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Experiments were performed to identify mechanisms underlying non-leakage and non-H+/HCO3--linked transmembrane Cl- transports in the slowly adapting stretch receptor neurone of the European lobster, using intracellular microelectrode and pharmacological techniques. In methodological tests, it was established that direct estimates of intracellular Cl- with ion-sensitive microelectrodes are statistically identical with indirect estimates by means of a GABA method, where 1-2 mM GABA is transforming the cell's membrane voltage into its Cl- equilibrium voltage from which the Cl- concentration is inferred by the Nernst equation. From experiments using sodium orthovanadate and ethacrynic acid, supposed to block primary Cl- pumps, and bumetanide, supposed to block Na-K-Cl co-transporters, it appeared that neither of the two Cl- transport systems exists in the stretch receptor neurone. It could be shown, however, that the cell is equipped with an electroneutral K-Cl co-transporter that (a) is blockable by furosemide in high (Km approximately 350 microM), by 4-acetamido-4'-isothiocyanato-stilbene-2,2-disulphonic acid (SITS) in medium-high (Km approximately 35 microM), and by 4, 4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) in low (Km approximately 15 microM) doses, (b) is (transiently) activatable by (1 mM) n-ethylmaleimide, (c) is not suppressed by extracellular Rb+ or NH4+, and (d) is not directly coupled to any transmembrane transports of Na+, H+ or HCO3-. From functional tests, with varying transmembrane K+ and Cl- gradients, evidence obtained that the K-Cl co-transporter is able to reverse its transport direction and to adjust its transport rate in a considerable range. As a whole, the results speak in favour of the K-Cl co-transporter being responsible (a) for normally keeping the intracellular Cl- concentration at low levels, for an optimization of the cell's inhibitory system, and (b) for achieving fast transmembrane shifts of K+ (and Cl-), as a means of stabilizing the cell's membrane excitability in conditions of varying extracellular K+ concentrations. |
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