Mechanism of magnesium dependent activation of large conductance potassium channels

Large conductance potassium channels (BK channels), encoded by the slowpoke gene, play important roles in many physiological processes including neurotransmitter release, vascular smooth muscle contraction, hearing and immunity. These multiple physiological functions are attributed to their activations by depolarizing voltages, intracellular Ca2+ in micromolar concentration and intracellular Mg2+ in millimolar concentration in an allosteric mechanism. BK channels are homotetramers where each subunit is composed of a transmembrane domain and a large intracellular domain. While the transmembrane domain contains its voltage sensor as well as its pore and gate, the intracellular domain is critical for its Ca 2+ and Mg2+ dependent activation. Previous studies have identified a site critical for its Mg2+ sensing, located in the intracellular RCK1 (Regulating Conductance of K+) domain, which presumably forms its Mg2+ binding site. But how the energy of Mg2+ binding to this site is propagated to activate the BK channel is not known. Using multi-disciplinary methods including site-directed mutagenesis, patch-clamping and chemical modification, we found that the S4 voltage sensor participated in the Mg2+dependent activation. Upon further examination, we found that the energy of Mg2+ binding to its site in the RCK1 domain is propagated to activate BK channels through its electrostatic interaction with gating charge R213 in the S4 voltage sensor. Mg2+ activates the voltage sensor primarily when the channel is open, revealing a state-dependent interaction between the voltage sensor domain and the RCK1 domain that accompanies the opening of the pore.;During our investigation of the mechanism of Mg2+ dependent activation in BK channels, we found a novel low affinity Mg2+ dependence on channel activation at high intracellular Mg2+ concentration ([Mg2+]i) (≥ 10 mM). The effects of the two Mg2+ dependent activation at [Mg2+] i around 10 mM overlap, and thus this novel Mg2+ dependence interferes with the study of Mg2+ dependent activation at physiological concentrations. Using mutagenesis and modeling methods, we found that an additional metal binding site may contribute to this activation. By mathematically subtracting the effect on channel activation through this novel low affinity site, we can focus on Mg2+ dependent activation associated with RCK1 domain at physiological concentrations.