Ion transport across the plasma membrane by proteins such as ion channels and ion pumps is vital to cellular functions such as signal transduction and volume control. Despite increased information about the molecular structure of transport proteins from the cloning of the respective genes, mechanisms underlying the cellular regulation of ion transport by channels and pumps are not well understood. This research will focus on the regulation of mechanosensitive ion channels and a proton pump in the plasma membrane of the model eukaryote, Saccharomyces cerevisiae. Mechanosensitive channels will be analyzed using patch-clamp electrophysiology to determine the relationship of membrane deformation and specific ions to the activation and adaptation of mechanosensitive channels by combined measurements of membrane capacitance and conductance. Another goal of this proposal is to determine the number and function of proteins which functionally interact with the proton pump, either as modulators or as parallel ion transport mechanisms such as channels. Isolation and genetic characterization of mutants affecting proton extrusion by the pump will be followed by biochemical, electrophysiological, and molecular genetic studies to determine exactly how pump regulation is altered in the mutants. Ion channels and ion pumps are of great fundamental importance to the physiology of cells since they control the traffic of specific ions across the membrane. The proposed combined application of the powerful techniques of molecular genetics and sophisticated electrophysiology to study ion channels and pumps in yeast will make it possible to develop parallel knowledge of their molecular structure of the channels and their function.
