Calcium-Dependent Operation of the Human BK Channel Gating Ring Apparatus

Abstract

The large cytoplasmic C-terminal domain (CTD) of the human BK channel forms a gating ring structure composed by two tandem RCK domains serving as a signal transducer for intracellular Ca2+ and other ligands. However, the mechanism of the gating ring's operation remains unknown. We have used steady-state and time-resolved spectroscopy in combination with dynamic light scattering to the Ca-induced conformational changes of the purified CTD of human BK channel. The CTD domain of the human BK channel assembles as a tetrameric gating ring structure (MW∼310 kDa) with a hydrodynamic radius (RH) ∼10.5 nm. In the presence of 35 μM free Ca2+, RH reversibly decreases to ∼7.5 nm. The modulation of the gating ring hydrodynamic shape suggests that it undergoes Ca2+-induced conformational transitions, which we further, characterized using time-correlated single-photon counting spectroscopy. Increasing free [Ca2+] up to 35 μM shortened the average Trp fluorescence lifetime (τavg) of the wild-type gating ring from ∼2.6ns to ∼1.9ns, while the neutralization of the high-affinity Ca-binding site within RCK2 (Ca bowl, D894-898N) attenuated the effect of Ca2+. Steady-state fluorescence analysis revealed that these ligand-dependent structural rearrangements of the gating ring possess strong divalent cation selectivity. The gating ring exhibited i) high-affinity Ca2+-binding (Khalf_1 ∼0.3μM and Khalf_2 ∼4μM); ii) significantly lowered affinity for Mg2+ (Khalf ∼200μM) and iii) no Ba2+ sensitivity up to 13mM, consistent with the lack of Ba2+-dependent BK channel activation. Interestingly, Ca2+ bowl neutralization eliminated Mg2+ sensing up to 12mM. In summary, under physiologically-relevant conditions, these ligand-induced conformational transitions are strongly ion-specific and associated with changes in the hydrodynamic properties of the BK gating ring and likely represent the ligand-induced molecular events underlying channel activation.