Faculty Research Interest

Molecular Mechanisms of Voltage-Dependent Ions Channels

Faculty Record

Research is aimed at understanding the mechanisms by which ion channels function (particularly channels which are contorlled by transmembrane voltage), and the mechanisms responsible for the movement of proteins across membranes. To this end, we are working with channels formed by various protein toxins, which lend themselves to genetic and chemical manipulation. Proteins and peptides are generally incorporated into planar lipid bilayers and studied by electrophysiological techniques. The primary project involves the bacterial colicins, a family of plasmid-encoded toxins that form voltage-dependent channels in both target cells and artificial membranes. We are constructing a series of mutations in these proteins, with the aim of 1) altering channel properities directly, and 2) creating target sites for various probe molecules that can be used to report on the structure of the channel in different states. For example, we have constructed a series of proteins carrying biotin moieties at unique sites, which serve as specific ligands that bind to the soluble protein streptavidin, thus allowing us to map out the position of the point mutation with respect to the membrane. We have consequently indentified a large region of the protein which crosses the membrane in conjunction with channel gating. These studies are now being extended to colicin A, for which a crystal structure has been published.

Another project concerns the so-called Cry toxins synthesyzed by B. thuringiensis. This large family of proteins is toxic to select insects, and some of them are actually used as insecticides. Their mechanism of action is not entirely understood, but they are clearly capable of disrupting normal cell membrane functions. Collaborating with Dr. L. English, of Ecogen, Inc., we have found that at least some of these Cry proteins can form channels in planar bilayers. The protein has three distinct domains, and we have expressed one of them, which consists almost entirely of seven nearly parallel helices, in E. coli, and shown that it, too, forms channels in planar membranes. About 15 of the Cry proteins have been sequenced, and the crystal structure of one of them has been solved (with 2 others in the pipeline), making these proteins promising targets for further research.