期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 116, 期 20, 页码 10150-10155出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1900261116
关键词
G protein-coupled receptors; kinetics; confocal patch-clamp fluorometry; metabotropic glutamate receptors; photouncaging
资金
- Deutsche Forschungsgemeinschaft [TR166]
- Bundesministerium fur Bildung und Forschung Grant OptiMAR
G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the light receptor rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within similar to 1 ms, while all other receptors are thought to become activated within similar to 50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of tau(1) similar to 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (tau(2) similar to 20 ms). Receptor deactivation occurred with time constants of similar to 40 and similar to 900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states.
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