Participation of a chloride conductance in the subthreshold behavior of the rat sympathetic neuron.
The presence of a novel voltage-dependent chloride current, active in the subthreshold range of membrane potential, was detected in the mature and intact rat sympathetic neuron in vitro by using the two-microelectrode voltage-clamp technique. Hyperpolarizing voltage steps applied to a neuron held at -40/-50 mV elicited inward currents, whose initial magnitude displayed a linear instantaneous current-voltage (I-V) relationship; afterward, the currents decayed exponentially with a single voltage-dependent time constant (63.5 s at -40 mV; 10.8 s at -130 mV). The cell input conductance decreased during the command step with the same time course as the current. On returning to the holding potential, the ensuing outward currents were accompanied by a slow increase in input conductance toward the initial values; the inward charge movement during the transient ON response (a mean of 76 nC in 8 neurons stepped from -50 to -90 mV) was completely balanced by outward charge displacement during the OFF response. The chloride movements accompanying voltage modifications were studied by estimating the chloride equilibrium potential (E(Cl)) at different holding potentials from the reversal of GABA evoked currents. [Cl(-)](i) was strongly affected by membrane potential, and at steady state it was systematically higher than expected from passive ion distribution. The transient current was blocked by substitution of isethionate for chloride and by Cl(-) channel blockers (9AC and DIDS). It proved insensitive to K(+) channel blockers, external Cd(2+), intracellular Ca(2+) chelators [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)] and reduction of [Na(+)](e). It is concluded that membrane potential shifts elicit a chloride current that reflects readjustment of [Cl(-)](i). The cell input conductance was measured over the -40/-120-mV voltage range, in control medium, and under conditions in which either the chloride or the potassium current was blocked. A mix of chloride, potassium, and leakage conductances was detected at all potentials. The leakage component was voltage independent and constant at approximately 14 nS. Conversely, gCl decreased with hyperpolarization (80 nS at -40 mV, undetectable below -110 mV), whereas gK displayed a maximum at -80 mV (55.3 nS). Thus the ratio gCl/gK continuously varied with membrane polarization (2.72 at -50 mV; 0.33 at -110 mV). These data were forced in a model of the three current components here described, which accurately simulates the behavior observed in the "resting" neuron during membrane migrations in the subthreshold potential range, thereby confirming that active K and Cl conductances contribute to the genesis of membrane potential and possibly to the control of neuronal excitability.
Sacchi
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Department of Biology, Sezione di Fisiologia Generale, University of Ferrara, I-44100 Ferrara, Italy.
Rossi
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ML
Canella
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R
Fesce
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R
D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D002712:Chlorides / Q000502:physiology; D005071:Evoked Potentials / Q000187:drug effects; D066298:In Vitro Techniques; D008564:Membrane Potentials; D008839:Microelectrodes; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D017783:Superior Cervical Ganglion / Q000502:physiology*; D019789:Tetraethylammonium / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D000873:Anthracenes; D018118:Chloride Channels; D002712:Chlorides; D005680:gamma-Aminobutyric Acid; D019789:Tetraethylammonium; C010030:9-anthroic acid