The unique property of ion channels being highly selective pores, and their ability to catalyze ion translocation at extremely high rates, is the basis of a very effective labeling approach. It uses the physical flow of thallium (Tl+) ions through individual potassium channels to produce specific, weakly soluble, inorganic microcrystals with some halide anions and transition metals at the exact location of the channel pore. This crystallization method will be further developed to reveal unknown aspects of molecular organization and function of potassium channels in the membrane of living cells. Various approaches will be applied: a) to obtain detailed patterns of membrane localization and clustering of functional potassium channels in the living cell. Channel distribution will be mapped using the crystallization method, followed by conventional labeling of underlying cytoskeletal structure (microtubules, actin filaments, and associated proteins) with fluorescent indicators or specific antibodies, and the nature of their co-localization will be studied; b) to physically capture and analyze novel structural and regulatory proteins of potassium channels. The crystallization method may offer an opportunity to selectively immobilize, within the growing microcrystal, and then purify, proteins located in close vicinity to the channel pore. Isolated proteins will be microsequenced and cDNA clones obtained by conventional methods. Proteins will then be overexpressed and their activity reduced by transfected or microinjection with antisense sequences and properties of potassium channels analyzed to reveal novel protein-channel interactions; c) to get high resolution electron microscopy footprints and images of potassium channel complexes; the extremely high density of thallium containing microcrystals and their growth only at the exact locations of potassium channels will allow selective precipitation of intact membrane patches with high channel density, suitable for further electron microscopy analysis. Direct viewing of potassium channel complexes will allow resolution of their exact oligomeric structure and subunit organization.
| Lopatin, A N; Nichols, C G (2001) Inward rectifiers in the heart: an update on I(K1). J Mol Cell Cardiol 33:625-38 |
| Lopatin, A N; Shantz, L M; Mackintosh, C A et al. (2000) Modulation of potassium channels in the hearts of transgenic and mutant mice with altered polyamine biosynthesis. J Mol Cell Cardiol 32:2007-24 |
