RESUMO
The accessory ß1 subunit modulates the Ca2+- and voltage-activated K+ (BK) channel gating properties mainly by increasing its apparent Ca2+ sensitivity. ß1 plays an important role in the modulation of arterial tone and blood pressure by vascular smooth muscle cells (SMCs). 17ß-estradiol (E2) increases the BK channel open probability (Po) in SMCs, through a ß1 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 ß1 subunit, including the residues W163 and F166, as the binding site for E2. We further show that the increase in Po 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 ß1 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.
Assuntos
Estradiol/metabolismo , Ativação do Canal Iônico , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Miócitos de Músculo Liso/metabolismo , Cálcio/metabolismo , Células HEK293 , Humanos , Potenciais da Membrana , Técnicas de Patch-Clamp/métodos , Subunidades Proteicas , Receptores de Estradiol/metabolismoRESUMO
High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. The most remarkable effects of beta1 and beta2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by alpha and beta1 or beta2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of beta1 but not beta2. Here we reveal the molecular regions in these beta subunits that determine their differential functional coupling with the pore-forming alpha-subunit. We made chimeric constructs between beta1 and beta2 subunits, and BK channels formed by alpha and chimeric beta subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the beta1 and beta2 subunits demonstrates that the NH3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these beta subunits. Moreover, the intracellular domains of beta1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the alpha-subunit to be the target of the modulation by the beta1-subunit.