Electrogenic sodium/bicarbonate cotransporter (NBCe1) variants expressed in xenopus oocytes: Protein regions involved in function, expression, and ion translocation

Electrogenic Na/bicarbonate cotransporters (NBCes) are important regulators of intracellular pH in many tissues including the kidney, brain, and pancreas, and are members of a superfamily of [special characters omitted] transporters including Anion Exchangers (AEs) and Nadriven, Cl-bicarbonate exchangers (NDCBEs). There are three NBCe1 splice variants (NBCe1-A, -B, and -C) that only differ in their cytoplasmic N and C termini. The first study is a systematic comparison of the three variants expressed in Xenopus oocytes, and examination of the role of the N and C termini. NBC function of the constructs was assessed using the 2-electrode, voltage-clamp technique or the macropatch technique with NBC-expressing, voltage-clamped oocytes. The three variants had similar ion- and voltage-dependencies, but the A variant displayed a ∼4-fold higher current in whole-cell experiments due to its unique N terminus. Data from truncation mutants indicate that the cytoplasmic C termini influence function and the N termini can influence regulation of NBC activity. Whereas, the unique N terminus of the A variant stimulates NBC function and the different N termini of the B/C variants inhibit function. This finding was also observed in macropatch experiments. In the second study, I performed cysteine-scanning mutagenesis and Substituted-Cysteine Accessibility Method (SCAM) on NBCe1 TMD8, because positions in the homolgous region of AE1 are involved in ion translocation. 21 residues of TMD8 were each replaced with cysteine. Function and/or sulfhydryl sensitivity of these mutants were assessed using the 2-electrode, voltage-clamp technique with NBC-expressing oocytes clamped at -60 mV. Eight mutants are mildly sensitive to one or both sulfhydryl reagents, MTSEA and pCMBS. The L750C mutant is inhibited ≥ 85% by both reagents. pCMBS accessibility of L750C is (1) reduced in the presence of both Na⁺ and [special characters omitted] when the transporter is active, (2) reduced in the presence of an NBC inhibitor, and (3) stimulated at more positive holding potentials. In summary, TMD8 forms part of the anion translocation pathway, and accessibility of L750C is affected by the state of the transporter.