Identification of Sterol-Sensing Amino Acids in Transmembrane Domain 2 of the BK Channel Beta 1 subunit

Abstract

The activity of large conductance, voltage- and calcium-gated potassium (BK) channels in myocytes critically controls arterial diameter. At <300 μM in the aqueous phase (below critical micellar concentration), lithocholate (LC) and related steroids increase myocyte BK channel activity (Po) and cause arterial dilation. We showed that: 1) BK β1 TM2 is critical for the channel sensitivity to LC, and 2) BK β2-4 cannot substitute for β1 in providing LC-sensitivity to BK channels (Bukiya et al., 2008; 2009). Moreover, computational dynamics identified two LC-sensing amino acid clusters in BK β1TM2: L157,L158,T165 and T169,L172,L173. Here, we used pinpoint mutagenesis and patch-clamp electrophysiology to determine the relative contribution of each cluster to the LC-sensitivity of BK channels. BK α (cbv1) and β1 subunits cloned from rat cerebral artery myocytes (AY330293; FJ154955) were coexpressed in Xenopus oocytes, and BK currents were recorded from inside-out patches at Ca2+i=10 μM and Vm=-20 to −40mV. BK β1T165A and β1T165A,L157A,L158A mutants rendered LC-sensitive BK channels, with 150 μM LC-induced increase in Po (×1.7 times) being identical to that found with cbv1+wtβ1. In contrast, BK β1T165A,T169A and β1T169A mutants were absolutely unresponsive to ≥150 μM LC. Data unveil a critical role for β1T169 in providing LC-sensitivity to BK channels. While the β1T169S mutation was able to rescue the channel's LC-sensitivity, the β1T169S,L172A,L173A mutation failed to do so. Moreover, BK β1L172A,L173A was also LC-insensitive. These data validate our computational prediction that hydrogen bonding between T/S169 and the steroid hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for successful docking of LC onto the T169,L172,L173 cluster. Thus, T169, L172 and L173 in BK β1TM2 provide a docking surface that is essential for the LC-sensitivity of BK channels.Support: HL104631 (AMD).