Functionally Important Residues in the Predicted 3rd Transmembrane Domain of the Type IIa Sodium-phosphate Cotransporter (NaPi-IIa)
| Autor: | Juerg Biber, Heini Murer, Andrea Bacconi, Ian C. Forster, Leila V. Virkki |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
Gene isoform
Physiology Voltage clamp Molecular Sequence Data Biophysics Xenopus Sodium-Phosphate Cotransporter Proteins Type IIa Membrane Potentials Divalent Cell membrane Structure-Activity Relationship Xenopus laevis medicine Animals Amino Acid Sequence Binding site Cells Cultured chemistry.chemical_classification biology Cell Membrane Cell Biology biology.organism_classification Recombinant Proteins Protein Structure Tertiary Transmembrane domain medicine.anatomical_structure Amino Acid Substitution chemistry Biochemistry Mutagenesis Site-Directed Oocytes Cotransporter Ion Channel Gating |
| Popis: | The type IIa Na(+)/P(i), cotransporter (NaPi-IIa) mediates electrogenic transport of three Na(+) and one divalent P(i) ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and P(i) transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of P(i) transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the P(i)-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na(+)-dependent P(i) transport. Since electrogenic NaPi-II transports 3 Na(+)/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na(+) binding sites in NaPi-IIa. |
| Databáze: | OpenAIRE |
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