Effects of chemical group specific reagents on sodium entry and the amiloride binding site in frog skin: Evidence for separate sites

Abstract

Previously we have shown that the inhibition of active transport by amiloride is noncompetitive with sodium in Rana catesbeiana skin, suggesting that amiloride acts at a site separate from the sodium entry site (Benos, D.J., Mandel, L.J., Balaban, R.S. 1979, J. Gen Physiol. 73:307). In the present study, the effects of a number of sulfhydryl, amino, and carboxyl group selective reagents were studied on short-circuit current (Isc) as well as the efficacy of amiloride in bullfrog skin, to determine those functional ligands which may be involved with either of these processes.Addition of the sulfhydryl reagent PCMBS (1 raM) to the outside bathing medium produced biphasic effects, initially reversibly increasing Iso by an average 56% followed by a slower, irreversible decay to levels below baseline. In contrast, the addition of 0.1 m~ PCMB always resulted in a rapid, irreversible decrease in Isc. When a 40,000 tool wt dextran molecule was attached to PCMB, a stable, reversible increase in I~c was observed. These observations suggest that at least two populations of -SH groups are involved in Na translocation through the entry step. Amiloride was equally effective in inhibiting Is~ before and after treatment with PCMBS both during the stimulatory as well as the inhibitory phase. The sulfhydryl reducing agent DTT, and oxidizing agent DTNB had only minor influence on I~c and did not alter the effectiveness of amiloride.Similarly, the amino reagents, SITS and TNBS did not affect I~c. However, TNBS decreased the ability of amiloride to inhibit Na entry. These results suggest that an amino group may be involved in the interaction of amiloride and its site, without affecting Na entry.The carboxyl reagents EEDQ, TMO, and three separate carbodiimides were without effect on Is~ or amitoride inhibition, Methylene blue (MB), a molecule that interacts with both carboxyl and hydroxylspecific groups, inhibited Isc by 20% and decreased amiloride's ability to inhibit I~. These effects, however, are likely to occur from the cytoplasmic side as MB appears to enter into the cells.These results support the notion that amiloride and Na interact with the entry protein at different regions on the membrane.

Electrogenic sodium absorption in rabbit cecum in vitro.

“…The present data are most consistent with significant alterations of the binding sites associated with the classical Na channel rather than a fundamentally distinct channel. Work by several investigators studying electrogenic Na absorption in a variety of tight epithelia has suggested that there are specific loci associated with the Na channel that modify its behavior: an amiloride-binding site (20), a Na self-inhibition site (21,22), a sulfhydryl group sensitive to mercurial agents ( 12), a trypsin-sensitive site (23), and a methyl receptor locus (24). There may be overlap among these sites; however, it appears that the amiloride-binding and Na self-inhibition sites are distinct (20).…”

“…In rabbit distal colon, PCMBS "freezes" the Na conductance of the apical membrane at a mean value of 2-3 ,ueq cm-2 h-' and renders the transport amiloride-insensitive (25). Sulfhydryl reagents also stimulate electrogenic Na absorption in frog skin (12,26); however, in contrast to distal colon, this Na transport is blocked by amiloride. The response of the cecum to PCMBS and phenamil is similar to that found in frog skin rather than distal colon: i.e., sulfhydryl reagent stimulation of Na transport that retains its sensitivity to an amiloride analogue.…”

Ion selectivity of epithelial Na channels

“…The effects of agents which modify carboxyl groups have been studied by several laboratories with mixed results. Carbodiimides were found to block Na transport by the skin of Rana temporaria (Zeiske & Lindemann, 1975) but not by that of other frog species (Benos et al, 1980a) or by the toad bladder (Harms & Fanestil, 1977). EEDQ, a more amphipathic carboxyl-modifying reagent, has been shown to inhibit Na transport in toad bladder, and the action of this reagent is protected against by amiloride (Park, Kipnowski & Fanestil, 1983):…”

Sulfhydryl-reactive heavy metals increase cell membrane K+ and Ca2+ transport in renal proximal tubule

“…Ag § has also been shown to increase the cation conductance of toad bladder (Walser, 1970) and rat ileum (Clarkson & O'Toole, 1964). Similarly, pCMBS has been shown to increase the cation conductance of total urinary bladder (Spooner & Edelman, 1976) and to stimulate short-circuit current (and by inference Na + transport) across frog skin (Benos, Mandel & Simon, 1980). In addition to the Hg2+-induced increase in cell membrane K § permeability, we also observed a delayed inhibition of both ouabain-sensitive and nystatin-stimulated QO2.…”