Elevated [Cl-](i) and [Na+](i) inhibit Na+,K+,Cl- cotransport by different mechanisms in squid giant axons

Bumetanide-sensitive (BS) unidirectional fluxes of Cl-36(-) or Na-22(+) were measured in internally dialyzed squid giant axons while varying the intra- or extracellular concentrations of Na+ and/or Cl-. Raising either [Cl-](i) or [Na+](i) resulted in a concentration-dependent reduction of the BS influx of both Cl-36(-) and Na-22(+). Raising [Cl-](i) above 200 mM completely blocked BS influxes. However, raising [Na+](i) to 290 mM resulted in saturable but incomplete inhibition of both BS Na+ influx and BS Cl- influx. The consequences of varying intracellular Cl- on cotransporter effluxes were complex. At lower [Cl-](i) values (below 100 mM) intracellular Cl- activated cotransporter effluxes. Surprisingly, however, raising [Cl-](i) levels >125 mM resulted in a [Cl-](i)- dependent inhibition of BS effluxes of both Na+ and Cl-. On the other hand, raising [Na+]i resulted only in the activation of the BS Na+ efflux; intracellular Na+ did not inhibit BS efflux even at 290 mM. The inhibitory effects of intracellular Na+ on cotransporter-mediated influxes, and lack of inhibitory effects on BS effluxes, are consistent with the trans-side inhibition expected for an ordered binding/release model of cotransporter operation. However, the inhibitory effects of intracellular Cl- on both influxes and effluxes are not explained by such a model. These data suggest that Cl- may interact with an intracellular site (or sites), which does nor mediate Cl- transport, but does modulate the transport activity of the Na+, K+, Cl- cotransporter.