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Papers In Press, published online ahead of print November 1, 2008
J. Lipid Res., doi:10.1194/jlr.M800286-JLR200

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Journal of Lipid Research, Vol. 49, 2441-2451, November 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

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

Anna N. Bukiya^*, Jacob McMillan, Abby L. Parrill and Alejandro M. Dopico^1,*

^* Department of Pharmacology, The University of Tennessee Health Science Center, Memphis, TN 38163
Department of Chemistry and Computational Research on Materials Institute, University of Memphis, Memphis, TN 38152

^* This work was supported by National Institutes of Health Grant HL-77424 (A.M.D.). A.L.P. gratefully acknowledges the Chemical Computing Group for the MOE software license.

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of five figures.

Published, JLR Papers in Press, June 23, 2008.

¹ To whom correspondence should be addressed. e-mail: adopico{at}utmem.edu

Lithocholate (LC) (10–300 µM) in physiological solution is sensed by vascular myocyte large conductance, calcium- and voltage-gated potassium (BK) channel β₁ accessory subunits, leading to channel activation and arterial dilation. However, the structural features in steroid and target that determine LC action are unknown. We tested LC and close analogs on BK channel (pore-forming cbv1+β₁ subunits) activity using the product of the number of functional ion channels in the membrane patch (N) and the open channel probability (Po). LC (5β-cholanic acid-3-ol), 5-cholanic acid-3-ol, and 5β-cholanic acid-3β-ol increased NPo (EC₅₀ 45 µM). At maximal increase in NPo, LC increased NPo by 180%, whereas 5-cholanic acid-3-ol and 5β-cholanic acid-3β-ol raised NPo by 40%. Thus, the -hydroxyl and the cis A-B ring junction are both required for robust channel potentiation. Lacking both features, 5-cholanic acid-3β-ol and 5-cholenic acid-3β-ol were inactive. Three-dimensional structures show that only LC displays a bean shape with clear-cut convex and concave hemispheres; 5-cholanic acid-3-ol and 5β-cholanic acid-3β-ol partially matched LC shape, and 5-cholanic acid-3β-ol and 5-cholenic acid-3β-ol did not. Increasing polarity in steroid rings (5β-cholanic acid-3-sulfate) or reducing polarity in lateral chain (5β-cholanic acid 3-ol methyl ester) rendered poorly active compounds, consistent with steroid insertion between β₁ and bilayer lipids, with the steroid-charged tail near the aqueous phase. Molecular dynamics identified two regions in β₁ transmembrane domain 2 that meet unique requirements for bonding with the LC concave hemisphere, where the steroid functional groups are located.

Supplementary key words steroids • protein-ligand interaction • computational modeling • maxiK channel • vascular smooth muscle • vasodilation

Abbreviations: BK, large conductance, calcium- and voltage-gated potassium; CMC, critical micellar concentration; LC, lithocholate; N, number of functional ion channels in the membrane patch; Po, open channel probability; RMSG, root mean square gradient; TM2, transmembrane domain 2

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