Ion sensing in the RCK1 domain of BK channels

Abstract

BK-type K + channels are activated by voltage and intracellular Ca 2+ , which is important in modulating muscle contraction, neural transmission, and circadian pacemaker output. Previous studies suggest that the cytosolic domain of BK channels contains two different Ca 2+ binding sites, but the molecular composition of one of the sites is not completely known. Here we report, by systematic mutagenesis studies, the identification of E535 as part of this Ca 2+ binding site. This site is specific for binding to Ca 2+ but not Cd 2+. Experimental results and molecular modeling based on the X-ray crystallographic structures of the BK channel cytosolic domain suggest that the binding of Ca 2+ by the side chains of E535 and the previously identified D367 changes the conformation around the binding site and turns the side chain of M513 into a hydrophobic core, providing a basis to understand how Ca 2+ binding at this site opens the activation gate of the channel that is remotely located in the membrane. ] i . Because of this function, BK channels are important modulators of muscle contraction (1), neuronal spike frequency adaptation (2), neurotransmitter release (3), and circadian pacemaker output (4). BK channels are formed by four Slo1 subunits (5, 6). Each Slo1 contains a membrane-spanning domain, which comprises the pore-gate domain (PGD) and the voltage sensing domain (VSD), and a cytosolic domain (CTD) (7,8), which is made of two regulating domains for K + conductance (RCK1 and RCK2) (9, 10). Intracellular Ca 2+ binds to the CTD to activate the channel by enhancing the open probability of the activation gate located in the membrane-spanning PGD.Previous studies have identified two putative Ca 2+ binding sites in the CTD of BK channels, one is the Ca 2+ bowl located in the RCK2 domain (10-13) and the other is located in the RCK1 domain including the residue D367 (14). The existence of two distinctively different high-affinity Ca 2+ binding sites that are responsible for Ca 2+ -dependent activation of BK channels has been demonstrated in various experimental studies (15). These studies demonstrated that Ca 2+ binding to the two sites activates channel independently with only a small cooperativity (14,16,17), and the two sites show differences in various properties including affinities for Ca 2+ (14, 17), voltage dependence (17), and the molecular mechanisms of coupling to the activation gate (18). The distinction between the properties of the two putative Ca 2+ binding sites may lead to different physiological roles of these sites. For instance, a mutation in Slo1 that is associated with epilepsy and dyskinesia in human (19) specifically enhances the coupling of the RCK1 site to the activation gate to increase Ca 2+ sensitivity of channel activation (18). Although previous studies showed that the Ca 2+ binding site in RCK1 is important for physiological functions, its molecular identity is less certain than that of the Ca 2+ bowl. In the Ca 2+ bowl, previous mutagenesis studies (13) and a recently published X-ray cr...

Interactions of divalent cations with calcium binding sites of BK channels reveal independent motions within the gating ring

“…These results suggest that changes in FRET signals might originate from Mg 2+ interacting with the gating ring at the high-affinity binding site in the RCK1 domain and/or the Ca 2+ bowl located in the RCK2 domain. To explore these possibilities, we first introduced mutations D362A and D367A, which are known to alter Ca 2+ binding to the RCK1 site (4,31). Because in our previous work we used another mutation for this purpose (M513I), we first verified that in our construct the D362A/D367A 2 E and F), which remained very similar to the unaltered BK667CY constructs ( Fig.…”

“…1 A and B, red circles) (2). The first site identified, named the Ca 2+ bowl (28)(29)(30), is contained within the RCK2 domain (mainly D895 and D897 in the human BK channels), whereas another site was described in the RCK1 domain (including residues D362, D367, and E535) (2,4,31). Additionally, Mg 2+ ions bind specifically, but with low affinity, to a site formed by four amino acids: two from the transmembrane region (D99 and N172) and two located in the RCK1 (E374 and E399) (Fig.…”

Omega-3 fatty acids lower blood pressure by directly activating large-conductance Ca 2+ -dependent K + channels

“…3G), including ∼10 nM at which the Ca 2+ sensors of Slo1 are unoccupied and also 100 μM at which the high-affinity Ca 2+ sensors should be largely saturated with Ca 2+ (20)(21)(22). However, the ΔV 0.5 induced by DHA at 100 μM Ca 2+ was smaller than that at 10 nM (P = 0.03), indicating a potential influence by Ca 2+ .…”

Structural Determinants of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulation of BK Channel Activity through the RCK1 Ca2+ Coordination Site

“…Subsequently, the side chain of Glu 535 (Fig. 1C, right) was reported to be a part of the RCK1 Ca 2ϩ coordination site together with Asp 367 (31). High-resolution crystal structures of the cytosolic domain have been obtained either with the RCK2 Ca 2ϩ -bowl occupied by Ca 2ϩ (PDB 3 code 3MT5) (32) or in the absence of Ca 2ϩ (PDB code 3NAF) (33).…”