McAlear Suzanne D, Bevensee Mark O
Department of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, AL 35294, USA.
J Biol Chem. 2006 Oct 27;281(43):32417-27. doi: 10.1074/jbc.M607253200. Epub 2006 Aug 26.
Na/HCO(3) cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557-3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed <10% wild-type activity: P743C, A744C, L746C, D754C, and T758C. For the remaining 16 mutants, we compared transporter-mediated inward currents elicited by removing external Na(+) before and after exposing oocytes to either 2-aminoethylmethane thiosulfonate (MTSEA) or pCMBS. MTSEA inhibited NBC mutants T748C, I749C, I751C, F752C, M753C, and Q756C by 9-19% and stimulated mutants A739C, A741C, L745C, V747C, Q755C, and I757C by 11-21%. pCMBS mildly inhibited mutants A739C, A740, V747C, and Q756C by 5 or 8%, and stimulated I749C by 10%. However, both sulfhydryl reagents strongly inhibited the L750C mutant by > or =85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na(+) and HCO(3)(-), likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.
诸如NBCe1之类的Na/HCO(3)共转运体（NBCs）是包括阴离子交换体在内的碳酸氢盐转运体超家族的成员。阴离子交换体1的假定跨膜结构域8（TMD8）内的残基参与离子转运（Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557 - 3564），并且NBCe1变体中的相应结构域高度同源。我们进行了半胱氨酸扫描诱变，以研究TMD8残基在大鼠NBCe1 - A离子转运中的作用。我们使用双电极电压钳技术，检测了非洲爪蟾卵母细胞中表达的21种半胱氨酸取代的NBC突变体的功能和/或巯基敏感性以及对对氯汞苯磺酸盐（pCMBS）的可及性。五个NBC突变体表现出<10%的野生型活性：P743C、A744C、L746C、D754C和T758C。对于其余16个突变体，我们比较了在将卵母细胞暴露于2 - 氨基乙硫代磺酸（MTSEA）或pCMBS之前和之后，通过去除外部Na(+)引发的转运体介导的内向电流。MTSEA抑制NBC突变体T748C、I749C、I751C、F752C、M753C和Q756C达9 - 19%，并刺激突变体A739C、A741C、L745C、V747C、Q755C和I757C达11 - 21%。pCMBS轻微抑制突变体A739C、A740、V747C和Q756C达5%或8%，并刺激I749C达10%。然而，两种巯基试剂均强烈抑制L750C突变体达≥85%。使用取代半胱氨酸可及性方法，我们检测了NBC突变体L750C在不同转运体条件下的可及性。当转运体在Na(+)和HCO(3)(-)均存在时处于活性状态时，pCMBS的可及性（i）降低，这可能是由于底物与pCMBS竞争；（ii）在存在芪抑制剂时降低；以及（iii）在更正的膜电位下受到刺激。总之，NBCe1的TMD8残基特别是L750参与离子转运，并且可及性受转运体活性状态的影响。
