The electrogenicity of the rat sodium-bicarbonate cotransporter NBCe1 requires interactions among transmembrane segments of the transporter.
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 microS (compared with 12.5 microS 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 microS), the cytoplasmic C-terminal domain (Ct; -123.8 mV/9.7 microS) or Nt + L + Ct (-120.9 mV/3.7 microS). 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.
Choi
Inyeong
I
Department of Physiology, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA. ichoi@emory.edu
Soo Yang
Han
H
Boron
Walter F
WF
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004594:Electrophysiology; D005110:Extracellular Space / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000502:physiology*; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000502:physiology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000502:physiology*; D014981:Xenopus
D001639:Bicarbonates; D050506:Mutant Chimeric Proteins; C422004:Slc4a4 protein, rat; D027982:Sodium-Bicarbonate Symporters