Gating mechanism of potassium channels studied by electron paramagnetic resonance spectroscopy.

摘要

Ion channels are gated by transducing a variety of physical stimuli into structural rearrangements. The presence of significant conformational changes in intracellular regions of voltage-dependent potassium channels has been supported by extensive functional evidence. To better understand the gating conformational changes of the tetrameric channels, we constructed tandem dimers and tandem tetramers to measure inter- and intra-subunit distances of Streptomyces K+ channel KcsA. Both function and structure of the tandem constructs with protease-cut linkers were examined and they are conserved to those of the wild type channel. Based on the inter-subunit distance constraints of tandems, we deduced the open structure of inner helical bundle at backbone resolution by ReDCaT computation (Restraint-Driven Cartesian Transformations). The movement of TM2 creates a larger vestibule for ion permeating with the increase in diameters by minimum 2A at the pivot point and as much as 10A in the C-terminus. Moreover, dramatically increased distances between cytoplasmic helices indicate the release of the bundle upon opening. We also found that the possible helical hinges at positions Gly99 and Gly104 are critical for channel opening. Most intra-subunit distance changes between TM1 and TM2 upon channel opening are within 4A, suggesting the concerted movements of these two transmembrane helices. The inconsistency between the intra-subunit distances in the open state obtained by Electron Paramagnetic Resonance (EPR) and ones calculated by MMCM-MD computation (Metropolis Monte Carlo Minimization and Molecular Dynamics) based on the crystal structure of MthK (potassium channel from Methanobacterium thermoautotrophicum ) suggests MthK-based open structure is not the open model for KcsA. Techniques developed here provide not only the structural information of open KcsA but also the general methods to study the dynamic structures of symmetric oligomeric proteins.