Voltage and Cosubstrate Dependence of the Na-HCO3 Cotransporter Kinetics in Renal Proximal Tubule Cells

Abstract

The voltage dependence of the kinetics of the sodium bicarbonate cotransporter was studied in proximal tubule cells. This electrogenic cotransporter transports one Na+, three HCO3-, and two negative charges. Cells were grown to confluence on a permeable support, mounted on a Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, di-nitro-stilbene-di-sulfonate-sensitive current was shown to be sodium and bicarbonate dependent and therefore was taken as flux through the cotransporter. Voltage-current relations were measured as a function of Na+ and HCO3- concentrations between -160 and +160 mV under zero-trans and symmetrical conditions. The kinetics could be described by a Michaelis-Menten behavior with a Hill coefficient of 3 for HCO3- and 1 for Na+. The data were fitted to six-state ordered binding models without restrictions with respect to the rate-limiting step. All ordered models could quantitatively account for the observed current-voltage relationships and the transinhibition by high bicarbonate concentration. The models indicate that 1) the unloaded transporter carries a positive charge; 2) the binding of cytosolic bicarbonate to the transporter "senses" 12% of the electric field in the membrane, whereas its translocation across the membrane "senses" 88% of the field; 3) the binding of Na+ to the cotransporter is voltage independent.

The renal Na-HCO 3 -cotransporter expressed in Xenopus laevis oocytes: change in stoichiometry in response to elevation of cytosolic Ca 2+ concentration

“…As can be seen in Fig. 7, these results fit nicely to the concentration dependence published by Gross and Hopfer [16], indicating a Hill coefficient near 3.…”

“…In an elegant study Gross and Hopfer [16] demonstrated that the charge transport by rkNBC of primary monolayer cultures of rat proximal tubule exhibits a sigmoidal dependence on [HCO 3 -] i with a Hill coefficient near 3, which agrees with the flux coupling ratio of q=3:1 observed in the same preparation [15]. Although our measurements could not be completed, 1 we succeeded in changing [HCO 3 -] i from 17.5 to either 10.8 or 35.0 mmol/l in three patches bathed with 0.5 µmol/l [Ca 2+ ] i that exhibited q=3:1 (see Table 5).…”

“…3. Recently Gross and Hopfer [16] presented an alternative model in which rkNBC binds 3 HCO 3 ions + 1 Na + ion but they lumped the binding of all 3 HCO 3 ions into one single reaction step [16].…”

Cation‐Coupled Bicarbonate Transporters

“…The uptake rate was insensitive to presumed depolarization by increasing extracellular [K + ] and it was suggested that this could reflect an electroneutral Na,HCO 3 -cotransport. As pointed out by Gross and Hopfer (124), the rate of an electrogenic transport is not necessarily strongly dependent on the membrane potential because the effect of the membrane potential depends on how the transport molecule operates, i.e. whether the rate limiting step of the translocation is voltage sensitive.…”

“…The question of the kinetic model of the electrogenic Na,HCO 3 -cotransporter was investigated in a detailed study by Gross and Hopfer (124). Based on a comparison of experimental data and a model for ordered binding of Na + and HCO 3 − , it was concluded that an ordered model is possible and that the major voltage dependence of the transporter is associated with the binding and translocation of HCO 3 − , whereas the binding of Na + is voltage independent.…”