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Cerebrovascular dilation via selective targeting of the cholane steroid-recognition site in the BK channel ?1-subunit by a novel nonsteroidal agent.

ABSTRACT: The Ca(2+)/voltage-gated K(+) large conductance (BK) channel ?1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK ?1 allows the BK channels to reduce myogenic tone, facilitating vasodilation. The endogenous steroid lithocholic acid (LCA) dilates cerebral arteries via BK channel activation, which requires recognition by a BK ?1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate ?1-containing BK channels and evoke vasodilation remain unknown. We performed a chemical structure database similarity search using LCA as a template, along with a two-step reaction to generate sodium 3-hydroxyolean-12-en-30-oate (HENA). HENA activated the BK (cbv1 + ?1) channels cloned from rat cerebral artery myocytes with a potency (EC?? = 53 ?M) similar to and an efficacy (×2.5 potentiation) significantly greater than that of LCA. This HENA action was replicated on native channels in rat cerebral artery myocytes. HENA failed to activate the channels made of cbv1 + ?2, ?3, ?4, or ?1T169A, indicating that this drug selectively targets ?1-containing BK channels via the BK ?1 steroid-sensing site. HENA (3-45 ?M) dilated the rat and C57BL/6 mouse pressurized cerebral arteries. Consistent with the electrophysiologic results, this effect was larger than that of LCA. HENA failed to dilate the arteries from the KCNMB1 knockout mouse, underscoring BK ?1's role in HENA action. Finally, carotid artery-infusion of HENA (45 ?M) dilated the pial cerebral arterioles via selective BK-channel targeting. In conclusion, we have identified for the first time a nonsteroidal agent that selectively activates ?1-containing BK channels by targeting the steroid-sensing site in BK ?1, rendering vasodilation.

SUBMITTER: Bukiya AN

PROVIDER: S-EPMC3629834 | biostudies-other | 2013 May

REPOSITORIES: biostudies-other

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Cerebrovascular dilation via selective targeting of the cholane steroid-recognition site in the BK channel β1-subunit by a novel nonsteroidal agent.

Bukiya Anna N AN McMillan Jacob E JE Fedinec Alexander L AL Patil Shivaputra A SA Miller Duane D DD Leffler Charles W CW Parrill Abby L AL Dopico Alex M AM

Molecular pharmacology 20130301 5

The Ca(2+)/voltage-gated K(+) large conductance (BK) channel β1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK β1 allows the BK channels to reduce myogenic tone, facilitating vasodilation. The endogenous steroid lithocholic acid (LCA) dilates cerebral arteries via BK channel activation, which requires recognition by a BK β1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate β1-containing BK ...[more]

PMID: 23455312

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Project description:In most mammalian tissues, Ca2+i/voltage-gated, large conductance K+ (BK) channels consist of channel-forming slo1 and auxiliary (β1-β4) subunits. When Ca2+i (3-20 µM) reaches the vicinity of BK channels and increases their activity at physiological voltages, β1- and β4-containing BK channels are, respectively, inhibited and potentiated by intoxicating levels of ethanol (50 mM). Previous studies using different slo1s, lipid environments, and Ca2+i concentrations-all determinants of the BK response to ethanol-made it impossible to determine the specific contribution of β subunits to ethanol action on BK activity. Furthermore, these studies measured ethanol action on ionic current under a limited range of stimuli, rendering no information on the gating processes targeted by alcohol and their regulation by βs. Here, we used identical experimental conditions to obtain single-channel and macroscopic currents of the same slo1 channel ("cbv1" from rat cerebral artery myocytes) in the presence and absence of 50 mM ethanol. First, we assessed the role five different β subunits (1,2,2-IR, 3-variant d, and 4) in ethanol action on channel function. Thus, two phenotypes were identified: (1) ethanol potentiated cbv1-, cbv1+β3-, and cbv1+β4-mediated currents at low Ca2+i while inhibiting current at high Ca2+i, the potentiation-inhibition crossover occurring at 20 µM Ca2+i; (2) for cbv1+β1, cbv1+wt β2, and cbv1+β2-IR, this crossover was shifted to ∼3 µM Ca2+i Second, applying Horrigan-Aldrich gating analysis on both phenotypes, we show that ethanol fails to modify intrinsic gating and the voltage-dependent parameters under examination. For cbv1, however, ethanol (a) drastically increases the channel's apparent Ca2+ affinity (nine-times decrease in Kd) and (b) very mildly decreases allosteric coupling between Ca2+ binding and channel opening (C). The decreased Kd leads to increased channel activity. For cbv1+β1, ethanol (a) also decreases Kd, yet this decrease (two times) is much smaller than that of cbv1; (b) reduces C; and (c) decreases coupling between Ca2+ binding and voltage sensing (parameter E). Decreased allosteric coupling leads to diminished BK activity. Thus, we have identified critical gating modifications that lead to the differential actions of ethanol on slo1 with and without different β subunits.

Dataset Information

Cerebrovascular dilation via selective targeting of the cholane steroid-recognition site in the BK channel ?1-subunit by a novel nonsteroidal agent.

Publications

Cerebrovascular dilation via selective targeting of the cholane steroid-recognition site in the BK channel β1-subunit by a novel nonsteroidal agent.

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