Functional evidence for a pH sensor of erythrocyte K-Cl cotransport through inhibition by internal protons and diethylpyrocarbonate.
The sidedness of proton modulation of K-Cl cotransport (K-Cl COT) was studied in low K sheep red blood cells stripped of cellular Mg, Mgi, at alkaline medium pH, pHo, by the divalent ionophore A23187 and a chelator. This procedure activates K-Cl COT, presumably, by inhibition of MgATP-dependent kinases. Ouabain-resistant K efflux and Rb influx were measured in Cl or NO3 either at variable pHi and fixed pHo, or vice versa, in erythrocytes pH- and volume-clamped with the anion exchange inhibitor 4,4'-diisothiocyanato-2,2'-disulfonic stilbene (DIDS) and sucrose. Between pHi 9 and 6, and at constant pHo 9, K effluxes decreased hyperbolically in Cl and linearly in NO3 whereas Rb influxes fell almost linearly in Cl and asymptotically in NO3. Thus, saturation of outward and inward K-Cl COT, the calculated difference of the fluxes in Cl and NO3, occurred at slightly different pHi values. Hill plots revealed pKa values of 6.5 and 7.0, and Hill coefficients of > 1 for outward and inward K-Cl COT, respectively. Raising pHo from 6 to 9 at fixed pHi slightly increased K and Rb fluxes in both Cl or NO3, but not K-Cl COT. The histidine reagent diethylpyrocarbonate (DEPC) inhibited low Mgi-activated K-Cl COT at approximately 4 mM, an effect partially reversible by subsequent treatment with hydroxylamine. It is concluded that protons inhibit erythrocyte K-Cl COT through internal histidine(s) which may be part of a pH sensor.
Lauf
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Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, Ohio 45401-0927, USA. plauf@wright.edu
Adragna
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D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D048429:Cell Size; D002712:Chlorides / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D008274:Magnesium / Q000378:metabolism; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D011522:Protons; D012413:Rubidium; D012756:Sheep; D027981:Symporters; D000096922:K Cl- Cotransporters
D002352:Carrier Proteins; D002712:Chlorides; D004047:Diethyl Pyrocarbonate; D006639:Histidine; D008274:Magnesium; D010042:Ouabain; D011188:Potassium; D011522:Protons; D012413:Rubidium; D027981:Symporters; D000096922:K Cl- Cotransporters