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Effect of simultaneously replacing putative TM6 and TM12 of human NBCe1-A with those from NBCn1 on surface abundance in Xenopus oocytes.

ABSTRACT: HCO?? translocation across the plasma membrane via the electrogenic Na/HCO?? cotransporter NBCe1 plays an important role in intracellular pH regulation and transepithelial HCO?? transport. However, the structural determinants of transporter function remain largely unknown. A previous study showed that the putative fourth extracellular loop (EL4) plays an essential role in determining the electrogenicity of NBCe1. In the present study, we generated eight new chimeras of human NBCe1-A and NBCn1-A. All possess the putative NBCe1 EL4 and are electrogenic. Chimera O, in which the putative sixth transmembrane segment (TM6) and EL5 through the C terminus (Ct) of NBCe1 was replaced by corresponding NBCn1 sequence, produces the smallest hyperpolarization (1-2 mV) when CO?/HCO?? is added to the extracellular solution. Biotinylation experiments show that O has a very low abundance at the plasma membrane. However, chimeras in which we simultaneously replaced the putative TM6 and smaller subdomains of the EL5-Ct region for the NBCn1 sequence were strongly electrogenic except for chimera T, in which we replaced TM6 and TM12 of NBCe1 with the corresponding regions of NBCn1. T exhibited greatly reduced transporter surface expression compared to wild-type NBCe1-A, while retaining at least some electrogenic character. We hypothesize that putative TM6 and TM12 are part of a functional unit and that if the two TMs are replaced by those of the same transporter type, high surface expression would require that the surrounding TMs are also from the same transporter type.

SUBMITTER: Chen LM

PROVIDER: S-EPMC4636127 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Effect of simultaneously replacing putative TM6 and TM12 of human NBCe1-A with those from NBCn1 on surface abundance in Xenopus oocytes.

Chen Li-Ming LM Qin Xue X Moss Fraser J FJ Liu Ying Y Boron Walter F WF

The Journal of membrane biology 20120302 3

HCO₃⁻ translocation across the plasma membrane via the electrogenic Na/HCO₃⁻ cotransporter NBCe1 plays an important role in intracellular pH regulation and transepithelial HCO₃⁻ transport. However, the structural determinants of transporter function remain largely unknown. A previous study showed that the putative fourth extracellular loop (EL4) plays an essential role in determining the electrogenicity of NBCe1. In the present study, we generated eight new chimeras of human NBCe1-A and NBCn1-A. ...[more]

PMID: 22383045

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Project description:Key pointsAccording to the HCO3- metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO3 cotransporter NBCe1-A speeds transport by regenerating/consuming HCO3- . The present work addresses published discrepancies as to whether cytosolic CAs stimulate NBCe1-A, heterologously expressed in Xenopus oocytes. We confirm the essential elements of the previous experimental observations, taken as support for the HCO3- metabolon hypothesis. However, using our own experimental protocols or those of others, we find that NBCe1-A function is unaffected by cytosolic CAs. Previous conclusions that cytosolic CAs do stimulate NBCe1-A can be explained by an unanticipated stimulatory effect of the CAs on an endogenous Na-H exchanger. Theoretical analyses show that, although CAs could stimulate non- HCO3- transporters (e.g. Na-H exchangers) by accelerating CO2 / HCO3- -mediated buffering of acid-base equivalents, they could not appreciably affect transport rates of NBCe1 or other transporters carrying HCO3- , CO3= , or NaCO3- ion pairs.AbstractThe HCO3- metabolon hypothesis predicts that cytosolic carbonic anhydrase (CA) binds to NBCe1-A, promotes HCO3- replenishment/consumption, and enhances transport. Using a short step-duration current-voltage (I-V) protocol with Xenopus oocytes expressing eGFP-tagged NBCe1-A, our group reported that neither injecting human CA II (hCA II) nor fusing hCA II to the NBCe1-A carboxy terminus affects background-subtracted NBCe1 slope conductance (GNBC ), which is a direct measure of NBCe1-A activity. Others - using bovine CA (bCA), untagged NBCe1-A, and protocols keeping holding potential (Vh ) far from NBCe1-A's reversal potential (Erev ) for prolonged periods - found that bCA increases total membrane current (ΔIm ), which apparently supports the metabolon hypothesis. We systematically investigated differences in the two protocols. In oocytes expressing untagged NBCe1-A, injected with bCA and clamped to -40 mV, CO2 / HCO3- exposures markedly decrease Erev , producing large transient outward currents persisting for >10 min and rapid increases in [Na+ ]i . Although the CA inhibitor ethoxzolamide (EZA) reduces both ΔIm and d[Na+ ]i /dt, it does not reduce GNBC . In oocytes not expressing NBCe1-A, CO2 / HCO3- triggers rapid increases in [Na+ ]i that both hCA II and bCA enhance in concentration-dependent manners. These d[Na+ ]i /dt increases are inhibited by EZA and blocked by EIPA, a Na-H exchanger (NHE) inhibitor. In oocytes expressing untagged NBCe1-A and injected with bCA, EIPA abolishes the EZA-dependent decreases in ΔIm and d[Na+ ]i /dt. Thus, CAs/EZA produce their ΔIm and d[Na+ ]i /dt effects not through NBCe1-A, but endogenous NHEs. Theoretical considerations argue against a CA stimulation of HCO3- transport, supporting the conclusion that an NBCe1-A- HCO3- metabolon does not exist in oocytes.

Dataset Information

Effect of simultaneously replacing putative TM6 and TM12 of human NBCe1-A with those from NBCn1 on surface abundance in Xenopus oocytes.

Publications

Effect of simultaneously replacing putative TM6 and TM12 of human NBCe1-A with those from NBCn1 on surface abundance in Xenopus oocytes.

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