This project aims to determine closed and opened structures of three physiologically important K+ ion channels to develop a physical model of their function as molecular devices.
The aims will be addressed through (1) development of expression and purification protocols, (2) reconstitution for functional analysis using high time resolution electrical recordings, and finally (3) application of new advances in cryo-electron microscopy for structure determination. We propose methods to control the conformational state - closed versus opened - of each channel during structural analysis. The three channels belong to the same architectural family, but each is activated in the cell by unique stimuli: Na+ in one case, membrane voltage in another, and membrane voltage plus Ca2+ in the third. To control a voltage-dependent channel's conformation while determining structure we will use a spider toxin to hold the channel closed while it is embedded in a nanodisc-mediated lipid membrane. The contrast and comparison of these channels' distinct signal responses, given that they are structurally related, should yield a deeper mechanistic understanding of each. The three channels under focus mediate smooth and skeletal muscle contraction, regulate neuronal firing rates, and propagation of action potentials along axons. Heritable defects within these channels underlie asthma and various forms of epilepsy in humans. I believe that the deeper mechanistic understanding that these experiments will yield will bring us closer to pharmacological control of these ion channels for the benefit of human health. Furthermore, the methods developed for membrane protein preparation and structure determination should be applicable to other membrane proteins, which account for 25% of proteins encoded in the human genome, and thus advance a broader field of medical science.

Public Health Relevance

Heritable defects in ion channels under study in this proposal underlie asthma and various forms of epilepsy including malignant migrating partial seizures of infancy and autosomal dominant nocturnal frontal lobe epilepsy. Ion channels are fundamental to neuronal as well skeletal, smooth and cardiac muscle function. A deeper understanding of the structure and chemistry of ion channels I believe will lead to new pharmacological therapies for epilepsy, cardiac arrhythmia and skeletal and smooth muscle dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043949-29
Application #
9594152
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Nie, Zhongzhen
Project Start
1990-04-01
Project End
2019-11-30
Budget Start
2018-12-01
Budget End
2019-11-30
Support Year
29
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Biology
Type
Graduate Schools
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Lee, Chia-Hsueh; MacKinnon, Roderick (2018) Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures. Science 360:508-513
Wang, Weiwei; MacKinnon, Roderick (2017) Cryo-EM Structure of the Open Human Ether-à-go-go-Related K+ Channel hERG. Cell 169:422-430.e10
Lee, Chia-Hsueh; MacKinnon, Roderick (2017) Structures of the Human HCN1 Hyperpolarization-Activated Channel. Cell 168:111-120.e11
Tao, Xiao; Hite, Richard K; MacKinnon, Roderick (2017) Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel. Nature 541:46-51
Hite, Richard K; Tao, Xiao; MacKinnon, Roderick (2017) Structural basis for gating the high-conductance Ca2+-activated K+ channel. Nature 541:52-57
Hite, Richard K; MacKinnon, Roderick (2017) Structural Titration of Slo2.2, a Na+-Dependent K+ Channel. Cell 168:390-399.e11
Whicher, Jonathan R; MacKinnon, Roderick (2016) Structure of the voltage-gated K? channel Eag1 reveals an alternative voltage sensing mechanism. Science 353:664-9
Su, Zhenwei; Brown, Emily C; Wang, Weiwei et al. (2016) Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels. Proc Natl Acad Sci U S A 113:5748-53
Touhara, Kouki K; Wang, Weiwei; MacKinnon, Roderick (2016) The GIRK1 subunit potentiates G protein activation of cardiac GIRK1/4 hetero-tetramers. Elife 5:
Wang, Weiwei; Touhara, Kouki K; Weir, Keiko et al. (2016) Cooperative regulation by G proteins and Na(+) of neuronal GIRK2 K(+) channels. Elife 5:

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