Inhibition by mercuric chloride of Na-K-2Cl cotransport activity in rectal gland plasma membrane vesicles isolated from Squalus acanthias

摘要

The rectal gland of the dogfish shark is a model system for active transepithelial transport of chloride. It has been shown previously that mercuric chloride, one of the toxic environmental pollutants, inhibits chloride secretion in this organ. In order to investigate the mechanism of action of HgCl_2 at a membrane-molecular level, plasma membrane vesicles were isolated from the rectal gland and the effect of mercury on the activity of the Na-K-2Cl cotransporter was investigated in isotope flux studies. During a 30 s exposure HgCl_2 inhibited cotransport activity in a dose-dependent manner with an apparent K_i of approx. 50 μM. The inhibition was complete after 15 s, partly reversible by dilution of the incubation medium and completely attenuated upon addition of reduced glutathione. The extent of inhibition by mercury depended on the ionic composition of the medium. The sensitivity of the cotransporter was highest when only the high affinity binding sites for sodium and chloride were saturated. Organic mercurials such as p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid at 100 μM did not inhibit the cotransporter, similarly exposure of the vesicles to 10 mM H_2O_2 or 1 mM dithiothreitol for 30 min at 15 ℃ did not change cotransport activity. Transport activity was, however, reduced by 45.9 ± 2.5% after an incubation with 3 mM N-ethylmaleimide for 20 min. Blocking free amino groups by N-hydroxysuccinimide or biotinamidocapronate-N-hydroxysulfosuccinimide had no effect. Investigations on the sidedness of the plasma membrane vesicles, employing the asymmetry of the (Na+k)-ATPase, demonstrated a right-side-out orientation in which the former extracellular face of the membrane is exposed to the incubation medium. In addition, extracellular mercury (5 * 10~(-5) M) inhibited bumetanidesensitive rubidium uptake into T84 cells by 48.5 ± 7.1% after a 2 min incubation period. This inhibition was reversible in a manner similar to that observed in the plasma membrane vesicles. These studies suggest that in isolated rectal gland plasma membrane vesicles the Na-K-2Cl cotransporter (sNkCC1) exposes functionally relevant mercury binding sites at its external surface. These sites represent probably cysteines, the accessibility and/or sensitivity of which depends on the functional state of the transporter.