Probing molecular structure and structural changes of voltage-gated channel by expressing mutant channels in yeast and reconstituting them into planar membranes

X-Ray crystallography, although extremely powerful, yields the static structure of a protein in a crystal, which may be quite different from its functional state. This chapter presents an example of the insights that can be gleaned from molecular genetics and electrophysiology. For channel-forming membrane proteins, the structure must include a polar protein surface that allows water to penetrate an extremely small tunnel within the protein, forming an aqueous channel. The use of selectivity changes to determine which portions of the protein form the water-filled pore, can yield a wealth of information about the protein structure and involves changing the charge at a specific location by site-directed mutagenesis. For membrane channels that form large aqueous pores, amino acid side chains form a part of the polar wall of the aqueous pore. The nature and especially the charge of these side chains influence the ion selectivity of the channel. Thus, changing the appropriate side chain should change the ion selectivity of the channel. Conversely, amino acid substitutions that change the ion selectivity are good indicators of locations within the protein that form part of the wall of the aqueous pore.