Cl^– transport in the lobster stretch receptor neurone

Abstract

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.

The K–Cl Cotransporter in the Lobster Stretch Receptor Neurone—A Kinetic Analysis

“…Solutions containing non-standard K + concentrations were prepared by varying the KCl content in the standard physiological saline without osmotic compensation, thus keeping the osmolality of the saline close to that of the cytosol following any transmembrane KCl exchange (cf. Theander et al, 1999). To all solutions 480 nM tetrodotoxin, 10.0 mM tetraethylammonium, 0.5 mM 4-aminopyridine, and 2.0 mM Cs + were added to eliminate both action potential generating currents, and hyperpolarizationactivated Q-current components (Edman et al, 1987b).…”

“…In a recent study, an electroneutral K-Cl cotransporter, efficiently suppressed by the stilbene derivative 4, 4 0 -diisothiocyanostilbene-2, 2 0 -disulphonic acid (DIDS) and activated by the thiol reagent n-ethylmaleimide, was identified in the lobster stretch receptor neurone (Theander et al, 1999). Functionally, the cotransporter was characterized by mediating a KCl efflux across the plasma membrane in steady state, and by being capable of changing its transport direction transiently in the presence of rapidly reversing transmembrane K + and Cl À gradients.…”

“…

Experiments were performed to define quantitatively the substrate (K + and Cl À ) dependence of the transport function (production of equally large and oppositely directed K + and Cl À flows/currents) of an earlier (Theander et al, 1999) identified electroneutral K-Cl cotransporter in the slowly adapting stretch receptor neurone of the European lobster. The experiments were based on microelectrode techniques.

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Random Stimulation of Spider Mechanosensory Neurons Reveals Long-Lasting Excitation by GABA and Muscimol

“…However, this is unlikely since both antagonists have previously been extensively used in many other invertebrate species, including Drosophila (Ianowski and O'Donnell 2004), moth (Gillen et al 2006), cockroach (Hille and Waltz 2006), crayfish (Deisz and Lux 1982), and lobster (Theander et al 1999), suggesting that similar Cl Ϫ transporters in a number of different species are sensitive to these drugs. However, it is also important to remember that in addition to Cl Ϫ , ionotropic GABA receptor channels are permeable to other anions, such A: an original recording shows that although the membrane potential was clamped to Ϫ84 mV, the spike amplitude decreased from 44 to 8 mV in response to application of 100 M muscimol.…”

“…To learn whether the excitatory GABA response in VS-3 neurons could also be regulated by Cl Ϫ transporters we used the "loop" diuretics furosemide and bumetanide. In addition to NKCC1 these diuretics also inhibit KCC1, a K ϩ -Cl Ϫ cotransporter that actively extrudes Cl Ϫ from cells in vertebrate and invertebrate preparations (Cheung et al 2006;Deisz and Lux 1982;Gillen et al 2006;Hille and Walz 2006;Ianowski and O'Donnell 2004;Theander et al 1999). Furosemide has about equal potency for NKCC1 and KCC1, but bumetanide has a significantly higher affinity for NKCC1 (Payne et al 2003) and would therefore be more likely to affect the response of VS-3 neurons.…”