The second transmembrane domain of the large conductance, voltage‐ and calcium‐gated potassium channel β₁ subunit is a lithocholate sensor

Abstract

Bile acids and other steroids modify large conductance, calcium-and voltage-gated potassium (BK) channel activity contributing to non-genomic modulation of myogenic tone. Accessory BK b 1 subunits are necessary for lithocholate (LC) to activate BK channels and vasodilate. The protein regions that sense steroid action, however, remain unknown. Using recombinant channels in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/ 1-palmitoyl-2-oleoyl-phosphatidylserine bilayers we now demonstrate that complex proteolipid domains and cytoarchitecture are unnecessary for b 1 to mediate LC action; b 1 and a simple phospholipid microenvironment suffice. Since b 1 senses LC but b 4 does not, we made chimeras swapping regions between these subunits and, following channel heterologous expression, demonstrate that b 1 TM2 is a bile acid-recognizing sensor.

Structural determinants of monohydroxylated bile acids to activate β1 subunit-containing BK channels

“…In hepatobiliary disease, not only overall increase in systemic bile acid levels but also changes in composition may occur, with drastic increase in LC levels (11). LC causes endothelium-independent relaxation of small arteries via activation of myocyte BK channels (6), a bile acid action that requires LC sensing by the channel b 1 TM2 domain (17). Remarkably, among naturally occurring bile acids, LC is the most effective activator of vascular myocyte native BK channels (4).…”

“…Second, LC itself does not decrease but actually increases the cholesterol/ phospholipid ratio of red blood cell membranes (41). Finally, LC activation of cbv11b 1 channels remains when studied on channels reconstituted into cholesterol-free phospholipid bilayers (17).…”

“…Data are shown as mean 6 SEM. likely that this domain represents or at least contributes to the LC binding site (17). We proposed that the LC-target interaction required the steroid to insert normally to the bilayer plane: the hydrophobic hemisphere of the planar amphiphile faces the bilayer lipids while the hemisphere containing the polar hydroxyl faces polar amino acids provided by the b 1 -subunit, with the carboxylate/carboxylic acid of the lateral chain residing in or near the aqueous solution (6,17). If this model is correct, substitution of highly ionized groups for the polar hydroxyl group should decrease steroid insertion in the bilayer and, thus, reduce steroid access to the putative target site.…”

“…Having explored the contribution of A/B cis junction bend, a conformation of the hydroxyl group, steroidal hydrophobic concavity, and polarity of the lateral chain to recombinant BK channel activation by monohydroxylated bile acids, and having advanced that the b 1 TM2 contributes to the LC binding pocket (6,17), we used molecular dynamics simulations to model the LC-b 1 TM2 interaction. The primary amino acid sequence of the b 1 TM2 domain was modeled three-dimensionally as an ideal a-helix.…”

“…Notably, channel accessory subunits of the b 4 type, which prevail in nervous tissue, fail to render the BK channel complex sensitive to LC (6). Furthermore, recent data from BK channels that include b 1 -b 4 chimeric subunits demonstrate that the b 1 transmembrane domain 2 (TM2) distinctly behaves as a bile acid-sensing element (17). Whether b 1 TM2 provides a docking area that allows selective interaction with LC, a structural hypothesis for this steroid effectiveness on channel function, remains unknown.…”