W0446

The Structure and Function of Ion Channels using Spherical Reconstruction. David W. Chester, Qiu Xing Jiang, Fred J. Sigworth, Yale Univ. School of Medicine, Dept. of Cell and Molecular Physiology, 333 Cedar Street, New Haven, CT 06520

Spherical Reconstruction is a single particle reconstruction (SPR) approach for determining the structure/ function relationship for ion channels asymmetrically reconstituted into large unilamellar vesicles. This approach has a number of significant advantages: (a) enhanced resolution limits typically associated with SPR, (b) evaluation of structures in a more 'native' and fully hydrated environment, (c) correlation of the structure with a pharmacological demonstration of ion channel function (e.g., ion flux assays, bilayer recording), (d) development of biological and chemical 'tools' for increasing the fidelity of particle picking for proteins bound into liposomal membranes. This latter goal is hoped to augment the process of automating data collection for reconstituted membrane proteins.

One additional advantage of this approach is that, via vectoral insertion of these channels into vesicles of defined size, we can begin to examine the structural interaction of auxiliary/regulatory proteins under more native conditions. This comes as a result of intra- and extravesicular compartmentalization of the reconstituted channel. I believe that these kinds of studies represent the principle niche for Cryo-EM between x-ray crystallography and Cell Biology.