A functional study of AMPA and kainate receptors

The ionotropic glutamate receptors are localized in the pre- and postsynaptic membrane of neurons and mediate the majority of fast excitatory synaptic transmission. These receptors are divided into three subtypes: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainic acid, and N-methyl-D-aspartic acid (NMDA) receptors. My thesis work focused on AMPA and kainate receptor subtypes.;Native AMPA receptors are predominantly heteromeric channels, and most of them contain the edited isoform of GluA2 AMPA receptor subunit or GluA2R. The Q/R editing occurs uniquely in the GluA2 subunit, and the Q isoform can form homomeric channels by itself, whereas the R isoform cannot. In Chapter 1 I characterize how these GluA2R-containing heteromeric complex channels work, using a laser-pulse photolysis technique with ∼60 microsecond time resolution. The hypothesis to be tested is that GluA2R plays a key role in affecting the gating properties of the GluA2R-containing channels. Specifically, I choose to study GluA2Q/2R and GluA1/2R, and two other L→Y mutant channels, i.e., the GluA1L497Y/GluA2R and the GluA1/GluA2RL504Y mutants. My results show that (a) in all four heteromeric channels, the insertion of GluA2R in the heteromeric channel assembly reduces the channel-opening and channel-closing rate constants; (b) binding of two glutamate molecules is sufficient to open an complex channel; and (c) the glutamate molecule most likely binds to the R isoform with an EC50 value of ∼0.6 mM. Most interestingly, I found that the same mutation, i.e., the L→Y mutation, which reduces the EC50 value of a homomeric channel (i.e., the GluA2Q or GluA1 homomeric channels), has a greater impact on the GluA2R than other participating subunits in a tetrameric assembly. Thus, my results suggest that a fractional occupancy of glutamate binding sites in a tetramer is sufficient to change the channel activity. Specifically, it is the binding of glutamate to the two GluA2R subunits in a tetramer that plays the key role in shaping the kinetic properties of the GluA2R-containing AMPA receptor complex channels.;As a kainate receptor subunit, GluK2 from the rat origin is one of the most studied glutamate receptors to date. In Chapter 2, I investigated whether the human ortholog of the rat GluK2 is the same in terms of its channel-opening kinetic properties, and how a particular mutation affects the channel-opening properties. The mutation in this case is a methionine-to-isoleucine replacement at amino acid residue 867 (M867I), and it is presumably linked to autism by exerting a gain-of-function. Using the laser-pulse photolysis technique, I investigated the channel-opening kinetic properties of the wild-type and mutant human GluK2, using rat GluK2 as the control. My results show that the M867I mutation does not affect either the rate or the equilibrium constants of the channel opening but does reduce the maximum channel desensitization rate by ~1.6-fold. These results rule out the possibility that the gain-of-function is due to modification of the channel-opening kinetic properties. Rather, the mutation may promote the receptor trafficking and membrane expression. Furthermore, I find that the human GluK2 is a relatively slow activating channel but is more sensitive to glutamate, as compared to the rat ortholog, despite the fact that the human and rat forms share 99% sequence homology.;GluK1 is another kainate receptor subunit and can form homomeric channels by itself. However, GluK1 is a less understood kainate receptor. In Chapter 3, I characterized the channel-opening kinetic mechanism for a surface-expression enhanced mutant of GluK1-2b (i.e., R896A, R897A, R900A, K901A) whose endoplasmic reticulum (ER) retention signal is disrupted and the wild-type GluK1-2a that lacks the ER-retention signal located in the C-terminal domain. Thus, both receptors are expected to traffic well to the surface, thereby generating a larger whole-cell current than the wild-type GluK1-2b. My results demonstrate that both channels show identical channel kinetic properties, including channel desensitization rate, dose-response relationships, channel-opening and channel-closing rate constants. Therefore, these data suggest that the ER retention signal sequence that regulates the receptor trafficking and surface expression does not affect the channel-gating properties. My results also provide much needed understanding of this kainate receptor channels.