Ion transport across posterior gills of hyperosmoregulating shore crabs (Carcinus maenas): amiloride blocks the cuticular Na+ conductance and induces current-noise

Abstract

SUMMARYSplit gill lamellae and gill cuticles of shore crabs (Carcinus maenas) adapted to 10 ‰ salinity were mounted in a modified Ussing-type chamber. With NaCl saline on both sides, split gill lamellae generated a short-circuit current (Isc) of –301±16 μA cm–2 at a conductance (Gte) of 40±2 mS cm–2. The net influxes of Na+ and Cl– were 8.3±2.6 and 18.2±2.7 μmol cm–2 h–1, respectively. External amiloride (100 μmol l–1) reduced Gte to approximately 50 % of the original value at unchanged Isc; Cl– fluxes remained unaffected, whereas Na+ fluxes were markedly reduced by 70–80 %. The Isc in the presence of external amiloride was almost completely inhibited by internal ouabain. At a clamp voltage of 50 mV (outside-positive), a positive current was measured at unchanged Gte. Under these conditions, amiloride reduced the current and conductance at half-maximal concentrations of 3.6 and 2.0 μmol l–1, respectively. At outside-positive voltages, but not under short-circuit conditions, external amiloride induced Lorentzian components in the power density spectra. The amiloride-dependent changes in the corner frequency (linear) and of the low-frequency plateau (‘bell-shaped’) were as expected for channel blockade by amiloride with pseudo-first-order kinetics. With an outside-positive clamp voltage of 50 mV across isolated cuticles, a positive cuticular current (Icut) of 25 188±3791 μA cm–2 and a cuticular conductance (Gcut) of 547±76 mS cm–2 were measured. External amiloride reduced Icut and Gcut at half-maximal concentrations of 0.7 and 0.6 μmol l–1, respectively. Amiloride-induced current-noise analysis gave similar results to those observed with split gill lamellae. Ion-substitution experiments with isolated cuticles further support inhibition by external amiloride of the cuticular Na+ conductance of shore crab gills and not amiloride-sensitive transporters (Na+ channels or Na+/H+ antiports) in the apical membrane.