The focus of this proposal is the nicotinic-receptor superfamily of ion channels. Our long-term goal is to elucidate how the biophysical properties of these receptor-channels shape their function under both normal and pathological conditions. To this end, we will apply a multi-faceted approach that includes single-channel and ensemble electrophysiology, protein engineering, X-ray crystallography and theoretical calculations. Most of the experiments will be conducted on the muscle nicotinic acetylcholine receptor (AChR) and its bacterial counterparts, ELIC and GLIC. Because of its large single-channel conductance, robust stoichiometry, tractable kinetics and high expression levels in heterologous expression systems, the muscle AChR remains an unparalleled model for the entire superfamily. Furthermore, throughout the years, research on this particular receptor has provided answers to fundamental questions on the physics, chemistry and biology of ion channels that cannot be addressed with (probably) any other channel. In this application, we propose to address the three pillars of ion-channel function, namely, ion conduction, gating, and modulation of gating. Our three Specific Aims are: 1) To characterize the ring of glutamates in the charge selectivity filter of the AChR in terms of number of total negative charges and acid-base behavior;2) To identify the nature of the gate(s) and its rearrangement upon channel opening, closing and desensitization;and 3) To characterize the regions of the AChR hypothesized to be involved in the metabolic and intramembrane modulation of function. Because of its role as a paradigmatic neurotransmitter-gated ion channel, the knowledge derived from the proposed experiments on the muscle AChR is poised to have a broad impact on our understanding of postsynaptic receptors and fast synaptic transmission in general. It should also be emphasized that AChR-mediated neurotransmission supports not only motor, but also, autonomic and cognitive function. As a result, in addition to being compromised in myasthenic syndromes, cholinergic neurotransmission is also impaired in cardiovascular diseases and neurodegenerative disorders such as Alzheimer's disease and schizophrenia. Even more broadly, AChRs are also expressed in the airways, keratinocytes, lymphocytes and endothelial cells where they are mediators of cell differentiation, proliferation and resistance to apoptosis, and hence, of cancer.

Public Health Relevance

The main subject of this research proposal is the biophysical characterization of the nicotinic acetylcholine receptor. In the central and autonomic nervous system, nicotinic receptors play important roles in learning, memory and the modulation of cardiac function. It follows then a full understanding of not only motor, but also, cardiovascular and cognitive pathological conditions (such as Alzheimer's disease and schizophrenia) requires a thorough knowledge of the molecular underpinnings of these receptors. Furthermore, there is growing evidence that nicotinic receptors are also expressed in the lung, skin, platelets, immune cells and the cells that line the blood vessels, where they mediate cell growth and, eventually, the development of malignant tumors.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MDCN-J (04))
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Silberberg, Shai D
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University of Illinois Urbana-Champaign
Schools of Arts and Sciences
United States
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Papke, David; Grosman, Claudio (2014) The role of intracellular linkers in gating and desensitization of human pentameric ligand-gated ion channels. J Neurosci 34:7238-52
Harpole, Tyler J; Grosman, Claudio (2014) Side-chain conformation at the selectivity filter shapes the permeation free-energy landscape of an ion channel. Proc Natl Acad Sci U S A 111:E3196-205
Gonzalez-Gutierrez, Giovanni; Cuello, Luis G; Nair, Satish K et al. (2013) Gating of the proton-gated ion channel from Gloeobacter violaceus at pH 4 as revealed by X-ray crystallography. Proc Natl Acad Sci U S A 110:18716-21
Gonzalez-Gutierrez, Giovanni; Lukk, Tiit; Agarwal, Vinayak et al. (2012) Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals. Proc Natl Acad Sci U S A 109:6331-6
Cymes, Gisela D; Grosman, Claudio (2011) Tunable pKa values and the basis of opposite charge selectivities in nicotinic-type receptors. Nature 474:526-30
Cymes, Gisela D; Grosman, Claudio (2011) Estimating the pKa values of basic and acidic side chains in ion channels using electrophysiological recordings: a robust approach to an elusive problem. Proteins 79:3485-93
Gonzalez-Gutierrez, Giovanni; Grosman, Claudio (2010) Bridging the gap between structural models of nicotinic receptor superfamily ion channels and their corresponding functional states. J Mol Biol 403:693-705
Elenes, Sergio; Decker, Michael; Cymes, Gisela D et al. (2009) Decremental response to high-frequency trains of acetylcholine pulses but unaltered fractional Ca2+ currents in a panel of "slow-channel syndrome" nicotinic receptor mutants. J Gen Physiol 133:151-69
Cymes, Gisela D; Grosman, Claudio (2008) Pore-opening mechanism of the nicotinic acetylcholine receptor evinced by proton transfer. Nat Struct Mol Biol 15:389-96
Purohit, Yamini; Grosman, Claudio (2006) Block of muscle nicotinic receptors by choline suggests that the activation and desensitization gates act as distinct molecular entities. J Gen Physiol 127:703-17

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