LOCAL ANESTHETICS--RECEPTOR TOPOLOGY AND DRUG DESIGN

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The goals of this project are (1) to map the topology of the local anesthetic (IA) receptor in voltage-gated Na+ channels, (2) to determine the primary structure of the LA receptor, and (3) to design better Na+ channel blockers for this LA receptor. To date, the action of LAs on Na+ channels has been extensively studied. The mechanism underlying the LA action, however, remains elusive mainly because Na+ channels exhibit rapid time- and voltage-dependent conformational changes upon depolarization. In this proposal, we plan to map the topology of the LA receptor in single batrachotoxin (BTX)-activated muscle Na+ channels at equilibrium conditions using a simple planar lipid bilayer technique. The structure/activity relationship of various LAs and their quaternary derivatives as well as quaternary ammonium compounds will be studied. Both hydrophilic and hydrophobic binding domains of the LA receptor will be explored in detail. This study will then be extended to the macroscopic current level in clonal GH3 cells with and without BTX present. In addition, we plan to resolve the primary structure of the LA receptor in Na+ channels first by photoaffinity labelling, peptide mapping, and sequencing. The putative LA receptor will be subsequently delineated by deletion and point mutation of the Na+ channel cDNA. The mutated Na+ channels will be expressed in Xenopus oocytes and their sensitivity toward LAs will be assayed under voltage clamp conditions. Concurrently, we plan to synthesize a variety of amphipathic LA derivatives which supposedly will display high affinities for the LA receptor due to the enhanced hydrophobic interactions. In return, these compounds may be used for further probing the receptor topology and for photoaffinity labelling. Together, these experiments should give us a clearer view if the LA receptor in Na+ channels and possibly provide us better Na+ channel blockers. Such high affinity blockers may be potentially beneficial for the management of chronic as well as intractable cancer pain.