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CELLULAR |
1 Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA
2 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA
The electrogenic Na+–HCO3– cotransporter (NBCe1) plays a central role in intracellular pH (pHi) regulation as well as HCO3– secretion by pancreatic ducts and HCO3– reabsorption by renal proximal tubules. To understand the structural requirements for the electrogenicity of NBCe1, we constructed chimeras of NBCe1-A and the electroneutral NBCn1-B, and used two-electrode voltage clamp to measure electrogenic transporter current in Xenopus oocytes exposed to 5% CO2–26 mM HCO3–(pH 7.40). The chimera consisting of NBCe1-A (i.e. NBCe1-A ‘background’) with the cytoplasmic N-terminal domain (Nt) of NBCn1-B had a reversal potential of –156.3 mV (compared with a membrane potential Vm of –43.1 mV in a HCO3–-free solution) and a slope conductance of 3.0 µS (compared with 12.5 µS for NBCe1-A). Also electrogenic were chimeras with an NBCe1-A background but with NBCn1-B contributing the extracellular loop (L) between transmembrane segment (TM) 5 and 6 (–140.9 mV/11.1 µS), the cytoplasmic C-terminal domain (Ct; –123.8 mV/9.7 µS) or Nt + L + Ct (–120.9 mV/3.7 µS). Reciprocal chimeras (with an NBCn1 background but with NBCe1 contributing Nt, L, Ct or Nt + L + Ct) produced no measurable electrogenic transporter currents in the presence of CO2–HCO3–. pHi recovered from an acid load, but without the negative shift of Vm that is characteristic of electrogenic Na+–HCO3– cotransporters. Thus, these chimeras were electroneutral, as were two others consisting of NBCe1(Nt–L)/NBCn1(TM6–Ct) and NBCn1(Nt–L)/NBCe1(TM6–Ct). We propose that the electrogenicity of NBCe1 requires interactions between TM1–5 and TM6–13.
(Received 7 June 2006;
accepted after revision 5 October 2006;
first published online 12 October 2006)
Corresponding author I. Choi: Department of Physiology, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA. Email: ichoi{at}emory.edu
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