A Unified View of the Role of Electrostatic Interactions in Modulating the Gating of Cys Loop Receptors

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on γ-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily. In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on γ-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily. The Cys loop superfamily of neurotransmitter-gated ion channels plays a prominent role in mediating fast synaptic transmission. Receptors for acetylcholine (nicotinic ACh receptor, nAChR), 2The abbreviations used are: nAChRnicotinic acetylcholine receptor5-HT3serotoninGABAAγ-aminobutyric acid type AAChacetylcholineHAhemagglutininSuChsuccinylcholine.2The abbreviations used are: nAChRnicotinic acetylcholine receptor5-HT3serotoninGABAAγ-aminobutyric acid type AAChacetylcholineHAhemagglutininSuChsuccinylcholine. serotonin (5-HT3 receptor), γ-aminobutyric acid (GABA, types A and C receptors), and glycine are known, and the receptors are classified as excitatory (cation-conducting; nAChR and 5-HT3) or inhibitory (anion-conducting; GABA and glycine). Malfunctions in these receptors are responsible for a number of “channelopathies,” and the receptors are targets of pharmaceutical efforts toward treatments for a wide range of neurological disorders, including Alzheimer disease, Parkinson disease, addiction, schizophrenia, and depression (1Leite J.F. Rodrigues-Pinguet N. Lester H.A. J. Clin. Investig. 2003; 111: 436-437Crossref PubMed Google Scholar, 2Paterson D. Nordberg A. Prog. Neurobiol. 2000; 61: 75-111Crossref PubMed Scopus (690) Google Scholar). The receptors share a common architecture, are significantly homologous, and are known to have evolved from a single ancestral gene that coded for an ACh receptor. nicotinic acetylcholine receptor serotonin γ-aminobutyric acid type A acetylcholine hemagglutinin succinylcholine. nicotinic acetylcholine receptor serotonin γ-aminobutyric acid type A acetylcholine hemagglutinin succinylcholine. The gating mechanism for the Cys loop superfamily is one of the most challenging questions in molecular neuroscience. At issue is how the binding of a small molecule neurotransmitter can induce a structural change in a large, multisubunit, integral membrane protein sufficient to open (gate) a previously closed ion channel contained within the receptor (3Changeux J.P. Edelstein S.J. Curr. Opin. Neurobiol. 2001; 11: 369-377Crossref PubMed Scopus (99) Google Scholar, 4Lester H.A. Dibas M.I. Dahan D.S. Leite J.F. Dougherty D.A. Trends Neurosci. 2004; 27: 329-336Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). All evidence indicates that the neurotransmitter-binding site is quite remote (50-60 Å) from the channel gate, the region that blocks the channel when the neurotransmitter is absent and that must move to open the channel. The quest for a gating mechanism has been greatly aided by several recent structural advances. First, crystal structures of the soluble acetylcholine-binding protein (AChBP) (5Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; PubMed Scopus Google Scholar, Klaassen R.V. van van van Smit A.B. Sixma T.K. J. Full Text Full Text PDF PubMed Scopus Google Scholar, Dijk K. Smit A.B. Sixma T.K. 2004; Full Text Full Text PDF PubMed Scopus Google is to the domain of the nAChR by of the a of the of the site and to the of the receptor. of of the nAChR by and N. A. J. J. PubMed Scopus Google Scholar, A. N. Nature. 2003; PubMed Scopus Google Scholar, N. J. PubMed Scopus Google from the has a of the is to from the that although quite is not a crystal of the is a from data and a for the and to molecular on the and of Cys loop receptors must as a The of the nAChR N. J. PubMed Scopus Google in the site to the transmembrane region and gating. in and from the are toward the transmembrane and in these of the transmembrane that loop is the Cys The transmembrane region of is that or most of the channel. of the transmembrane region toward the a is from the of the protein in recent a in is A. M. M. N. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In the C of a region we can the A is the a the transmembrane have loop at the interface the transmembrane and by and have that loop an role in gating. recent Lester H.A. Dougherty D.A. Nature. PubMed Scopus Google has that a at the of the loop the conformational that the channel in the receptor. have to in the interface the domain and the transmembrane and we of these results interface a number of charged and most efforts have on to electrostatic that gating. have been A. N. Nature. 2003; PubMed Scopus Google Scholar, Neurosci. 2004; PubMed Scopus Google Scholar). interacting pairs have been in receptors J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Nature. 2003; PubMed Scopus Google and specific gating based on electrostatic have been A. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar, J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar). 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