Characterization of essential sulfhydryl groups of rat renal Na(+)-Pi cotransporter

Sulfhydryl (SH) groups are essential for the function of the Na(+)-Pi cotransporter of renal brush-border membrane (BBM) as determined by inhibition of Na(+)-Pi cotransport by HgCl2. Recent studies suggest that essential SH groups may be present on the cytoplasmic side of the BBM. We used various maneuvers to differentiate between external and internal SH groups on the Na(+)-Pi cotransporter in renal BBM vesicles (BBMV). The inhibitory potency of p-chloromercuriphenylsulfonic acid (PCMBS), a poorly permeable SH reagent, was about one-half that of p-chloromercuribenzoic acid (PCMB), a highly permeable reagent (half-maximal inhibitory concentrations of 625 and 350 microM, respectively). 5,5'-Dithio-bis-(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) were additive to HgCl2 for inhibition of Pi transport. The highly permeable NEM gave a more pronounced additive effect (+30%) than the less permeable DTNB (+15%). When the intravesicular pH (pHi) and extravesicular pH (pHo) were varied independently, NEM (which reacts mainly at an alkaline pH) inhibited Pi transport only at pHi = 8.5, regardless of pHo. When internal SH groups were blocked by NEM at pH 8.5, PCMB and PCMBS produced similar additive effects. The binding of 14C-labeled phosphonoformic acid was inhibited by both reagents and to the same extent. Both PCMB and PCMBS increased 32Pi efflux from BBMV. The findings are consistent with the presence of essential SH groups on the cytoplasmic side of the BBM, with possible conformational changes induced by modification of the external SH groups.