The Role of Met-691 in Heme-Dependent Regulation Supports the Presence of a Cytochrome-C-Like Structure in Human BK Channels

Abstract

We have recently proposed that the linker region between RCK domains in the cytoplasmic portion of BK channels consists of a Cytochrome-c-like domain, which encompasses a conserved heme regulatory motif (HRM, 612CKACH616 (Tang et al., Nature 2003) and endows BK channels with catalytic properties (Yusifov et al., Biophys J 2710-Pos 2014). Alignment of the BK Cytochrome-c-like domain and the human Cytochrome C (CytC) sequences revealed that they share, in addition to the HRM, key structural and functional elements. Notably, Met-80 in CytC, which is the second axial ligand to the heme iron, aligns with BK Met-691. In fact, mutation M691A dramatically reduced the Heme-dependent peroxidase activity of the purified BK channel Gating Ring. We assessed the involvement M691 in the heme modulation of BK channel conducting properties by inside-out patch clamp in WT and M691A channels expressed in Xenopus oocytes. The K+ current from WT BK channels was reduced by 61.6±14.6% (140mV) in 100nM heme. On the contrary, under identical conditions, K+ current was reduced by only 13.2±2.23% in BK channels carrying the M691A mutation. Moreover, the conductance half-activation potential (Vhalf) of WT BK shifted from 81.2±3.03 to 126±1.67mV after the addition of 100nM heme. Conversely, in M691A channels, the Vhalf was largely unperturbed by 100nM heme (from 98.2±4.13 to 100±8.76mV). Thus, M691A essentially eliminated the BK channel sensitivity to heme. These results reinforce the view that a Cytochrome-c-like domain, which associates heme via the HRM and a second axial ligand (Met-691) is an integral part of BK channels.