Exploring the impact of sequence on the mechanistic properties of voltage-sensing domains

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The presence of the plasma membrane makes it possible for cells to maintain a specialized intracellular environment compatible with cell survival and proper function. Communication with the external environment requires signals to be passed back and forth across this barrier, and this transfer must be tightly regulated so as to maintain cellular conditions. One class of integral membrane proteins has evolved voltage-sensing domains (VSDs) so that their action can be regulated by changes in membrane potential. These domains comprise four transmembrane segments, typically labeled S1-S4, which contain charged residues capable of sensing changes in the electric field across the membrane. Negative potentials 'hold' the sensor in a configuration in which the charges are closer to the intracellular space, termed the 'down state', and more positive voltages drive the sensor outward across the membrane interface into an 'up state'. VSDs are highly conserved; however, VSD sequence variations are responsible for fast gating of voltage-gated sodium channels (Nav) and slower gating of voltage-gated potassium channels (Kv). Moreover within the past year and a half, the number and diversity of proteins that have co-opted VSDs to regulate their function has grown to include proton channels and membrane bound phosphatases. All of these proteins must respond to voltage differently to ensure specificity. Two ways of doing this are by controlling the voltage range over which the VSDs switch from the down state to the up state and by tuning the timescale with which these transitions occur. For instance, the large difference in opening kinetics between Nav and Kv channels is essential for the generation of action potentials. Given the highly charged nature of the VSD and its extreme sensitivity to electric fields, both of these mechanisms will be probed using molecular models of the VSD combined with continuum electrostatic calculations. Down state molecular models of the VSD developed during Dr. Grabe's NSF postdoctoral fellowship will be further refined and these models then will be used to construct gating trajectories of the opening transition. This will be done by comparing the down state model with the up state X-ray crystal structure of the Kv1.2 potassium channel. Realistic intermediates along this pathway will be created by combining incremental rigid body interpolation of helices between the end point states with molecular dynamics simulations at each increment. The structures will then be used in Poisson-Boltzmann calculations, as developed in Grabe et al. (2004), to quantify the electrostatics of gating. This energetic analysis will allow determination of the voltage range over which different voltage sensors activate, as well as the estimation of activation kinetics. These are both important steps in developing a unified view of voltage sensing in this important class of proteins, and this work will provide new insights into how cells sense their environments. The broader impacts of this project will be on education and training, as well as the career development of a new investigator. The PI has already recruited a graduate student and an undergraduate student into his lab, and support for these students as well as another undergraduate student is requested in the budget. The PI also has introduced new course material into graduate and undergraduate curricula, including a molecular modeling course for undergraduate students that incorporates material from his lab project and a new cross-disciplinary mathematical/computational biology course for graduate and advanced undergraduate students to be offered Fall 2007.