Invertebrate epithelial Na + channels: amiloride-induced current-noise in crab gill

Abstract

Epithelial sheets (including cuticle) from posterior gills of the freshwater-adapted euryhaline crab Eriocheir sinensis were obtained according to the method of Schwarz and Graszynski ((1989) Comp. Biochem. Physiol. 92A, 601–604; (1989) Verh. Dtsch. Zool. Ges. 82, 211 and (1989) Arch. Int. Physiol. Biochim. 97, C45). With external NaCl-saline, the outward-directed short-circuit current ( I sc) could hardly be influenced by external amiloride up to 100 μmol/l but was, on the contrary, strictly dependent on apical Cl (Onken, Graszynski and Zeiske (1991) J. Comp. Physiol. B 161, 293–301). In absence of external chloride an inward-directed, amiloride-inhibitable I sc was observed which depended on external Na + (thus, I sc ≈ I Na) in a two-step, saturating mode. The I sc-block by amiloride obeyed saturation kinetics (half-maximal at ⩽1 μmol/l, suggesting apical Na +-channels). Only for Na + concentrations below 100 mmol/l we found an indication for a competitive interaction between Na + and amiloride at the channel. Current fluctuation analysis revealed the presence of an amiloride-induced relaxation (Lorentzian) component in the I sc-noise (so-called ‘blocker-noise’). The Lorentzian parameter-shifts with increasing amiloride concentration indicate first-order kinetics of the blocker with its apical receptor. Using a ‘two-state’ blocking model we calculated, for amiloride concentrations between 2 and 5 μmol/l, a mean single-channel current of 0.46 pA and a mean channel density of 250 · 10 6 cm 2.