| Autor: |
Ilyaskin AV; Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstr. 6, 91054, Erlangen, Germany., Kirsch SA; Computational Biology, Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany., Böckmann RA; Computational Biology, Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany., Sticht H; Abteilung für Bioinformatik, Institut für Biochemie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany., Korbmacher C; Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstr. 6, 91054, Erlangen, Germany., Haerteis S; Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstr. 6, 91054, Erlangen, Germany. silke.haerteis@fau.de., Diakov A; Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Waldstr. 6, 91054, Erlangen, Germany. |
| Abstrakt: |
The bile acid-sensitive ion channel (BASIC) is a member of the ENaC/degenerin family of ion channels. It is activated by bile acids and inhibited by extracellular Ca 2+ . The aim of this study was to explore the molecular mechanisms mediating these effects. The modulation of BASIC function by extracellular Ca 2+ and tauro-deoxycholic acid (t-DCA) was studied in Xenopus laevis oocytes heterologously expressing human BASIC using the two-electrode voltage-clamp and outside-out patch-clamp techniques. Substitution of aspartate D444 to alanine or cysteine in the degenerin region of BASIC, a region known to be critically involved in channel gating, resulted in a substantial reduction of BASIC Ca 2+ sensitivity. Moreover, mutating D444 or the neighboring alanine (A443) to cysteine significantly reduced the t-DCA-mediated BASIC stimulation. A combined molecular docking/simulation approach demonstrated that t-DCA may temporarily form hydrogen bonds with several amino acid residues including D444 in the outer vestibule of the BASIC pore or in the inter-subunit space. By these interactions, t-DCA may stabilize the open state of the channel. Indeed, single-channel recordings provided evidence that t-DCA activates BASIC by stabilizing the open state of the channel, whereas extracellular Ca 2+ inhibits BASIC by stabilizing its closed state. In conclusion, our results highlight the potential role of the degenerin region as a critical regulatory site involved in the functional interaction of Ca 2+ and t-DCA with BASIC. |
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