The goal of this project is to define the sites and molecular mechanisms of local anesthetic (LA) drugs acting to block impulses. Blockade of impulses in peripheral nerve is known to occur through the """"""""state-dependent"""""""" inhibition of voltage-gated sodium channels in the axons, but the precise location and the number of LA binding sites are unknown. In the proposed experiments, we will examine the effects of structurally diverse local anesthetics, investigating at fast time resolution the inhibition of single Na channels in cells and in planar lipid bilayers. One hypothesis is that lipids participate directly in the dynamics of the drug:channel interaction and our approach is to investigate the effects of altering lipids around the channel and to assess directly the drug:lipid interactions. Solvent-free planar bilayers will be reconstituted with purified Na channels using mixtures of lipids, to provide a particular membrane fluidity, """"""""melting point"""""""" for phase transition, or electric surface charge. Synthetic vesicles from the same lipids and proteins will be used to study the dynamic distribution of LA among different regions of a particular membrane, using magnetic spin resonance. The kinetics of gating of Na channels catalyzed by several different """"""""activator"""""""" drugs, ranging from high-affinity, irreversible (batrachotoxin) to low-affinity, reversible (veratridine) agents, will be quantified in the presence and absence of local anesthetics. Direct binding of activators and the effects thereon of LA will be assayed in parallel experiments to determine if LA blockade requires the total displacement of activator drugs from the channel. The activators will be covalently bound to channels to examine LA inhibition of irreversibly conjugated channels. We will thus identify the membrane loci and mechanisms for anesthetics that selectively inhibit resting channels and those that block activated or inactivated channels.
