Site and mechanism of BK channel inhibition and cerebral artery constriction by pregnenolone.

Authors

Kelsey C North, Shiwani Thapa, Steven A Mysiewicz, Andrew Shaw, Gregg E Homanics, Anna N Bukiya, Alex M Dopico

Published in

Pharmacological research. Pages 108409. Aug 23, 2026. Epub Aug 23, 2026.

Abstract

Circulating vasoactive steroids including estrogens, androgens, progesterone, and bile acids modulate the activity of voltage/Ca²⁺-gated, big conductance potassium (BK) channels, an action that requires regulatory BK β subunits. In contrast, pregnenolone at local/therapeutic concentrations has recently been shown to modulate BK channel activity in absence of such subunits. The site and mechanism of pregnenolone action remain unknown. Thus, we decided to test the hypothesis that BK channel-forming α subunits (slo1 proteins encoded by KCNMA1 or Slo1 in mammals) contain a specific residue(s) that recognizes pregnenolone and mediates modulation of smooth muscle BK channel activity and diameter of cerebral arteries by this steroid. We used microscale thermophoresis to demonstrate that pregnenolone at local/therapeutic concentrations binds to slo1 proteins at concentrations that inhibit BK channel activity and decrease cerebral artery diameter (K_D=3.5µM). Based on a slo1 cryo-EM structure (PDB 6V38) computational modeling identified Tyr450, Hys409 and Asp896 as participants in pregnenolone recognition, with hydrogen-arene interactions between steroid and Tyr450. Consistently, slo1Y450F proteins failed to bind pregnenolone. Following heterologous expression, this mutant was resistant to pregnenolone-induced inhibition, an action that was coupled to Ca²⁺-driven gating and enabled by the RCK1 high-affinity Ca²⁺-binding site. In contrast to their wild-type counterparts, middle cerebral arteries from Kcnma1 Y450F knock-in mice failed to constrict to pregnenolone, whether probed ex vivo or in vivo. Thus, metabolic, circulating, or endothelial factors cannot override pregnenolone cerebrovascular action, which mainly results from direct interaction with Tyr450 within the proximal CRAC motif in the slo1 cytosolic tail domain. Our study provides a new paradigm in vasoactive steroid-BK channel interactions that can be used in developing pharmacophores/new agents to replace/regulate pregnenolone effects in clinical scenarios, and underscores that CRAC motifs may mediate ionotropic receptor regulation by steroids other than cholesterol.

PMID:
42633875
Bibliographic data and abstract were imported from PubMed on 24 Aug 2026.

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