1. Cardiac Bioelectricity Research and Training Center and Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106-7207, USA
2. Structural Biology Program, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA

Correspondence to: Jianmin Cui¹ Correspondence and requests for materials should be addressed to J.C. (e-mail: Email: jxc93@cwru.edu).

Abstract

Large-conductance (BK type) Ca²⁺-dependent K⁺ channels are essential for modulating muscle contraction and neuronal activities such as synaptic transmission and hearing^{1, 2, 3, 4, 5}. BK channels are activated by membrane depolarization and intracellular Ca²⁺ and Mg²⁺ (refs 6–10). The energy provided by voltage, Ca²⁺ and Mg²⁺ binding are additive in activating the channel, suggesting that these signals open the activation gate through independent pathways^{9, 11}. Here we report a molecular investigation of a Mg²⁺-dependent activation mechanism. Using a combined site-directed mutagenesis and structural analysis, we demonstrate that a structurally new Mg²⁺-binding site in the RCK/Rossman fold domain—an intracellular structural motif that immediately follows the activation gate S6 helix^{12, 13, 14, 15}—is responsible for Mg²⁺-dependent activation. Mutations that impair or abolish Mg²⁺ sensitivity do not affect Ca²⁺ sensitivity, and vice versa. These results indicate distinct structural pathways for Mg²⁺- and Ca²⁺-dependent activation and suggest a possible mechanism for the coupling between Mg²⁺ binding and channel opening.