Mechanism of Na+/proline symport inEscherichia coli: Reappraisal of the effect of cation binding to the Na+/proline symport carrier |
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Authors: | Ichiro Yamato Yasuhiro Anraku |
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Affiliation: | (1) Department of Physiology and Biophysics, Wright State University, School of Medicine, 45401-0927 Dayton, Ohio |
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Abstract: | Summary Recently we proposed that cytoplasmic acidification of low K+ (LK) sheep erythrocytes may stimulate ouabain-resistant Cl–-dependent K+ flux (K+Cl– cotransport), 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-3,2-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 pHipHo. 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. |
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Keywords: | ouabain-resistant K fluxes sheep erythrocytes pH effects DIDS N-ethylmaleimide cotransport membranes |
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