Assuntos
Acetamidas/uso terapêutico , Anemia Hemolítica Congênita/sangue , Eritrócitos/metabolismo , Mutação com Ganho de Função , Hidropisia Fetal/sangue , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Transporte de Íons/efeitos dos fármacos , Mutação de Sentido Incorreto , Bloqueadores dos Canais de Potássio/uso terapêutico , Potássio/sangue , Compostos de Tritil/uso terapêutico , Acetamidas/farmacologia , Anemia Hemolítica Congênita/genética , Contagem de Células , Avaliação Pré-Clínica de Medicamentos , Deformação Eritrocítica , Eritrócitos/efeitos dos fármacos , Heterozigoto , Humanos , Hidropisia Fetal/genética , Técnicas In Vitro , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Fragilidade Osmótica , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Compostos de Tritil/farmacologiaRESUMO
Expression of the polycystin-1 C-terminal cytoplasmic tail increases Cl(-) channel activity in Xenopus oocytes. Background. Cyst expansion in autosomal-dominant polycystic kidney disease (ADPKD) is characterized by active Cl(-) secretion in excess of solute reabsorption. However, the connections between elevated epithelial Cl(-) secretion and loss-of-function or dysregulation of either ADPKD gene polycystin-1 (PC1) or polycystin-2 (PC2) remain little understood. Methods. Cl(-) transport in Xenopus oocytes expressing the CD16.7-PKD1 (115-226) fusion protein containing the final 112 amino acid (aa) of the PC1 C-terminal cytoplasmic tail, or in oocytes expressing related PC1 fusion protein mutants, was studied by isotopic flux, two-electrode voltage clamp, and outside-out patch clamp recording. Results. Expression in oocytes of CD16.7-PKD1 (115-226) increased rates of both influx and efflux of (36)Cl(-), whereas CD16.7-PKD1 (1-92) containing the initial 92 aa of the PC1 C-terminal cytoplasmic tail was inactive. The increased Cl(-) transport resembled CD16.7-PKD1 (115-226)-stimulated cation current in its sensitivity to ADPKD-associated missense mutations, to mutations in phosphorylation sites, and to mutations within or encroaching upon the PC1 coiled-coil domain, as well as in its partial suppression by coexpressed PC2. The NS3623- and 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive (36)Cl(-) flux was not blocked by injected ethyleneglycol tetraacetate (EGTA) or by the cation channel inhibitor SKF96365, and was stimulated by the cation channel inhibitor La(3+), suggesting that CD16.7-PKD1 (115-226)-associated cation conductance was not required for (36)CI(-) flux activation. Outside-out patches from oocytes expressing CD16.7-PKD1 (115-226) also exhibited increased NS3623-sensitive Cl(-) current. Conclusion. These data show that CD16.7-PKD1 (115-226) activates Cl(-) channels in the Xenopus oocyte plasma membrane in parallel with, but not secondary to, activation of Ca(2+)-permeable cation channels.
Assuntos
Canais de Cloreto/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Proteínas/genética , Proteínas/metabolismo , Animais , Cálcio/metabolismo , Cátions/metabolismo , Membrana Celular/metabolismo , Cloretos/metabolismo , Citoplasma/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Oócitos/citologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Fosforilação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/fisiopatologia , Estrutura Terciária de Proteína , Proteínas/química , RNA Complementar , Radioisótopos , Serina/metabolismo , Canais de Cátion TRPP , Tirosina/metabolismo , XenopusRESUMO
The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared. Transport was studied as unidirectional fluxes of (36)Cl(-), [(14)C]oxalate, and [(35)S]sulfate; as net fluxes of HCO(3)(-) by fluorescence ratio measurement of intracellular pH; as current by two-electrode voltage clamp; and as net Cl(-) flux by fluorescence intensity measurement of relative changes in extracellular and intracellular [Cl(-)]. Four human SLC26A6 polypeptide variants each exhibited rates of bidirectional [(14)C]oxalate flux, Cl(-)/HCO(3)(-) exchange, and Cl(-)/OH(-) exchange nearly equivalent to those of mouse slc26a6. Cl(-)/HCO(3)(-) exchange by both orthologs was cAMP-sensitive, further enhanced by coexpressed wild type cystic fibrosis transmembrane regulator but inhibited by cystic fibrosis transmembrane regulator DeltaF508. However, the very low rates of (36)Cl(-) and [(35)S]sulfate transport by all active human SLC26A6 isoforms contrasted with the high rates of the mouse ortholog. Human and mouse orthologs also differed in patterns of acute regulation. Studies of human-mouse chimeras revealed cosegregation of the high (36)Cl(-) transport phenotype with the transmembrane domain of mouse slc26a6. Mouse slc26a6 and human SLC26A6 each mediated electroneutral Cl(-)/HCO(3)(-) and Cl(-)/OH(-) exchange. In contrast, whereas Cl(-)/oxalate exchange by mouse slc26a6 was electrogenic, that mediated by human SLC26A6 appeared electroneutral. The increased currents observed in oocytes expressing either mouse or human ortholog were pharmacologically distinct from the accompanying monovalent anion exchange activities. The human SLC26A6 polypeptide variants SLC26A6c and SLC26A6d were inactive as transporters of oxalate, sulfate, and chloride. Thus, the orthologous mouse and human SLC26A6 proteins differ in anion selectivity, transport mechanism, and acute regulation, but both mediate electroneutral Cl(-)/HCO(3)(-) exchange.