IBN 98-07941 KORN Heart, brain, endocrine and muscle cells are called excitable cells, and rely on the precisely coordinated action of many ion channels for their function. These channels in the plasma membrane of the cell open and close in response to physiological stimulation, and allow the flux of ions into or out of the cell. Flux of calcium and sodium ions through their respective channels tends to subserve excitatory functions, and flux of potassium through potassium channels underlies many inhibitory functions. Although control of excitable behavior is extremely complex, the magnitude and time-course of potassium flux through potassium channels is often a critical factor in determining the duration of excitatory events. There are many different potassium channels, with subtly different structures. This subtle structural variation among channels results in a large degree of functional diversity. Both the magnitude and time-course of potassium channel activity is sensitive to the concentration of potassium and sodium ion in the extracellular space (the region just outside of a cell), and this sensitivity also varies among channels. Two potassium binding sites have been functionally isolated, a low affinity site that underlies some channel functions and a high affinity site that underlies others. In particular, the high affinity site, located at the "selectivity filter," underlies the ability of potassium to prevent sodium from passing through the cell. The low affinity site is not involved in selectivity but is involved in several other channel functions. Recent structural studies have identified the molecular location of the selectivity filter, and the associated high affinity site(s). These studies were unable to identify a cation binding site external the site of selectivity. Consequently, either one of these putative high affinity sites contributes to the low affinity site functions or there is an as yet unknown cation binding site, external to the st ructurally described sites, which cannot be observed in structural studies. The primary focus of these studies will be to determine whether the structurally described selectivity filter site(s) interconvert between low affinity and high affinity sites, or whether a low affinity site exists external to and independent from the high affinity site. The studies will combine molecular biological techniques with electrophysiological recordings and biophysical analysis to determine whether the different functionally- identified binding sites are structurally distinct. Results from this project will enhance our understanding of the nature of cation binding sites in the channel pore, and will thus enhance our knowledge of the molecular mechanisms that underlie phenotypic diversity in potassium channels.
