A cysteine-scanning mutagenesis study of transmembrane domain 8 of the electrogenic sodium/bicarbonate cotransporter NBCe1
A cysteine-scanning mutagenesis study of transmembrane domain 8 of the electrogenic sodium/bicarbonate cotransporter NBCe1
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DOI：
10.1074/jbc.m607253200
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发表时间：
2006-10-27
影响因子：
4.8
通讯作者：
Bevensee, Mark O.
中科院分区：
文献类型：
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作者：
McAlear, Suzanne D.;Bevensee, Mark O.
Na/HCO3共转运体（NBCs）如NBCe1是碳酸氢盐转运体超家族的成员，包括阴离子交换体。阴离子交换器1的假定跨膜结构域8 （TMD8）残基参与离子转运(Tang， X. B., Kovacs， M., Sterling， D., and Casey， J. R.（1999）。化学，274,3557 -3564)，NBCe1变体中相应的结构域是高度同源的。我们通过半胱氨酸扫描诱变来检测TMD8残基在大鼠NBCe1-A离子易位中的作用。我们使用双电极电压钳技术研究了爪蟾卵母细胞中表达的21个半胱氨酸取代的NBC突变体的功能和/或巯基敏感性以及对氯汞苯磺酸（pCMBS）的可及性。5个NBC突变体显示出< 10%的野生型活性：P743C、A744C、L746C、D754C和T758C。对于剩下的16个突变体，我们比较了将卵母细胞暴露于2-氨基乙基甲烷硫代磺酸盐（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对NBC突变体T748C、I749C、I751C、F752C、M753C和Q756C的抑制作用为9-19%，对突变体A739C、A741C、L745C、V747C、Q755C和I757C的抑制作用为11-21%。pCMBS对突变体A739C、A740、V747C和Q756C的抑制作用为5%或8%，对突变体I749C的抑制作用为10%。然而，两种巯基试剂对L750C突变体的抑制作用均为>= 85%。采用替代半胱氨酸可达性方法，研究了NBC突变体L750C在不同转运体条件下的可达性。当转运体在Na+和HCO3-存在下都活跃时，pCMBS的可及性(i)降低，可能是由于底物与pCMBS的竞争；（ii）在二苯乙烯抑制剂存在下还原；(3)膜电位更正。综上所述，NBCe1的TMD8残基，特别是L750，参与了离子易位，其可及性受转运体活性状态的影响。
Na/HCO3 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-chloro-mercuribenzene 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 >= 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 HCO3-, 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.