Ion channels underlie physiological functions from nerve excitation to blood-cell volume control as well as pathological conditions such as cystic fibrosis and lupus erythromatosis. Osmoregulation is important in animal cells in general and in blood and kidney cells in particular. Both ion channels and osmoregulation evolved early and have been found in the bacterium Escherichia coli. The long-term goal of this projected is to use E. coli to understand the structures and functions of ion channels and the molecular mechanism of osmotaxis. The patch-clamp survey will be continued to uncover the electric currents through different types of ion channels on the inner or on the outer membranes of E. coli. Their unit conductances, ion specificities, gating mechanisms and kinetic properties will be described. To find out the genes that correspond to the ion currents, existing mutants which may code for channels (phoE-, ompA, envZ-, trkB-, trkC-, osmoregulatory mutants, chemotactic mutants) will be examined for their electrophysiological phenotypes. Ion-resistant, ion-sensitive and osmotactic mutants will be isolated and subjected to the same scrutiny. Once the mutations which correspond to the loss of ion currents are identified, they will be grouped and mapped. The genes suspected to code for ion channel structures will be cloned. Structural models will be built based on the amino-acid sequences. Random mutations selected in vivo as well as mutations directed to specific sites in vitro will be induced and their bioelectric consequences analyzed to test the proposed models. To understand osmotaxis, the existing mutants defective in sensory transduction (flaI-, che-, mot-) will be examined for their osmotactic ability. Osmotactic mutants to be isolated will be used to find the genes and gene products used in this taxis. The possibility of repulsion by low osmolarity besides high osmolarity will be explored. The point at which the osmotactic and chemotactic information merge will be identified through mutational analyses and the possibility that taxis and/or motility requires a proper turgor pressure will be examined. The relation between pressure-activated ion channels and osmotaxis will be examined by electrical studies of osmotactic mutants and behavioral studies of the ion-channel mutants.
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