Coupled proteolytic and mass spectrometry studies indicate a novel topology for the glycine receptor

Members of the heteropentameric ligand-gated ion channel superfamily rapidl y mediate signaling across the synaptic cleft. Sequence analysis and limite d experimental studies have yielded a topological model containing four tra nsmembrane alpha-helices, labeled M1 to M4, and a large soluble, extracellu lar N-terminal domain. This model persists to date despite some recent stru ctural studies that suggest it may be inappropriate. In this study, the top ology of the glycine receptor was probed by limited proteolysis coupled to mass spectrometry, Of particular note, accessible cleavage sites within the putative M1 and M3 transmembrane helices were identified. Membrane-associa ted fragments within the postulated globular extracellular N-terminal domai n were also observed. This report presents several key details incorporated in a new topological model and is the first direct experimental evidence t hat a subset of the transmembrane regions are too short to be membrane-span ning alpha-helices; rather, these regions are proposed to be a mix of alpha -helices and beta-sheets, This report is also the first to exploit the capa bility of mass spectrometry to probe critically the topology of a class of membrane proteins of unknown structure.