Progesterone activation of β1-containing BK channels involves two novel binding sites

Abstract Progesterone at ≥1 µM is used in recovery of cerebral ischemia, an effect likely contributed to by cerebrovascular dilation. The targets of this progesterone action are unknown. Activation of cerebrovascular myocyte BK channels, which include channel-forming α and regulatory β1 subunits, causes cerebrovascular dilation. We report that µM progesterone activates mouse cerebrovascular myocyte BK channels; this action is lost in β1-/- mice and α subunit homotetramers reconstituted into lipid bilayers but sustained in β1/β4-containing heteromers. Progesterone binds to both regulatory subunits, and two novel steroid binding sites, conserved in β1-β4 are unveiled: high affinity (sub-µM), which involves Trp87 in β1 loop, and low affinity (µM) defined by TM1 Tyr32 and TM2 Trp163. Probing mutant channels with progesterone and its oxime-analog indicates that progesterone-loop binding facilitates binding to the low-affinity site where a single progesterone molecule bridges Tyr32-Trp163 to activate BK channels, thus unveiling a novel model of steroid-ion channel interactions.