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

Abstract

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

Bile acids inhibit human purinergic receptor P2X4 in a heterologous expression system

“…Therefore, we conclude that t-DCA stabilizes the closed state of P2X4. In contrast, we have shown that t-DCA stabilizes the open state of BASIC, which explains the observed stimulatory effect of t-DCA on BASIC (Ilyaskin et al, 2018). In addition, t-DCA significantly changed the single-channel current amplitude of ENaC/DEG ion channels, indicating that t-DCA may interact with a region close to the channel pore (Ilyaskin et al, 2016, 2017, 2018).…”

“…Isolation of oocytes and two-electrode voltage-clamp experiments were performed essentially as described previously (Diakov and Korbmacher, 2004;Haerteis et al, 2009;Diakov et al, 2010;Ilyaskin et al, 2016Ilyaskin et al, , 2017Ilyaskin et al, , 2018. Defolliculated stage V-VI oocytes were collected from ovarian lobes of female Xenopus laevis with approval of the animal welfare officer of the University of Erlangen-Nürnberg, in accordance with the principles of German legislation and under state veterinary health inspectorate governance.…”

“…Single-channel recordings in outside-out membrane patches of P2X4-expressing oocytes were performed 48 h after injection of 2 or 5 ng WT P2X4 cRNA using a conventional patch-clamp technique essentially as described previously (Diakov and Korbmacher, 2004;Lefèvre et al, 2014;Ilyaskin et al, 2016Ilyaskin et al, , 2017Ilyaskin et al, , 2018. Patch pipettes were pulled from borosilicate glass capillaries and had a tip diameter of ∼1-1.5 µm after fire polishing.…”

“…The amphiphilic bile acids are also membrane active substances, which may incorporate into the membrane and interact with the transmembrane domain of P2X4. Recently, we have demonstrated that bile acids can modify the function of the acid-sensing ion channel (ASIC) 1a (ASIC1a) and other members of the epithelial sodium channel (ENaC)/degenerin (DEG) ion channel family (Ilyaskin et al, 2016, 2017, 2018). Moreover, we described a putative bile acid binding region at the intersubunit interface within the transmembrane domain of ENaC/DEG channels (so-called degenerin region).…”

A polycystin-2 protein with modified channel properties leads to an increased diameter of renal tubules and to renal cysts

“…Isolation of oocytes and two-electrode voltage-clamp experiments were performed essentially as described previously ( Haerteis et al, 2009 ; Ilyaskin et al, 2018 ; Rauh et al, 2010 ). Female Xenopus laevis were anesthetized with 0.2% ethyl 3-aminobenzoate methanesulfonate (MS-222) (Merck, cat.…”