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Articles in PresS, published online ahead of print January 16, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00589.2001
Submitted on December 12, 2001
Accepted on January 9, 2002
1 Department of Cellualr and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA
2 University Laboratory of Physiology, University of Oxford, Oxford, United Kingdom
* To whom correspondence should be addressed. E-mail: leila.virkki{at}yale.edu.
We have functionally characterized NBC4, originally cloned from human heart by Pushkin et al (Biochem Biophys Acta 1493: 215, 2000). Of the four NBC4 variants currently present in the GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession # AF293337) in Xenopus laevis oocytes and assayed membrane potential (Vm) and pH-regulatory function using microelectrodes. Exposing an NBC4c-expressing oocyte to a solution containing 5% CO2/33 mM HCO3 elicited a large hyperpolarization, indicating that the transporter is electrogenic. The initial CO2-induced decrease in intracellular pH (pHi) was followed by a slow recovery that was reversed by removing external Na+. Two-electrode voltage clamp of NBC4c-expressing oocytes revealed large HCO3-and Na-dependent currents. When we voltage clamped Vm far from NBC4c's estimated reversal potential (Erev), the pHi-recovery rate increased substantially. Both the currents and pHi recovery were blocked by 200 µM DIDS. We estimated the transporter's HCO3:Na+ stoichiometry by measuring Erev at different [Na]o values. A plot of Erev against log[Na]o was linear, with a slope of 54.8 mV/log[Na]o. This observation, as well as the absolute Erev values, are consistent with a 2:1 stoichiometry. In conclusion, the behavior of NBC4c, which we propose to call NBCe2-c, is similar to that of NBCe1, the first electrogenic NBC.
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