Effects of pH on kinetic parameters of the Na-HCO3 cotransporter in renal proximal tubule.
The effects of pH on cotransporter kinetics were studied in renal proximal tubule cells. Cells were grown to confluence on permeable support, mounted in an Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, dinitrostilbene-disulfonate-sensitive current (DeltaI) was Na+ and HCO3- dependent and therefore was taken as flux through the cotransporter. When the pH of the perfusing solution was changed between 6.0 and 8.0, the conductance attributable to the cotransporter showed a maximum between pH 7.25 and pH 7.50. A similar profile was observed in the presence of a pH gradient when the pH of the apical solutions was varied between 7.0 and 8.0 (basal pH lower by 1), but not when the pH of the basal solution was varied between 7.0 and 8.0 (apical pH lower by 1 unit). To delineate the kinetic basis for these observations, DeltaI-voltage curves were obtained as a function of Na+ and HCO3- concentrations and analyzed on the basis of a kinetic cotransporter model. Increases in pH from 7.0 to 8.0 decreased the binding constants for the intracellular and extracellular substrates by a factor of 2. Furthermore, the electrical parameters that describe the interaction strength between the electric field and substrate binding or charge on the unloaded transporter increased by four- to fivefold. These data can be explained by a channel-like structure of the cotransporter, whose configuration is modified by intracellular pH such that, with increasing pH, binding of substrate to the carrier is sterically hindered but electrically facilitated.
Gross
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Department of Urology, Case Western Reserve University, Cleveland, Ohio 44106, USA. ezg@po.cwru.edu
Hopfer
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D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D055592:Biophysical Phenomena; D001703:Biophysics; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D017136:Ion Transport; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters; D013267:Stilbenes / Q000494:pharmacology
D001639:Bicarbonates; D002352:Carrier Proteins; D027982:Sodium-Bicarbonate Symporters; D013267:Stilbenes; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D012964:Sodium