Sodium permeability of dog red blood cell membranes. I. Identification of regulatory sites.

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Abstract
Divalent cations and group-specific chemical modifiers were used to modify Na efflux in order to probe the molecular structure of Na channels in dog red blood cells. Hg++, Ni++, Co++ and PCMBS (parachloromercuribenzene sulfonic acid), a sulfhydryl reactive reagent, induced large increases in Na+ permeability and their effects can be described by a curve which assumes 2:1 binding with the Na channel. The sequence of affinities, as measured by the dissociation constants, reflects the reactivity of these divalent cations with sulfhydryl [SH] groups. In addition, the effects of Hg++ and PCMBS can be reversed by the addition of dithiothreitol, an SH-containing compound, to the medium. Much smaller increases in Na+ permeability were produced by Zn++ and the amino-specific reagents, TNBS (2,4,6-trinitrobenzene sulfonic acid) and SITS (4-acetamido-4''-isothiocyano-stilbene-2-2''-disulfonic acid). The Zn++ effect can be described by a curve which assumes bimolecular binding with the channel, and its effect on Na+ permeability can be reversed by the addition of glycine to the medium. The effects of Ni++ and SITS can be completely reversed by washing the cells in 0.16 M NaCl while TNBS binding was partially irreversible. Measurements of mean cell volumes (MCV) indicate that the modifier-induced increases in Na+ permeability were not caused by shrinkage of the cells. The movement of Na ions through ionic channels in dog red blood cells can apparently be enhanced by modification of amino and sulfhydryl groups. Zn++, TNBS and SITS increased Na+ permeability by modifying amino groups in the channel while Hg++, Ni++, Co++ and PCMBS acted on SH groups.