{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Moss FJ"],"funding":["NIDDK NIH HHS"],"pagination":["5821-5856"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC7747792"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["598(24)"],"pubmed_abstract":["<h4>Key points</h4>According to the HCO3- metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO<sub>3</sub> 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 CO<sub>2</sub> / 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.<h4>Abstract</h4>The 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 (G<sub>NBC</sub> ), which is a direct measure of NBCe1-A activity. Others - using bovine CA (bCA), untagged NBCe1-A, and protocols keeping holding potential (V<sub>h</sub> ) far from NBCe1-A's reversal potential (E<sub>rev</sub> ) for prolonged periods - found that bCA increases total membrane current (ΔI<sub>m</sub> ), 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, CO<sub>2</sub> / HCO3- exposures markedly decrease E<sub>rev</sub> , producing large transient outward currents persisting for >10 min and rapid increases in [Na<sup>+</sup> ]<sub>i</sub> . Although the CA inhibitor ethoxzolamide (EZA) reduces both ΔI<sub>m</sub> and d[Na<sup>+</sup> ]<sub>i</sub> /dt, it does not reduce G<sub>NBC</sub> . In oocytes not expressing NBCe1-A, CO<sub>2</sub> / HCO3- triggers rapid increases in [Na<sup>+</sup> ]<sub>i</sub> that both hCA II and bCA enhance in concentration-dependent manners. These d[Na<sup>+</sup> ]<sub>i</sub> /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 ΔI<sub>m</sub> and d[Na<sup>+</sup> ]<sub>i</sub> /dt. Thus, CAs/EZA produce their ΔI<sub>m</sub> and d[Na<sup>+</sup> ]<sub>i</sub> /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."],"journal":["The Journal of physiology"],"pubmed_title":["Carbonic anhydrases enhance activity of endogenous Na-H exchangers and not the electrogenic Na/HCO<sub>3</sub> cotransporter NBCe1-A, expressed in Xenopus oocytes."],"pmcid":["PMC7747792"],"funding_grant_id":["R01 DK113197","T32 DK007470","R37 DK030344","R01 DK030344"],"pubmed_authors":["Moss FJ","Boron WF"],"additional_accession":[]},"is_claimable":false,"name":"Carbonic anhydrases enhance activity of endogenous Na-H exchangers and not the electrogenic Na/HCO<sub>3</sub> cotransporter NBCe1-A, expressed in Xenopus oocytes.","description":"<h4>Key points</h4>According to the HCO3- metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO<sub>3</sub> 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 CO<sub>2</sub> / 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.<h4>Abstract</h4>The 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 (G<sub>NBC</sub> ), which is a direct measure of NBCe1-A activity. Others - using bovine CA (bCA), untagged NBCe1-A, and protocols keeping holding potential (V<sub>h</sub> ) far from NBCe1-A's reversal potential (E<sub>rev</sub> ) for prolonged periods - found that bCA increases total membrane current (ΔI<sub>m</sub> ), 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, CO<sub>2</sub> / HCO3- exposures markedly decrease E<sub>rev</sub> , producing large transient outward currents persisting for >10 min and rapid increases in [Na<sup>+</sup> ]<sub>i</sub> . Although the CA inhibitor ethoxzolamide (EZA) reduces both ΔI<sub>m</sub> and d[Na<sup>+</sup> ]<sub>i</sub> /dt, it does not reduce G<sub>NBC</sub> . In oocytes not expressing NBCe1-A, CO<sub>2</sub> / HCO3- triggers rapid increases in [Na<sup>+</sup> ]<sub>i</sub> that both hCA II and bCA enhance in concentration-dependent manners. These d[Na<sup>+</sup> ]<sub>i</sub> /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 ΔI<sub>m</sub> and d[Na<sup>+</sup> ]<sub>i</sub> /dt. Thus, CAs/EZA produce their ΔI<sub>m</sub> and d[Na<sup>+</sup> ]<sub>i</sub> /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.","dates":{"release":"2020-01-01T00:00:00Z","publication":"2020 Dec","modification":"2024-11-08T18:23:35.074Z","creation":"2022-02-11T13:15:21.799Z"},"accession":"S-EPMC7747792","cross_references":{"pubmed":["32969493"],"doi":["10.1113/jp280143","10.1113/JP280143"]}}