Thiol-dependent passive K: Cl transport in sheep red blood cells: IX. Modulation by pH in the presence and absence of DIDS and the effect of NEM

Abstract

Recently we proposed that cytoplasmic acidification of low K+ (LK) sheep erythrocytes may stimulate ouabain-resistant Cl(-)-dependent K+ flux (K+: Cl- contransport), also known to be activated by cell swelling, treatment with N-ethylmaleimide (NEM), or removal of cellular bivalent cations. Here we studied the dependence of K+ transport on intracellular and extracellular pH (pHi, pHo) varied either simultaneously or independently using the Cl-/HCO3- exchange inhibitor 4,4, diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In both control and NEM-treated LK cells volumes were kept near normal by varying extracellular sucrose. Using DIDS as an effective pH clamp, both K+ efflux and influx of Rb+ used as K+ congener were strongly activated at acid pHi and alkaline pHo. A small stimulation of K+ (Rb+) flux was also seen at acid pHi in the absence of DIDS, i.e., when pHi approximately pHo. Anti-Ll serum, known to inhibit K+: Cl-cotransport, prevented the pHi-stimulated K+ (Rb+) fluxes. Subsequent to NEM treatment at pH 6, K+ (Rb+) fluxes were activated only by raising pH, and thus were similar to the pH activation profile of K+ (Rb+) fluxes in DIDS-treated cells with pHo varied at constant physiologic pHi. Anti-Ll, which inhibited NEM-stimulated K+ (Rb+) fluxes, failed to do so in NEM-plus DIDS-treated cells. Thus, NEM treatment interferes with the internal but not with the external pH-sensitive site.