Sr > Ba > Mg and the mean closed-time is also clearly dependent on the nature ofthe permeant ion, a property which has only been described for a few other channels (the opening rate constant seems to be ion dependent in the case of the anomalous K rectifier (Hagiwara et al 1976) and in the case of the Aplysia hyperpolarizationactivated Cl channel (Chesnoy-Marchais, 1983) In most cases where such a conclusion was proposed (see the above references), the substitution of permeant ions by other ions of identical valency did not affect the activation time constant by a factor larger than 2 or 3. In the present paper, it has been shown that the mean open-time can be affected by a factor of about 9 for the Ca-Ba substitution and by a factor of about 40 for the Ca-Mg substitution.…”

“…9B: the Ca and Ba curves might cross above +15 mV). (The idea that there could be differences between a 'permeability' sequence and a 'conductance' sequence had already been proposed earlier (see Hagiwara, Toyama & Hayashi, 1971;Hille, 1984 Van Helden, Hamill & Gage, 1977;Gage & Van Helden, 1979 for the frog muscle end-plate acetylcholine-activated cationic channel; Ascher, Marty &Neild, 1978 andMarty, 1979 for the Aplysia acetylcholine-activated cationic channel; Swenson & Armstrong, 1981 for the squid giant axon K current; Hagiwara, Miyazaki &Rosenthal, 1976 andHestrin, 1981 for the starfish egg and frog muscle K anomalous rectifier; Chesnoy- Marchais, 1983 for the Aplysia hyperpolarization-activated Cl current). In the case of Ca channels too, the activation kinetics have sometimes been considered as dependent on the nature of the permeant ion.…”