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.
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