Covalent modification and inhibition of an epithelial sodium channel by tyrosine-reactive reagents

This study sought to elucidate the molecular mechanism involved in the Na+ entry across the apical membrane of the urinary bladder of the toad. Na+ transport, as measured by short-circuit current (SCC), was irreversibly inhibited by three tyrosine-specific reagents: N-acetylimidazole (ID50, 4.6 x 10(-2)M), tetranitromethane (1.8 x 10(-4) M), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl; 3.4 x 10(-5) M). The functional specificity of NBD-Cl to block Na+ entry via Na+ channels was attested by the following: 1) NBD-Cl produced comparable inhibition of SCC and Na+ influx under aerobic and anaerobic conditions; 2) amphotericin B produced complete recovery of inhibited SCC; 3) vasopressin increased SCC only in proportion to the uninhibited SCC; 4) Km for Na+ was not changed; and 5) the half time for the inhibition varied as a function of amiloride concentration or pharmacologic activity of its analogues. On the basis of the above findings, these tyrosine-specific reagents are believed to be useful chemical probes for the identification and characterization of Na+ channel protein.