Intersubunit Coupling in the Pore of BK Channels

Ying Wu,Yu Xiong,Sheng Wang,Hong Yi,Hui Li,Na Pan,Frank T Horrigan,Yingliang Wu,Jiuping Ding

doi:10.1074/jbc.m109.027789

Abstract

The structural basis underlying the gating of large conductance Ca(2+)-activated K(+) (BK) channels remains elusive. 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 Ca(2+) or voltage sensors and the gate. Some Phe-315 mutations also favor the open state, suggesting that Leu-312 and Phe-315 may interact in the closed state, forming a major energy barrier that the 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 closed. Additionally, single-channel recordings indicate 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 findings provide a basis for understanding the structure and gating of the BK channel pore.

Highlights

Symmetry of the side chain of leucine may produce a maximal effect on the aromatic ring of phenylalanine, named L-F coupling. Such intimate interaction reveals that a L-F coupling, existing in the closed structural conformation of BK channels, locks the channel to the closed state
Effect on single channel conductance of BK channels is consistent with the notion that they promote channel opening without altering the open conformation of the pore
Coupling structure can keep the pore of BK channel in the closed state. Once their connection was disrupted completely, channels open permanently under physiological conditions without changing single-channel conductance, suggesting that this coupling plays a critical role in the pore of BK channel

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis—Full-length cDNA for mSlo (accession number NP_034740) was subcloned into pcDNA3.1Zeo (ϩ) (Invitrogen). Expression in Xenopus Oocytes—After DNA was linearized with MluI, SP6 RNA polymerase (Roche Applied Science) was used to synthesize cRNA for oocyte injection. Intracellular solutions with different free Ca2ϩ were made by mixing 160 mM MeSO3K and 10 mM Hϩ-HEPES with Ca(MeS) and 5 mM HEDTA (for 10 ␮M) or 5 mM EGTA (for 0 ␮M). Represents the open-to-closed equilibrium constant when no voltage sensors are active and no Ca2ϩ binding sites are occu-. A cylindrical hole was made in the center of the bilayer by removing lipids whose P atoms fell within 2.0 Å of the above models. These whole systems were solvated (TIP3) and ionized in the presence of 160 mM KCl on both sides of the POPC membrane. The ff force field (Parm99) was applied throughout the energy minimization and MD simulations

RESULTS

VhC mV

To confirm that maximal activation

DISCUSSION