Electrogenic Na/HCO₃ cotransporters from rat and salamander kidney have similar external HCO₃ dependencies

The deduced amino-acid sequences of the electrogenic Na/HCO₃ cotransporters cloned from Ambystoma (aNBC) and rat (rNBC) are 86% identical. Here, we studied the extracellular HCO₃ dependencies of aNBC and rNBC expressed in Xenopus oocytes, injected with cRNA encoding aNBC or rNBC (in Xenopusexpression vector pTLN2). We monitored the shift in membrane potential (dV_m) or outward clamp current (I_out, V_hold = -60 mV) caused by switching the external buffer from HEPES to CO₂ /HCO₃ at a fixed pH_o of 7.5 (22°C). The major external anion was gluconate; we varied [HCO₃]_o from nominally 0 to 99 mM by exchanging gluconate for HCO₃, maintaining a constant [CO₂]/[HCO₃] ratio. In water-injected oocytes, 5% CO₂ /33 mM HCO₃ caused a small, slow depolarization or inward current. In NBC-expressing oocytes, CO₂ /HCO₃ caused a large, immediate dV_m of -30 to -100 mV (aNBC) or an immediate dI_out of 150 - 400 nA (rNBC). The dV_m and dI_out were 60% inhibited by 140 uM by DIDS, and 97% by 0 Na⁺. In aNBC-expressing oocytes, a non-linear, least-squares curve fit of the dV_m vs. [HCO₃]_o data by a normalized Michaelis-Menten equation yielded an apparent K_m for extracellular HCO₃ of 15.1 (SD=1.3) mM, and a V_max 1.54 (SD=0.05) fold greater than the dV_m caused by 33 mM HCO₃. In similar dV_m experiments with rNBC, the K_m was 12.9 (SD=3.1) mM, and the normalized V_max was 1.40 (SD=0.12). In rNBC oocytes, I_out was large enough for determining the HCO₃ dependence: K_m was 15.2 (SD=1.4) mM, and normalized V_max was 1.59 (SD=0.04). Our data shows that external HCO₃ dependence is similar for two NBCs from species in different classes (amphibian vs. mammal).