This proposal is designed to elucidate the molecular mechanisms underlying voltage-dependent gating of the sodium (Na) channels from adult human skeletal (hSkM1) or cardiac muscle (hH1), expressed heterologously either in a mammalian cell line or in Xenopus oocytes. The experiments will entail primarily electrophysiological recordings (single channels, macroscopic ionic currents, and gating currents) of wild-type and mutant channels, but will also include fluorescence measurements of single voltage-clamped cells. The first project is to study the relationship between the movement of S4 transmembrane segments and the voltage-dependent gating of hSkM1 channels. Initial studies will concentrate on the S4 segment of domain 4 (i.e. S4/D4). Cysteine replacements of each of the 8 basic residues of S4/D4 will be used to determine the accessibility, both extracellular and intracellular, of specific residues to cysteine reagents during the voltage-dependent movements of this transmembrane segment. The voltage dependence and kinetics of S4 movement will be determined from the reaction rates of these reagents. We will also obtain a lower-bound estimate for the distance between the extracellular mouth of the ion- conducting pore and the extracellular end of S4 segments, by testing whether extracellular pore blockers prevent access of 54 residues to chemical reagents. Later studies will include experiments on S4 segments in other domains and measurements of voltage-dependent changes in fluorescence intensity for fluorescently labelled S4 residues. The second project is to test the hypothesis of a physical connection between a putative cytoplasmic inactivation gate and an activation voltage sensor of the hH1 Na channel. We will also test the possibility that the cytoplasmic linker between S4 and S5 segments in D4 acts as a receptor for the inactivation gate.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR041691-09
Application #
6171279
Study Section
Physiology Study Section (PHY)
Program Officer
Lymn, Richard W
Project Start
1992-07-24
Project End
2001-05-31
Budget Start
2000-06-01
Budget End
2001-05-31
Support Year
9
Fiscal Year
2000
Total Cost
$309,017
Indirect Cost
Name
Thomas Jefferson University
Department
Physiology
Type
Schools of Medicine
DUNS #
061197161
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Cohen, Adi; Addesso, Vicki; McMahon, Donald J et al. (2006) Discontinuing antiresorptive therapy one year after cardiac transplantation: effect on bone density and bone turnover. Transplantation 81:686-91
Ahern, Christopher A; Horn, Richard (2005) Focused electric field across the voltage sensor of potassium channels. Neuron 48:25-9
Ding, Shinghua; Ingleby, Lindsey; Ahern, Christopher A et al. (2005) Investigating the putative glycine hinge in Shaker potassium channel. J Gen Physiol 126:213-26
Ahern, Christopher A; Zhang, Ji-Fang; Wookalis, Marilyn J et al. (2005) Modulation of the cardiac sodium channel NaV1.5 by Fyn, a Src family tyrosine kinase. Circ Res 96:991-8
Ahern, Christopher A; Horn, Richard (2004) Specificity of charge-carrying residues in the voltage sensor of potassium channels. J Gen Physiol 123:205-16
Ding, Shinghua; Horn, Richard (2003) Effect of S6 tail mutations on charge movement in Shaker potassium channels. Biophys J 84:295-305
Nguyen, Thao P; Horn, Richard (2002) Movement and crevices around a sodium channel S3 segment. J Gen Physiol 120:419-36
Ding, Shinghua; Horn, Richard (2002) Tail end of the s6 segment: role in permeation in shaker potassium channels. J Gen Physiol 120:87-97
Ding, S; Horn, R (2001) Slow photo-cross-linking kinetics of benzophenone-labeled voltage sensors of ion channels. Biochemistry 40:10707-16
Mitrovic, N; George Jr, A L; Horn, R (2000) Role of domain 4 in sodium channel slow inactivation. J Gen Physiol 115:707-18

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