This project has the long term objective of understanding at the molecular level the dynamics of voltage- dependent gating of voltage-dependent membrane proteins with emphasis in voltage-gated channels. In this proposal the experiments are designed to correlate structural changes with the function on voltage dependent Na, K and phospatase (Ci-VSP). Cloned and engineered proteins will be expressed in Xenopus oocytes or purified and reconstituted in lipid bilayers. Electrophysiological techniques will be used to follow the function and the structure will be probed with a combination of fluorescence spectroscopy and chemical modifications in the ensemble as well as at the single molecule level.
The specific aims are: 1) Description of the trajectory of the S4 segment during voltage sensing.
This aim will use many techniques including metal bridges in different states of the channel, replacement of critical residues in the hydrophobic plug of the sensor, fluorescence polarization and anisotropy, molecular modeling and the crystal structures available. By correlating the modification of the function with the structural measurements the position of several residues will be constrained in the resting, intermediate, active and relaxed states to propose the dynamic trajectory of the sensor including possible secondary structure changes. 2) Role of each subunit in the activation and inactivation of the K+ channel and the operation of one sensor in isolation. The objective is to understand the function of one sensor in virtual isolation. This will be done by studying ionic currents and conformational changes using fluorescence in tandem constructs with only one functional S4 segment and in Ci-VSP. 3) Structural and functional correlates of the voltage-gated Na+ channel. This will be approached by measuring intramolecular distances with lanthanide-based resonance energy transfer in each one of the domains of the channel and its voltage dependence and correlation with the function, including the beta subunit. 4) Description of conformational changes during gating at the single molecule level using single molecule fluorescence. The dynamics of channel gating studied at the single molecule level with fluorescence is expected to reveal structural changes that are hidden in macroscopic measurements and they are required to complete the description of molecular events in gating.
The experiments in this proposal are functional and structural studies using electrophysiological, fluorescence spectroscopy and protein and chemical modification done simultaneously in voltage dependent proteins such as Na and K channels. The emphasis is understanding the dynamics of the molecular events underlying the fundamental mechanism of voltage detection across the membrane and how those events can effect their action in the conduction of ions across the cell membrane. As voltage dependent mechanisms underlie basic biological processes such as the conduction of the nerve impulse, heart contraction, and cell homeostasis, these studies are expected to have impact in health and disease.
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