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.
|Kubota, Tomoya; Lacroix, Jérôme J; Bezanilla, Francisco et al. (2014) Probing ?-3(10) transitions in a voltage-sensing S4 helix. Biophys J 107:1117-28|
|Treger, Jeremy S; Priest, Michael F; Iezzi, Raymond et al. (2014) Real-time imaging of electrical signals with an infrared FDA-approved dye. Biophys J 107:L09-12|
|Dang, Bobo; Kubota, Tomoya; Correa, Ana M et al. (2014) Total chemical synthesis of biologically active fluorescent dye-labeled Ts1 toxin. Angew Chem Int Ed Engl 53:8970-4|
|Lacroix, Jérôme J; Hyde, H Clark; Campos, Fabiana V et al. (2014) Moving gating charges through the gating pore in a Kv channel voltage sensor. Proc Natl Acad Sci U S A 111:E1950-9|
|Villalba-Galea, Carlos A; Frezza, Ludivine; Sandtner, Walter et al. (2013) Sensing charges of the Ciona intestinalis voltage-sensing phosphatase. J Gen Physiol 142:543-55|
|Lacroix, Jerome J; Campos, Fabiana V; Frezza, Ludivine et al. (2013) Molecular bases for the asynchronous activation of sodium and potassium channels required for nerve impulse generation. Neuron 79:651-7|
|Dang, Bobo; Kubota, Tomoya; Mandal, Kalyaneswar et al. (2013) Native chemical ligation at Asx-Cys, Glx-Cys: chemical synthesis and high-resolution X-ray structure of ShK toxin by racemic protein crystallography. J Am Chem Soc 135:11911-9|
|Ruscic, Katarina J; Miceli, Francesco; Villalba-Galea, Carlos A et al. (2013) IKs channels open slowly because KCNE1 accessory subunits slow the movement of S4 voltage sensors in KCNQ1 pore-forming subunits. Proc Natl Acad Sci U S A 110:E559-66|
|Capes, Deborah L; Goldschen-Ohm, Marcel P; Arcisio-Miranda, Manoel et al. (2013) Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels. J Gen Physiol 142:101-12|
|Lacroix, Jerome; Halaszovich, Christian R; Schreiber, Daniela N et al. (2011) Controlling the activity of a phosphatase and tensin homolog (PTEN) by membrane potential. J Biol Chem 286:17945-53|
Showing the most recent 10 out of 116 publications