Ion channels, like other membrane proteins have resisted standard biochemical and structural analysis. Their structure has only recently begun to be elucidated by a combination of protein chemistry and molecular biology. On the other hand, we have a very detailed knowledge of the functioning of ion channels. Because each ion channel catalyzes the transport of millions of ions per second, we can measure electrically the current carried by just a single channel protein molecule. This technique of single channel recording has allowed us to make a detailed model for the conformational changes between open and closed states inducted by chemical ligands and changes in voltage, but we still have no knowledge of the protein structures that underlie these conformational changes.
My laboratory uses single channel biophysics and directed mutagenesis to relate ion channel function to structure. Our studies are focused primarily on voltage-activated potassium channels. By systematic mutagenesis, we have identified the region of the potassium channel protein that lines the pore through which ions cross the membrane. In conjunction with our biophysical studies on the mechanisms of K+ channel inactivation and blockade, these discoveries put us in a position to discover the basic mechanisms of ion selectivity and channel gating at the level of individual amino acids. Another strategy we use is to introduce individual cystein residues into the channel protein; these cysteins serve as targets for chemical modification. Our ability to modify the introduced cysteins in different conformational states gives specific information about the functional motions of the protein.
Other studies in the laboratory include:
Molecular determinants of general anesthetic inhibition of nicotinic acetylcholine receptors (in collaboration with Drs. Stuart Forman and Keith Miller of the Department of Anesthesia). We have identified specific residues in the pore of the nicotinic receptor that, when mutated, produce substantial changes in the ability of long-chain alcohols ad other general anesthetics to inhibit the channel. Biophysical studies support the idea that alcohols act primarily by blocking the open channel of the nicotinic receptor.
Electrophysiological studies of zebrafish mutants that affect cardiac rhythm (in collaboration with Dr. Mark Fishman of the Cardiovascular Research Center). Dr. Fishman's group has identified a number of mutant zebrafish that have modified cardiac rhythm. Many of these may affect specific cardiac ion channels. To elucidate the mechanisms involved, we are developing a primary dissociated cardiac myocyte preparation suitable for whole-cell electrophysiological characterization.