UCLA

Large-conductance voltage- and Ca2+-activated K+ channels (BK) have a broad distribution of expression in mammalian cells. BK channels are activated by the membrane depolarization and elevation of intracellular free calcium. When the channel is activated, it allows an increase in potassium permeability, which hyperpolarizes the membrane electrical potential, suppressing cellular excitability. This membrane hyperpolarization decreases the activity of voltage-gated channels, including Ca2+ channels: the latter confers a significant role of BK channels in the internal calcium homeostasis. BK channels are involved in various fundamental physiological processes including the control of blood pressure and neuronal excitability. The BK channel is a homotetramer consisting of four pore-forming alpha subunits, which encode for seven membrane-spanning domains (S0-S6) and two large intracellular domains, RCK1 and RCK2. S0-S4 segments are known as the voltage sensing domain (VSD); S5 and S6 form the pore region and are involved in ion selectivity, while the RCK domains assemble into the ligand-sensing Gating Ring superstructure. In different tissues, the activity of BK alpha subunits is tuned by their association with modulatory beta subunits, beta1-4. The first project investigated the voltage-dependent structural rearrangements of the human BK channel in the presence of beta1 subunit. The data showed that the association of beta 1 remodeled the VSD movements. It is postulated that the association with beta1 destabilizes the active conformation of the voltage sensor in the absence of Ca2+, thus producing an overall right shift in its voltage dependence. BK channels are also modulated by small cytosolic ligands such as heme. The RCK1-RCK2 linker possesses a conserved heme regulation motif (HRM) and exhibited structural homology to cytochrome C (CytC), a hemoprotein. In particular, BK Methionine-691 aligned with M80 of the CytC, the second axial ligand (outside the HRM) to the heme iron. The goal of the second project aimed to understand the role of M691 in the BK regulation by heme. The mutation M691A dramatically diminished the inhibition of BK channel opening by heme (100 nM) compared with the wild-type BK, suggesting that M691 is critical for heme binding.