Study of amino and sulfhydryl sites in the sodium pathway in dog red blood cell membranes

Summary Amino reactive TNBS (2,4,6-trinitrobenzene sulfonic acid), SITS (4-acetamido-4-isothiocyano-stilbene-2-2-disulfonic acid), and Zn⁺⁺, and SH reactive Hg⁺⁺ were employed to study sodium channels in dog red blood cells. Simultaneous modification of the membrane with both a SH and an amino modifier results in an increase in Na⁺ permeability which is equal to the sum of their individual effects. This indicates that SH and amino sites are separate units. Three lines of evidence indicate that the amino sites are more superficial than the SH sites. (1) Pretreatment with an amino modifier decreases the effectiveness of subsequent SH modification. (2) SITS, a nonpenetrating amino reagent, enhances Na⁺ permeability while DTNB, a nonpenetrating SH modifier, is ineffective. (3) Pretreatment of amino sites decreases the apparent affinity of Hg⁺⁺ for SH sites. In addition, three lines of evidence indicate that TNBS and Zn⁺⁺ modify different amino sites. First, simultaneous modification with TNBS and Zn⁺⁺ results in an increase in Na⁺ permeability equal to the sum of their individual effects. Secondly, Zn⁺⁺ causes an increase in Na⁺ permeability in cells previously treated with TNBS. Finally, the pH dependence of Zn⁺⁺ modification is opposite that for TNBS modification. These pH experiments suggest that Zn⁺⁺ enhances Na⁺ permeability by reacting with unprotonated amino sites while TNBS modifies protonated amino sites. It is concluded that the sodium permeability of dog red blood cells is normally limited by superficial amino sites and deeper sulfhydryl sites in the sodium channels.