The broad objectives of this project are: (1) to understand better the molecular basis of state-dependent interactions between voltage-gated Na channels and local anesthetics (LAs) and (2) to explore the interplay between LAs and the Na+ permeation pathway. This pathway, in part, consists of a selectivity filter, permenant ion binding sites and as inactivation gate. Among LAs included are putative inactivation enhancers, such as benzocaine and tricaine, putative open-channel blockers, such as cocaine, bupivacaine, and quaternary ammonium (QA) compounds, and putative dual blockers such as tetracaine and procaine. In this proposal, we plan to examine the structural basis that distinguishes these three distinct LA types. Two separate hypotheses will be tested: first, only one single receptor is present within the Na+ permeation pathway for all three types of LAs and second, the common amino group on the phenyl ring of inactivation enhancers and dual blockers preferentially stabilizes the inactivated state of the Na+ channel. Both whole-cell and single channel currents will be measured in order to obtain detailed kinetic information on the dynamic interactions between Na+ channels and LAs. Because ion-ion repulsion within the pore is a common trait for ion permeation, demonstration and further characterization of a knock-out phenomenon of LA/QA ions by the inflowing cations through the Na+ selectivity filter will be obtained to provide crucial evidence that the LA binding site is indeed located within the Na+ permeation pathway. Concurrently, we will delineate LA- channel interactions at the molecular level. At first, the LA binding toward cloned mu1 muscle Na+ channels will be studied with and without Beta1 subunit present. Subsequently, the roles of two separate regions of mu1 Na+ channels, including the internal QA binding site (probably within the pore and S6 regions) and the inactivation-related loop (between domain III and IV), on LA binding affinities will be examined by the macropatch technique in Xenopus oocytes injected with wild-type and mu1 mutant mRNAs. Together, these studies should provide a clearer understanding of LA-Na+ channel interactions as well as the whereabouts of the LA/QA binding site within the Na+ permeation pathway.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM035401-09A2
Application #
2177888
Study Section
Physiology Study Section (PHY)
Project Start
1985-09-06
Project End
1998-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
9
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Xiao, Yong-Fu; Ke, Qingen; Wang, Sho-Ya et al. (2004) Electrophysiologic properties of lidocaine, cocaine, and n-3 fatty-acids block of cardiac Na+ channels. Eur J Pharmacol 485:31-41
Sudoh, Yukari; Desai, Sukumar P; Haderer, Anna E et al. (2004) Neurologic and histopathologic evaluation after high-volume intrathecal amitriptyline. Reg Anesth Pain Med 29:434-40
Wang, Ging Kuo; Wang, Sho-Ya (2003) Veratridine block of rat skeletal muscle Nav1.4 sodium channels in the inner vestibule. J Physiol 548:667-75
Sudoh, Yukari; Cahoon, Elaine Elliott; Gerner, Peter et al. (2003) Tricyclic antidepressants as long-acting local anesthetics. Pain 103:49-55
Nau, Carla; Wang, Sho-Ya; Wang, Ging Kuo (2003) Point mutations at L1280 in Nav1.4 channel D3-S6 modulate binding affinity and stereoselectivity of bupivacaine enantiomers. Mol Pharmacol 63:1398-406
O'Reilly, J P; Wang, S Y; Wang, G K (2003) Methanethiosulfonate-modification alters local anesthetic block in rNav1.4 cysteine-substituted mutants S1276C and L1280C. J Membr Biol 193:47-55
Wang, G K; Wang, S Y (2002) Modifications of human cardiac sodium channel gating by UVA light. J Membr Biol 189:153-65
Xiao, Y F; Ke, Q; Wang, S Y et al. (2001) Point mutations in alpha-subunit of human cardiac Na+ channels alter Na+ current kinetics. Biochem Biophys Res Commun 281:45-52
Xiao, Y F; Ke, Q; Wang, S Y et al. (2001) Single point mutations affect fatty acid block of human myocardial sodium channel alpha subunit Na+ channels. Proc Natl Acad Sci U S A 98:3606-11
Wang, S Y; Barile, M; Wang, G K (2001) A phenylalanine residue at segment D3-S6 in Nav1.4 voltage-gated Na(+) channels is critical for pyrethroid action. Mol Pharmacol 60:620-8

Showing the most recent 10 out of 43 publications