Functional evidence for a pH sensor of erythrocyte K-Cl cotransport through inhibition by internal protons and diethylpyrocarbonate

Cell Physiol Biochem. 1998;8(1-2):46-60. doi: 10.1159/000016270.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Carrier Proteins / chemistry
  • Carrier Proteins / metabolism*
  • Cell Size
  • Chlorides / metabolism*
  • Diethyl Pyrocarbonate / pharmacology*
  • Erythrocytes / cytology
  • Erythrocytes / metabolism*
  • Histidine / metabolism
  • Hydrogen-Ion Concentration
  • Ion Transport
  • Magnesium / metabolism
  • Osmolar Concentration
  • Ouabain / pharmacology
  • Potassium / metabolism*
  • Protons*
  • Rubidium
  • Sheep
  • Symporters*

Substances

  • Carrier Proteins
  • Chlorides
  • Protons
  • Symporters
  • potassium-chloride symporters
  • Histidine
  • Ouabain
  • Magnesium
  • Diethyl Pyrocarbonate
  • Rubidium
  • Potassium