This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Voltage-gated potassium (Kv) channels ( integral membrane proteins present in all three domains of life. In a specializedclass of animal cell, known as excitable cells - including neurons, muscle cells, andendocrine cells - Kv channels work with other cation channels (sodium and calciumchannels) to regulate the electrical activity and signaling of the cell [1]. Kv channelsactivate (open and close) in response to changes in the electrical potential acrossthe cell membrane allowing passive and selective conduction of K+ ions through thechannel. Potassium conduction is directed by the electrochemical gradient acrossthe cell membrane and can achieve very high rates, while still discriminating againstall other cations (including the smaller Na+ ions) [1]. In addition to electrical signalingin nervous systems, Kv channels play an important role in the regulationof cardiac excitability and regulation of insulin release. In humans, malfunction ofthese channels can result in neurological or cardiovascular diseases such as long QTsyndrome or episodic ataxia [2].The crystal structures of Kv1.2 [3, 4], a member of the Shaker K+ channel family,have provided the first view of the molecular architecture of a mammalian potassiumchannel in a putative open state at 3.9 ?A resolution. However, the structure of thechannel in the closed state is still unknown.In collaboration with the Yarov-Yarovoy and Roux labs, the Resource has developedan atomic model for the closed state of the channel. The initial model was generatedusing the structure prediction program ROSETTA [5, 6]. The open and closedstate models of the channel [7] were refined in several stages of molecular dynamicssimulation. Each model was simulated in explicit water/membrane environment inthe presence of an electric field. A total of 400ns of simulations were required toobtain stable conformations of the channel, for the systems containing 100,000 or350,000 atoms.To study the gating mechanism of Kv1.2, the gating charge that is transferredacross the membrane upon activation of the channel is calculated from 900 ns ofall-atom MD simulation of the two protein states. The contribution of individualcharged residues of the channel to the total gating charge is determined, showingthat positions of four conserved arginines within the transmembrane region aretightly coupled to the membrane voltage, and that their movement drives the transitionof the channel between the two states. The results are in agreement withexperimental values obtained for the gating charge [8, 9], indicating that the refinedmodels of Kv1.2 are representatives of the two functional states of Kv channels.BIBLIOGRAPHY[1] B. Hille. Ionic channels of excitable membranes. Sinauer Associates, Sunderland,MA, 2nd edition, 1992.[2] G. J. Siegal, B. W. Agranoff, R. W. Albers, S. K. Fisher, and M. D. Uhler. Basicneurochemistry, molecular, cellular, and medical aspects. Lippincott Williams andWilkins, Philadelphia, 6th edition, 1999.[3] S. B. Long, E. B. Campbell, and R. MacKinnon. Crystal structure of a mammalianvoltage-dependent Shaker family K+ channel. Science, 309:897?903, 2005.[4] X. Tao and R. MacKinnon. Functional analysis of Kv1.2 and paddle chimera kvchannels in planar lipid bilayers. J. Mol. Biol., 382:24?33, 2008.[5] P. Bradley, K. M. S. Misura, and D. Baker. Toward high-resolution de novo structureprediction for small proteins. Science, 309:1868?1871, 2005.[6] V. Yarov-Yarovoy, D. Baker, and W. A. Catterall. Voltage sensor conformations inthe open and closed states in ROSETTA structural models of K+ channels. Proc.Natl. Acad. Sci. USA, 103:7292?7297, 2006.[7] M. M. Pathak, V. Yarov-Yarovoy, G. Agrawal, B. Roux, P. Barth, S. Kohout,F. Tombola, and E. Y. Isacoff. Closing in on the resting state of the Shaker K+channel. Neuron, 56:124?140, 2007.[8] S. Seoh, D. Sigg, D. M. Papazian, and F. Bezanilla. Voltage-sensing residues in theS2 and S4 segments of the Shaker K+ channel. Neuron, 16:1159?1167, 1996.[9] S. K. Aggarwal and R. MacKinnon. Contribution of the S4 segment to the gatingcharge in the Shaker K+ channel. Neuron, 16:1169?1177, 1996.

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