Charged Residues on the Intracellular Intersubunit Assembly Interface Contribute to Calcium-Sensitivity of BK Channels

The large-conductance calcium-activated K+ (BK) channel contains a large C-terminal intracellular Ca2+-sensing region formed by two RCK domains. The RCK2 domain harbors a Ca2+-binding site of clustered negatively charged residues, known as the Ca2+-bowl, while the RCK1 domain is believed to contain another Ca2+-binding site (RCK1-site). Recent X-ray crystallographic structures of the Ca2+-free and -bound intracellular regions revealed an interesting location of the Ca2+-bowl near the tertrameric intersubunit assembly interface. We investigated the role of the intersubunit assembly interface on Ca2+-gated BK channel activation through mutational analyses of charged residues Glu955, Glu956, Arg786, and Arg790 which may electrostatically stabilize RCK2 intersubunit interactions by formation of intersubunit salt-bridges. We observed that mutations E955Q, E956Q, and E955Q/E956Q caused ∼50%, ∼30%, and ∼70% loss, respectively, of Ca2+-sensitivity as determined at 0, 7.3, and 90.4 μM internal free Ca2+. Combined analyses with Ca2+-bowl or RCK1-site null mutations indicated that E955Q mutation eliminated most Ca2+-sensitivity from the Ca2+-bowl and ∼50% sensitivity from the RCK1-site. The charge neutralization mutation R786A/R790A on the interacting subunits also led to ∼50% loss of Ca2+-sensitivity, suggesting involvement of intersubunit salt-bridge(s) between residues E955 and R786 or R790. Furthermore, intersubunit charge reversal mutations induced recovery of Ca2+-sensitivity. In contrast to the drastic effects of these charged residue mutations, which are located distant (≥20A) to the bound Ca2+, mutation of the Asn449 residue, which may coordinate the RCK2 Ca2+ from a different subunit, displayed little effect on Ca2+-sensitivity. We conclude that the intersubunit interfacial electrostatic interactions play an important role in Ca2+-dependent BK channel gating. We propose that such interactions are unlikely to be involved in direct Ca2+-binding but may provide intersubunit supporting points to mediate Ca2+-gated RCK gating ring expansion or conformational coupling to channel activation.