The molecular nature of the 17β-Estradiol binding site in the voltage- and Ca2+-activated K+ (BK) channel β1 subunit (original) (raw)

the accessory β1 subunit modulates the Ca 2+-and voltage-activated K + (BK) channel gating properties mainly by increasing its apparent Ca 2+ sensitivity. β1 plays an important role in the modulation of arterial tone and blood pressure by vascular smooth muscle cells (SMCs). 17β-estradiol (E2) increases the BK channel open probability (P o) in SMCs, through a β1 subunit-dependent modulatory effect. Here, using molecular modeling, bioinformatics, mutagenesis, and electrophysiology, we identify a cluster of hydrophobic residues in the second transmembrane domain of the β1 subunit, including the residues W163 and F166, as the binding site for E2. We further show that the increase in P o induced by E2 is associated with a stabilization of the voltage sensor in its active configuration and an increase in the coupling between the voltage sensor activation and pore opening. Since β1 is a key molecular player in vasoregulation, the findings reported here are of importance in the design of novel drugs able to modulate BK channels. The large conductance, Ca 2+-and voltage-activated K + (BK) channel is a tetrameric transmembrane protein composed of α subunits that form the K + selective pore 1,2. The α subunit is broadly expressed in mammalian tissues 3 , where it is co-expressed with an accessory β subunits in a tissue-specific manner 4,5. The α subunit comprises seven transmembrane segments (S0-S6), leaving the amino terminus exposed to the external medium. The large intracellular carboxyl terminus domain consisting of two RCK domains contains the Ca 2+ binding sites. The different β subunits (β1-β4) are formed by two transmembrane segments linked by a large extracellular loop and are responsible for the modulation of the biophysical and pharmacological characteristics of the α subunit 4,6. The β1 subunit is abundantly expressed in smooth muscle cells and acts to increase the apparent sensitivity to Ca 2+ and dramatically slows down the activation and deactivation channel-gating kinetics 7-12. The presence of the BK α/β1 channel lowers the risk of pathologies associated with the vascular tone by causing a membrane hyperpolarization, decreasing vasoconstriction, and inducing a faster relaxation of the blood vessels 13,14. β subunits appear to regulate the BK channel activity by targeting specific gating mechanisms 11,14,15. In particular, by biasing the equilibrium resting-active of the voltage sensor towards its active configuration, β1 increases the apparent BK Ca 2+ sensitivity 14,16 Co-expression of BK α and β subunits can dramatically modify pharmacological responses of the channel 17-19 as potential targets for channel modulators such as alcohol 20 , estrogens 19 , hormones 21 and fatty acids 22,23. 17β-Estradiol (E2) is the main circulating oestrogen in women and reaches a plasma concentration of 30-400 pg/mL before menopause. E2 regulates growth and the development of the reproductive system, also, helps to maintain the osseous tissue, the central nervous system and the vasodilatation in the vascular tissue 24. The protective effect of E2 in the vasculature and against cardiovascular disease (CVD) has been demonstrated in several hormone replacement studies 25,26. E2 activates BK channels 19,27-30 via a process that requires the presence