The Effects Of Inter-subunit Coupling On Pore Structure And Gating Of BK Channel

Ion channels are one of the important functional proteins which can perform material and energy exchange with surrounding environment. Large-conductance voltage- and Ca²⁺-activated K⁺ (BK) channels encoded by the mslo1 exist abundantly in rat chromaffin cells, pancreaticβcells and DRG neurons. So far, BK channel is found in many kinds of excitable and unexcitable cells except for heart myocytes. The conductance of BK channel is ten times more than general potassium channels, so it plays the key role in physiological process. The permeation property, pore geometry and gating mechanism of BK channels has been an interesting project for long. In this study, with the help of several techniques such as electrophysiology, mutagenesis, immunofluorescence and molecular dynamics modeling, we studied the effects of an interaction between side-chain of Leu-312 and aromatic ring of Phe-315 in the neighboring subunits, which can maitain channel being closing conformation and modulate pore gating property.Based on the sequence alignment within the pore domain of potassium channels and their crystal structures, we focused on Leu-312 and Phe-315, which are near the putative gating hinge. We found that substitution of Leu-312 in the S6 transmembrane segment of mSlo1 BK channels with hydrophilic amino acids of smaller side-chain volume favored the open-state. The sensitivities of channels to calcium and voltage were modified by some mutations, and completely abolished by others. Interpretation of the results in terms of an allosteric model suggests that the calcium-insensitive mutants greatly destabilize the closed relative to the open conformation and may also disrupt the allosteric coupling between Ca²⁺- or voltage-sensors and the gate. Some of Phe-315 mutations also favor the open state, suggesting that Leu-312 and Phe-315 may have an interaction in the closed state, forming a major energy barrier that channel has to overcome to open. Homology modeling and molecular dynamic simulations further support that the side-chain of Leu-312 can couple strongly with the aromatic ring of Phe-315 in neighboring subunits (L-F coupling) to maintain the channel being closed. Additionally, single-channel recording indicated that the calcium-insensitive mutants, whose kinetics can be approximately characterized by a two-state closed-open (C-O) model, exhibit nearly 100% open probability under physiological conditions without alterations in single-channel conductance.These results of single-channel recording suggested that the mutants promote channel opening without altering the open conformation of the pore.We conclude that the inter-subunit coupling between Leu-312 and Phe-315 plays a critical role in pore gating and keeping the pore of channel to closed state. The Ca²⁺- and voltage-sensitivity deficiency of some mutant channels indicates that the disruption of their interaction may disrupt the allosteric coupling between Ca²⁺- or voltage-sensors and the gate. These findings provide a basis of understanding the structure and gating of the BK channel pore, and also give a model for us to gain an insight into the self-locking function in BK-type channels.