The long-term goal of the proposed research is to understand the molecular characteristics of ion movement through transmembrane channels. This goal will be pursued through studies on low-molecular channel formers, the linear gramicidins where structural information is available, and through studies on macromolecular channels, the voltage-dependent sodium channels. The kinetics of ion movement through gramicidin channels will be studied to delineate the steps involved in ion entry and ion exit. The major aim is to provide a dissection of the ion entry step. Linear gramicidins which have been site-specifically modified will be used to obtain a structure-function relation for a transmembrane channel. The emphasis will be on gramicidins where the polarity of the side chains are systematically varied at their N- and C-terminal ends, where the length is varied (from 13 to 17 amino acids), or where there are fixed negative charges at the channel entrances. The single-channel conductances and lifetimes will be studied as a function of permeant ion type and concentration, and the structural equivalence of the different compounds will be examined based on the formation of hybrid channels between the modified peptides and reference compounds, e.g. valine gramicidin A. The modulation of the channel behavior by extrinsic factors, such as changes in interfacial dipole potentials, will be studied. Voltage-dependent sodium channels from mammalian forebrain and cardiac muscle will be studied to get insight into the mechanism of ion entry into the channels, especially whether negative surface charges close to the channel entrance have a physiological function as guides for ion entry. Group-specific modification and proteolytic cleavage will be used to modify the channel entrance and examine the relation between the TTX binding site and the extracellular channel entrance. These studies will also contribute to a better understanding of the mechanism of neurotoxin (STX and TTX) induced block. The stationary voltage-activating characteristics will be examined, to characterize the slow """"""""state changes"""""""" which affect the gating (shift the midpoint of activation curves).
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