The long-term objective of this project is to understand better how the activation gate of voltage-gated Na+ channels works during state transitions. As a first step, we plan to delimit the whereabouts of an inner obstruction site critical for activation gating. We hypothesize that the cytoplasmic portions of Na+ channel S6 segments form such a constricted site. Our rationale is based on two S6-situated receptors and their """"""""gated"""""""" access for local anesthetics and batrachotoxin.
Our specific aims are (1) to create, express, and characterize a series of cysteine-substituted mutants at positions 15-28 of all four homologous S6 segments (D1-S6 to D4-S6), (2) to determine the accessibility of these cysteine-mutants with charged cysteine-modifying reagents, and (3) to create, express, and characterize additional mutants with residues of different size, hydrophobicity, and polarity at this putative constricted site. Mutants of the human heart a-subunit Na+ channel (hH1) clone wifi be expressed in human embryonic kidney cells by transient transfection. Mutant Na+ channels and their gating properties will be first characterized under whole-cell configuration. Cysteine-mutants will be then assessed after internal application of charged cysteine-modifying reagents with and without repetitive pulses to evaluate their """"""""gated"""""""" accessibility during state transitions. If needed, in-side-out patches will be used for direct measurements of chemical reactivity rate. Gated and ungated profiles of various cysteine-mutants will allow us to infer the clustered pore-lining residues along the S6 a-helical structures. In addition, UV irradiation of a tethered photo-activatable linker attached to cysteine-mutants may further reveal the S6 movement during channel opening. Subsequent characterizations of the junction between gated- and ungated-accessible region with additional single or double mutations may unravel how such a constricted site opens upon depolarization at the molecular level. This pore-lining site also governs the access of a variety of clinical drugs such as local anesthetics, antiarrhythmics, and anticonvulsants to their receptor(s) within the Na+ channel inner vestibule. Detailed mapping of the cytoplasmic S6 regions along with their linkage with the Na+ channel activation gating may provide insights for the design of new therapeutic drugs that target this important region.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL066076-02
Application #
6537915
Study Section
Cardiovascular and Renal Study Section (CVB)
Program Officer
Lathrop, David A
Project Start
2001-09-01
Project End
2005-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
2
Fiscal Year
2002
Total Cost
$257,510
Indirect Cost
Name
State University of New York at Albany
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Albany
State
NY
Country
United States
Zip Code
12222
Wang, Sho-Ya; Wang, Ging Kuo (2017) Single rat muscle Na+ channel mutation confers batrachotoxin autoresistance found in poison-dart frog Phyllobates terribilis. Proc Natl Acad Sci U S A 114:10491-10496
Wang, Ging Kuo; Russell, Gabriella; Wang, Sho-Ya (2013) Persistent human cardiac Na+ currents in stably transfected mammalian cells: Robust expression and distinct open-channel selectivity among Class 1 antiarrhythmics. Channels (Austin) 7:263-74
Wang, Ging Kuo; Edrich, Thomas; Wang, Sho-Ya (2006) Time-dependent block and resurgent tail currents induced by mouse beta4(154-167) peptide in cardiac Na+ channels. J Gen Physiol 127:277-89
Wang, Sho-Ya; Mitchell, Jane; Tikhonov, Denis B et al. (2006) How batrachotoxin modifies the sodium channel permeation pathway: computer modeling and site-directed mutagenesis. Mol Pharmacol 69:788-95
Wang, Sho-Ya; Wang, Ging Kuo (2005) Block of inactivation-deficient cardiac Na(+) channels by acetyl-KIFMK-amide. Biochem Biophys Res Commun 329:780-8
Wang, Sho-Ya; Russell, Corinna; Wang, Ging Kuo (2005) Tryptophan substitution of a putative D4S6 gating hinge alters slow inactivation in cardiac sodium channels. Biophys J 88:3991-9
Wang, Ging Kuo; Russell, Corinna; Wang, Sho-Ya (2004) Mexiletine block of wild-type and inactivation-deficient human skeletal muscle hNav1.4 Na+ channels. J Physiol 554:621-33
Wang, Sho-Ya; Mitchell, Jane; Moczydlowski, Edward et al. (2004) Block of inactivation-deficient Na+ channels by local anesthetics in stably transfected mammalian cells: evidence for drug binding along the activation pathway. J Gen Physiol 124:691-701
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
Wang, Sho-Ya; Bonner, Kaitlin; Russell, Corinna et al. (2003) Tryptophan scanning of D1S6 and D4S6 C-termini in voltage-gated sodium channels. Biophys J 85:911-20

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