Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

We recently cloned and reported the basic properties of rkNBC expressed in Xenopus oocytes [Romero et al, AJP 274:F425, 1998]. In the present study we used microelectrodes to voltage clamp or measure intracellular ion activities (H+ and Na+) of oocytes expressing rkNBC. Voltage clamp studies allow us to measure the voltage dependence of rkNBC-mediated currents, i.e., DIDS-inhibitable, HCO3 stimulated currents. At voltages more negative than the reversal poten-tial (Erev), we measure an inward current (NaHCO3 efflux). Conversely, at voltages more posi-tive than Erev, we measure an outward current (NaHCO3 influx). The magnitude of the current is a quantitative measure of rkNBC transport. To obtain a current-voltage profile (I-V), we stepped the cell membrane potential from a -60 mV holding potential to test voltages between -160 mV and +60 mV in 20 mV increments. The steady state current is recorded at each step. The rkNBC- current stimulated by 1.5% CO2 / 10 mM HCO3 (pH 7.5) reverses at -93±2 mV (n=19) and has an apparently linear I-V relationship (positive Vm elicits a greater outward current). 200 microM DIDS completely inhibits the HCO3 elicited current (outward and inward) at all voltages. Next, we examined if other monovalent cations (K+, Li+, or choline+) might be transported by rkNBC. Accordingly, we replaced Na+ with the test cation and used the voltage clamp to measure the HCO3 stimulated I-V relationships, i.e., the difference of currents with and without HCO3. Only Na+ elicited HCO-3 stimulated currents. Since Na+ was the only cation transported by rkNBC, we investigated the apparent Na+ affinity of rkNBC. First, we determined Vmax of rkNBC for extra-cellular Na+ by identifying the maximal I-V response by increasing Na+ at 1.5% CO2 / 10 mM HCO3 (pH 7.5). We tested CO2/HCO3 solutions of 96-120 mM Na+; solution osmolalities were matched with choline. 96 mM [Na+]o induced the maximal I-V response of rkNBC. Next after determining Vmax, we measured the apparent Na+ affinity using the following solution protocol: (i) 5 min non-HCO3- ringer. (ii) 10 min 1.5% CO2/10 mM HCO3 /96 mM Na+ (pH 7.5). (iii) In CO2/HCO3, pulses of 0 mM [Na+]o for 24s, test [Na+]o for 8s, 96 mM [Na+]o for 24s. This cycle is repeated for each subsequent test [Na+]o. Using this protocol, we varied [Na+]o from 0-96 mM (choline replacement). 10 min in 1.5% CO2/HCO3 allows equilibration of intracellular pH (pHi) after the CO2 addition (pHi = 7.28±0.02, [HCO3]i = 4.8±0.2 mM, n=9; intracellular Na+ 8-10 mM). For each solution switch, we measured the I-V relationships at the maximum induced cur-rent. For outward transport, the calculated apparent Na+ affinity decreases from 30 to 1 mM moving from -100 to -60 mV; whereas, for inward transport, this Na+ affinity increases from 32 to 80 mM moving from -20 to -80 mV. Finally, using the reversal potentials obtained from these voltage clamp data and intracellular pH and Na+ measurements, we calculated the Na+ : HCO3 stoichiometry as 1 Na+ to 2 HCO3 (n=24), independent of extracellular [Na+]. These data indicate that previous stoichiometry calculations of endogenous electrogenic Na/HCO3 cotransport from the renal proximal tubule may be an overestimate or that additional, yet unknown factors, are ca-pable of altering rkNBC transport stoichiometry. Thus, our data indicate that rkNBC is (i) Na+ selective, (ii) HCO3 activated, (iii) voltage dependent for NaHCO3 influx and efflux, (iv) regulated by extracellular Na+ in the physiologic voltage range (-20 to -100 mV), and (v) blocked by extracellular DIDS regardless of Na+ concentration or voltage.

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Presentation Number SAsciortino0797
Keywords: acid-base, intracellular pH, bicarbonate, voltage clamp, stoichiometry