This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ion-conductive channels are membrane proteins playing a vital regulatory role in all living organisms. Their failure to function properly results in a variety of diseases, such as neurological disorders. An essential function of channels is gating, activated by a variety of stimuli and accompanied by substantial structural changes. The X-ray data on 2-TM KcsA and MthK K+ channels led to a model based on a hinge-bending mechanism of gating. The 30? bending about the conserved Gly99 residue of the TM2-domain in MthK results in wide splay opening of the intracellular gating pore. KcsA has glycine 83 at the similar position of TM2. The model, however, contradicted an earlier study by cw-ESR that suggested smaller TM2 motion. Very recent data on gating in the Shaker channel has shown that the open conformation of MthK may not be applicable to other K+ channels. Thus, knowing the structure of KcsA in its open state is of utmost importance in our understanding of the mechanism of gating in K+ channels. Therefore, our current efforts in collaboration with Perozo are centered on the study of the pH induced global conformation rearrangement of KcsA using fully-labeled channels, tandem dimers, and tandem tetramers. This will provide both inter- and intra-domain distances to elucidate the global structural changes. Large structural changes require measuring distances of up to 50?. In our preliminary study on KcsA and MscL we have already demonstrated the power of DQC-ESR. We successfully applied DQC to WT KcsA fully-labeled at C28, and tandem dimer constructs, labeled at 87 and 119. For spin-labels in contact with lipids short phase relaxation times limits accurate measurements to ca. 35 ?. Therefore, in the cases when distances are large we will use lipids with perdeuterated acyl chains (i.e. DMPC-d54), which let us accurately measure distance up to 50 ?.
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