Of the dozen or so solved structures of voltage-gated ion channels (VGICs), only those of a weakly voltage-dependent Arabidopsis channel show the channel in its resting, de-activated state. Structures of VGIC voltage sensors in their resting ?down? position are lacking because when solubilized in detergent or even reconstituted into nanodiscs, the absence of a membrane potential always leaves the voltage sensors in an activated or inactivated-relaxed conformation. In the proposed work innovative single-particle cryo-EM methods will be applied to obtain structures VGIC proteins as reconstituted into lipid vesicles. In the previous funding period we have made many methodological advances and have progressed to the point of obtaining nanometer/sub-nanometer cryo-EM structures of the Kv1.2 wildtype and ?paddle chimera? potassium channel constructs, reconstituted into vesicles. In these structures no membrane potential is applied. We now propose to extend that work to higher resolution and to the resting-state structure of Kv1.2, the best-understood mammalian VGIC, by subjecting it to a large negative membrane potential. We will then acqure resting-state and activated-state structures of the Nav1.7 sodium channel, which is of intense interest as a central player and drug target in pain sensation. These structures will enable for the first time a quantitative understanding of VGIC voltage sensing, and mechanisms of pain disorders due to sodium channel mutations.

Public Health Relevance

Voltage-gated ion channels (VGICs) are responsible for electrical impulses in cells of the brain, heart and other tissues. We will use innovative electron cryo-microscopy techniques to obtain near-atomic-resolution structures of two VGICs in their resting and active states. This will provide an unprecedented view of the mechanism by which they control the electrical currents of ions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS021501-34
Application #
10105372
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Silberberg, Shai D
Project Start
1984-12-01
Project End
2025-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
34
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Yale University
Department
Physiology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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Kucukelbir, Alp; Sigworth, Fred J; Tagare, Hemant D (2014) Quantifying the local resolution of cryo-EM density maps. Nat Methods 11:63-5
Shigematsu, H; Sigworth, F J (2013) Noise models and cryo-EM drift correction with a direct-electron camera. Ultramicroscopy 131:61-9

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