Abstract
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 gamma-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.
Highlights
The gating mechanism for the Cys loop superfamily is one of the most challenging questions in molecular neuroscience
Crystal structures of the soluble acetylcholine-binding protein (AChBP) [5,6,7], which is homologous to the extracellular domain of the nAChR and, by extension, other members of the superfamily, provide a good sense of the layout of the agonist-binding site and its relationship to the rest of the receptor
It is important to appreciate from the outset that 2BG9, heuristically quite valuable, is not a crystal structure of the nAChR. It is a model built from low resolution data and homology modeling. It represents a substantial advance for the field, and all modern attempts to obtain molecular scale information on the structure and function of Cys loop receptors must consider this as a starting point
Summary
The gating mechanism for the Cys loop superfamily is one of the most challenging questions in molecular neuroscience. Crystal structures of the soluble acetylcholine-binding protein (AChBP) [5,6,7], which is homologous to the extracellular domain of the nAChR and, by extension, other members of the superfamily, provide a good sense of the layout of the agonist-binding site and its relationship to the rest of the receptor. It is a model built from low resolution data and homology modeling It represents a substantial advance for the field, and all modern attempts to obtain molecular scale information on the structure and function of Cys loop receptors must consider this as a starting point. Several groups have attempted to identify key interactions in the interface between the extracellular domain and the transmembrane domain, and we discuss some of these results below. We have been puzzled by the notion that in this closely related family of receptors, the mechanism of action of the essential function of the receptors seems to vary from system to system
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