Receptor occupancy and channel-opening kinetics: a study of GLUR1 L497Y AMPA receptor

AMPA glutamate ion channels are tetrameric receptors in which activation to form the open channel depends on the binding of possibly multiple glutamate molecules. However, it is unclear whether AMPA receptors bound with a different number of glutamate molecules (i.e. one being the minimal and four being the maximal number of glutamate molecules) open the channels with different kinetic constants. Using a laser pulse photolysis technique that provides microsecond time resolution, we investigated the channel-opening kinetic mechanism of a nondesensitizing AMPA receptor, i.e. GluR1Q(flip) L497Y or a leucine-to-tyrosine substitution mutant, in the entire range of glutamate concentrations to ensure receptor saturation. We found that the minimal number of glutamate molecules required to bind to the receptor and to open the channel is two (or n = 2), and that the entire channel-opening kinetics can be adequately described by just one channel-opening rate constant, k(op), which correlates to n = 2. This result suggests that higher receptor occupancy (n = 3 and 4) does not give rise to different k(op) values or, at least, not appreciably if the k(op) values are different. Furthermore, compared with the wild-type receptor (Li, G., and Niu, L. (2004) J. Biol. Chem. 279, 3990-3997), the channel-opening and channel-closing rate constants of the mutant are 1.5- and 13-fold smaller, respectively. Thus, the major effect of this mutation is to decrease the channel-closing rate constant by stabilizing the open channel conformation.