This proposal will focus on movements within voltage-gated ion channels and how these movements are coupled.
The first aim i s to study the relative movement between the S4 voltage sensors and the surrounding 'gating pore'. We will use a combination of mutagenesis, cysteine scanning, and photocrosslinking. Crosslinking will be done with our newly designed bifunctional photoactivatable reagent, benzophenone-4-carboxamidocysteine methanethiosulfonate (BPMTS), which can be attached to cysteines introduced by mutagenesis. UV irradiation causes the benzophenone moiety to insert into neighboring C-H bonds. Besides completing an accessibility scan of the S4 segment of domain 4, we will use these methods to examine voltage-dependent movements of S2 and S3 segments. We will also test for roles of S4-S5 linkers and of regions at the exterior of the 'pore domain', comprised of the S5 and S6 transmembrane segments, in the coupling between voltage sensors and gates.
The second aim i s to examine movements of the putative activation gates of sodium channels and of the N-type inactivation gate of Shaker potassium channels, using the same methods described in the first aim. The goals are to elucidate the conformational changes underlying gate movement and to test for cooperative interactions among gate participants.
The third aim i s to elucidate the kinetics of voltage sensor and gate movements by exploiting the kinetics of photocrosslinking after brief flashes of UV light.
The fourth aim i s to obtain structural insights by determining the insertion sites of the crosslinker, using Matrix-Assisted Laser Desorption/lonization Time-of-Flight mass spectrometry of peptide fragments obtained from BPMTS-labeled and irradiated channels. The goals are to determine orientations of voltage sensors and gates with respect to surrounding regions of the channel and to see how these orientations change in response to changes of membrane potential.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR041691-10
Application #
6325061
Study Section
Special Emphasis Panel (ZRG1-MDCN-3 (01))
Program Officer
Lymn, Richard W
Project Start
1992-07-24
Project End
2006-02-28
Budget Start
2001-06-01
Budget End
2002-02-28
Support Year
10
Fiscal Year
2001
Total Cost
$345,192
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
Ding, Shinghua; Horn, Richard (2002) Tail end of the s6 segment: role in permeation in shaker potassium channels. J Gen Physiol 120:87-97
Nguyen, Thao P; Horn, Richard (2002) Movement and crevices around a sodium channel S3 segment. J Gen Physiol 120:419-36
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

Showing the most recent 10 out of 29 publications