The long term objective of this project is the understanding of voltage-dependent gating of ion channels at the molecular level. In this proposal experiments are designed to describe structural aspects of Shaker and squid potassium channels and human skeletal muscle sodium channels. Cloned, engineered channels will be expressed in Xenopus oocytes and the function will be assessed with electrophysiological techniques while the structure will be probed with optical and chemical modification techniques. There are four specific aims. 1) Correlation of structural changes with the function of the voltage sensor. This will be approached using the technique of histidine scanning mutagenesis on the charges of the S4 and S2 segments. This technique utilizes protons to probe the accessibility of engineered histidine residues, usually replacing basic residues of the protein. In addition, fluorescent probes attached to specific sites of the channels (mutated to cysteine) will be used to assess changes in environment and correlate them with gating currents. 2) Measurements of distances in the channel molecule.
This aim will use the fluorescence resonance energy transfer and its variant, lanthanide-based resonance energy transfer, to measure distances between specific sites across subunits or between an specific site in the channel and an specific toxin sitting on the pore of the channel. The sites of attachment of the fluorophores and lanthanides are engineered cysteines in the channel molecule and Agitoxin II. Distance measurements will be done on sites in the S2, S3 and S4 segments using a newly developed optical setup that allows simultaneous voltage clamp and accurate measurements of gating currents. Distance measurements will be performed at different membrane potentials to assess possible distance during activation of the conductance. 3) Study of the activation and inactivation pathways. In this aim a study of the initial fast event of gating and a detailed characterization of the events leading to channel opening and slow inactivation will be studied with noise analysis of gating currents in the Shaker K channel and with gating currents in the Sodium channel to correlate them with the structural information obtained in aims 1 and 2. 4) Modeling. Kinetic modeling will be done to account for the results in electrophysiologyical and optical experiments. Simulations of fluctuations produced by voltage ramps will be compared to the noise analysis experiments of aim 3 to test models of activation and inactivation. In addition, molecular modeling will be done based on the results of distance measurements, including possible distance changes occurring during activation. These experiments are expected to give us insight on the molecular rearrangements concomitant with voltage-dependent gating, which is a basic property of many membrane channels and it has critical importance in excitability and cell homeostasis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM030376-23
Application #
6611360
Study Section
Special Emphasis Panel (ZRG1-MDCN-3 (01))
Program Officer
Shapiro, Bert I
Project Start
1981-08-01
Project End
2004-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
23
Fiscal Year
2003
Total Cost
$392,214
Indirect Cost
Name
University of California Los Angeles
Department
Physiology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Carrasquel-Ursulaez, Willy; Alvarez, Osvaldo; Bezanilla, Francisco et al. (2018) Determination of the Stoichiometry between ?- and ?1 Subunits of the BK Channel Using LRET. Biophys J 114:2493-2497
Bezanilla, Francisco (2018) Gating currents. J Gen Physiol 150:911-932
Nanazashvili, Mikheil; Sánchez-Rodríguez, Jorge E; Fosque, Ben et al. (2018) LRET Determination of Molecular Distances during pH Gating of the Mammalian Inward Rectifier Kir1.1b. Biophys J 114:88-97
Parameswaran, Ramya; Carvalho-de-Souza, João L; Jiang, Yuanwen et al. (2018) Photoelectrochemical modulation of neuronal activity with free-standing coaxial silicon nanowires. Nat Nanotechnol 13:260-266
Brugarolas, Pedro; Sánchez-Rodríguez, Jorge E; Tsai, Hsiu-Ming et al. (2018) Development of a PET radioligand for potassium channels to image CNS demyelination. Sci Rep 8:607
Kubota, Tomoya; Durek, Thomas; Dang, Bobo et al. (2017) Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET. Proc Natl Acad Sci U S A 114:E1857-E1865
Castillo, Juan P; Sánchez-Rodríguez, Jorge E; Hyde, H Clark et al. (2016) ?1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel. Proc Natl Acad Sci U S A 113:E3231-9
Jiang, Yuanwen; Carvalho-de-Souza, João L; Wong, Raymond C S et al. (2016) Heterogeneous silicon mesostructures for lipid-supported bioelectric interfaces. Nat Mater 15:1023-30
Labro, Alain J; Priest, Michael F; Lacroix, Jérôme J et al. (2015) Kv3.1 uses a timely resurgent K(+) current to secure action potential repolarization. Nat Commun 6:10173
Carvalho-de-Souza, João L; Treger, Jeremy S; Dang, Bobo et al. (2015) Photosensitivity of neurons enabled by cell-targeted gold nanoparticles. Neuron 86:207-17

Showing the most recent 10 out of 132 publications