The degenerin region of the human bile acid-sensitive ion channel (BASIC) is involved in channel inhibition by calcium and activation by bile acids

Alexandr V. Ilyaskin, Sonja A. Kirsch, Rainer A. Böckmann, Heinrich Sticht, Christoph Korbmacher, Silke Haerteis, Alexei Diakov

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 Ca2+. The aim of this study was to explore the molecular mechanisms mediating these effects. The modulation of BASIC function by extracellular Ca2+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 Ca2+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 Ca2+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 Ca2+and t-DCA with BASIC.