The long-term goal of the proposed research is to understand molecular characteristics of ion movement through transmembrane channels. This goal will be pursued through studies on voltage-dependent sodium channels that are incorporated into planar lipid bilayers of defined composition. Ion permeation, pharmacological modification, and voltage activation (gating), will be examined, with special emphasis on the role of fixed charges at the extra- and intracellular surface of the protein and host bilayer in modulating channel function. The mechanism of ion entry into the channels will be studied to determine whether negative charges close to the channel entrance have a physiological function as guides for ion entry. Ion permeability and block of channels modified by batrachotoxin, veratradine, pyrethroid insecticides, and group-specific modification will be compared in an attempt to clarify why channels that have a decreased single-channel conductance have a decreased ion selectivity. Group-specific modification and proteolytic cleavage will be used to modify the channel entrance and examine the relation between the guanidinium toxin binding site and the extracellular channel entrance. The kinetics of toxin-induced channel closures will be examined to determine whether the guanidinium toxin-induced channel closures occur as a two-step event, where the channel is closed thorough a conformational change subsequent to toxin binding. The stationary voltage-activation of single channels will be examined to define to what extent gating behavior is affected by lipid surface charges, and to further define the asymmetry in the apparent surface charge density at the extra- and intracellular surfaces of the channel. The slow """"""""mode changes"""""""" that affect gating will be studied.
The aim i s to characterize some of the stationary conformational fluctuations that occur in an integral membrane protein, as well as to determine whether the mode changes can be fully accounted for by discrete shifts in the midpoint potential of the activation curves.
