The goals of this proposal are (1) to map the topology of the local anesthetic (LA) receptor in voltage-gated Na+ channels, (2) to design better Na+ channel blockers toward this LA receptor, and (3) to utilize these blockers in vivo as long-acting LAs. The putative receptor of LAs and their quaternary ammonium (QA) derivatives has been assigned within the a-subunit of the voltage-gated Na+ channel. Various amino acid residues on this putative LA receptor will be mutated by the site-directed mutagenesis method. Mutants and wild-type muscle Na+ channels of rat mu1 clones will be expressed in transiently transfected mammalian cells, and their binding to conventional LAs and newly synthesized QA drugs will be assessed in patch membranes and/or in planar lipid bilayers at the single channel level with batrachotoxin present. The binding contacts to the drugs will be charted within the LA receptor. They will also continue to design a series of QA derivatives from various LAs; among LAs used are tonicaine, procaine, tetracaine, and etidocaine. These high-affinity Na+ channel blockers, in turn, will be employed to resolve further the topology of the LA receptor. Newly synthesized drugs will be tested first in vitro for their binding affinities in native Na+ channels and subsequently in vivo for their efficacy in rat sciatic nerve block and in rat spinal block. N-butyl tetracaine and its related QA derivatives that block sciatic sensory and motor functions for more than one week will be examined for their local anesthetic and neurolytic characteristics. Neurolytic compounds that destroy nerve fibers but retain the tissue integrity for later nerve regeneration will then be explored as potential ultralong-acting local anesthetics. Together, these experiments should give us a clearer view of the LA receptor at the molecular level and likely provide us better Na+ channel blockers with longer duration of block. These high-affinity Na+ channel blockers may be beneficial for patients with chronic as well as intractable cancer pain.
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