The long-term goal of this proposal is to determine the structural basis for allosteric mechanisms in pentameric ligand gated ion channels (LGIC). Upon neurotransmitter binding, LGIC rapidly switch from closed to open to desensitized channel conformations. Despite a substantial understanding of the physiological and pathophysiological role of desensitization, there is very little structural information available on this state. The fcus of this study is to use bacterial LGIC homologues, GLIC and ELIC, to identify the mechanism of desensitization and to determine how drugs modulate transitions between the closed, open and desensitized states. We propose to apply spectroscopic techniques to functionally defined states of GLIC and ELIC in a membrane environment. Our studies will involve a multidisciplinary approach that includes spectroscopic techniques (EPR and fluorescence), patch-clamp measurements in reconstituted proteoliposomes, and thermodynamic measurements using isothermal titration calorimetry. We will 1) Measure the conformational transitions underlying the desensitized conformation. 2) Determine the role of lipid-water-protein interface in gating transitions. 3) Identify the structural basis for allosteric modulation by general anesthetics and alcohols. These experiments will test the central hypothesis that membrane lipids play a key role in maintaining the conformational integrity, in driving gating transitions, and in governing drug modulation. The proposed studies are expected to be significant in that they will provide a dynamic view of LGIC gating in a physiologically- relevant membrane environment. These findings will establish a structural framework to enhance our understanding of LGIC function at the molecular level and thereby improve therapeutic strategies and drug design.

Public Health Relevance

Ligand-gated ion channel functions are crucial for synaptic transmission at the neuronal and neuromuscular junction. Dysfunctions in these channels underlie many neurological disorders. A molecular level understanding of channel function is a critical step towards design of new and safer therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM108921-03S1
Application #
9291772
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Nie, Zhongzhen
Project Start
2014-09-01
Project End
2019-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
3
Fiscal Year
2016
Total Cost
$158,500
Indirect Cost
$58,500
Name
Case Western Reserve University
Department
Physiology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Hughes, Taylor E T; Lodowski, David T; Huynh, Kevin W et al. (2018) Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM. Nat Struct Mol Biol 25:53-60
Chatterjee, Soumili; Vyas, Rajan; Chalamalasetti, Sreevatsa V et al. (2018) The voltage-gated sodium channel pore exhibits conformational flexibility during slow inactivation. J Gen Physiol 150:1333-1347
Basak, Sandip; Gicheru, Yvonne; Samanta, Amrita et al. (2018) Cryo-EM structure of 5-HT3A receptor in its resting conformation. Nat Commun 9:514
Basak, Sandip; Gicheru, Yvonne; Rao, Shanlin et al. (2018) Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT3A receptor. Nature 563:270-274
Gicheru, Yvonne; Chakrapani, Sudha (2018) Direct visualization of ion-channel gating in a native environment. Proc Natl Acad Sci U S A 115:10198-10200
Basak, Sandip; Schmandt, Nicolaus; Gicheru, Yvonne et al. (2017) Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel. Elife 6:
Maiti, Buddhadev; Manna, Arun K; McCleese, Christopher et al. (2016) Photoinduced Homolytic Bond Cleavage of the Central Si-C Bond in Porphyrin Macrocycles Is a Charge Polarization Driven Process. J Phys Chem A 120:7634-7640
Basak, Sandip; Chatterjee, Soumili; Chakrapani, Sudha (2016) Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels. J Vis Exp :
Guo, Xing; Sun, XiaoYan; Hu, Di et al. (2016) VCP recruitment to mitochondria causes mitophagy impairment and neurodegeneration in models of Huntington's disease. Nat Commun 7:12646
Chakrapani, Sudha (2015) EPR Studies of Gating Mechanisms in Ion Channels. Methods Enzymol 557:279-306

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