Sodium channels underlie the rapid depolarization of action potentials in most excitable cells and are involved in nervous conduction, voluntary muscle contraction, cardiac excitation-contraction coupling, as well as ion channel linked muscle diseases. They serve as receptors for cardiac antiarrhythmic agents, agents used to intervene in episodes of skeletal muscle derived periodic paralysis, as well as anticonvulsant agents. Thus, these important excitability proteins are essential to normal physiological behavior and are important targets for therapeutic intervention. There is considerable evidence for an inter-dependence between Na+ channel gating and channel block by local anesthetic-like agents. However at present little is known about the molecular determinants of channel gating and pharmacology. The long term aims of this proposal are to determine the role of the two principal protein subunits of human Na+ channel (alpha and beta1 subunits) and how their interactions modulate channel gating and pharmacology. Specific regions of the Na= channel (amino acid domains) will be manipulated through protein engineering and recombinant DNA methods with the goal of identifying their role in channel gating (opening and inactivation), interactions with the beta1 subunit, and in drug binding. The methods include patch clamp and high speed cut-open oocyte voltage clamp of channels expressed in Xenopus oocytes or in mammalian cells. A major strategy will be to capitalize on the natural functional and structural diversity of distinct Na+ channels to guide experiments. Three distinct human Na+ channels will be studied; the human cardiac Na+ channel, hH1; the human skeletal muscle Na+ channel, hSkm1; and a newly identified channel cloned from human ventricle, hNav2.1. One rationale for using these channels is that although highly conserved, functional differences combined with sequence differences provide clues for identifying and manipulating important domains in the protein. The need to understand state dependent drug block and channel gating mode changes is fundamental. The Na+ channel is the simplest system to begin these investigations at the molecular level. It offers significant advantages for successful protein structure-function studies. Ion channels have a functional signature (the single channel current) that can be measured with excellent, and functionally relevant temporal resolution at the level of a single protein molecule. The results will improve understanding of the function of these newly identified human proteins and will help identify protein domains involved with channel gating and binding of therapeutically relevant pharmacological agents.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL051197-01
Application #
2227783
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1994-04-09
Project End
1999-03-31
Budget Start
1994-04-09
Budget End
1995-03-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Johnson Jr, J P; Balser, J R; Bennett, P B (2001) A novel extracellular calcium sensing mechanism in voltage-gated potassium ion channels. J Neurosci 21:4143-53
Johnson Jr, J P; Mullins, F M; Bennett, P B (1999) Human ether-a-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors. J Gen Physiol 113:565-80
Po, S S; Wang, D W; Yang, I C et al. (1999) Modulation of HERG potassium channels by extracellular magnesium and quinidine. J Cardiovasc Pharmacol 33:181-5
Wang, D W; VanDeCarr, D; Ruben, P C et al. (1999) Functional consequences of a domain 1/S6 segment sodium channel mutation associated with painful congenital myotonia. FEBS Lett 448:231-4
London, B; Wang, D W; Hill, J A et al. (1998) The transient outward current in mice lacking the potassium channel gene Kv1.4. J Physiol 509 ( Pt 1):171-82
Wang, D W; Yazawa, K; Makita, N et al. (1997) Pharmacological targeting of long QT mutant sodium channels. J Clin Invest 99:1714-20
Wang, D W; George Jr, A L; Bennett, P B (1996) Comparison of heterologously expressed human cardiac and skeletal muscle sodium channels. Biophys J 70:238-45
Makita, N; Bennett Jr, P B; George Jr, A L (1996) Multiple domains contribute to the distinct inactivation properties of human heart and skeletal muscle Na+ channels. Circ Res 78:244-52
Wang, D W; Nie, L; George Jr, A L et al. (1996) Distinct local anesthetic affinities in Na+ channel subtypes. Biophys J 70:1700-8
Wang, D W; Yazawa, K; George Jr, A L et al. (1996) Characterization of human cardiac Na+ channel mutations in the congenital long QT syndrome. Proc Natl Acad Sci U S A 93:13200-5

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