Channel-Opening Kinetic Mechanism for Human Wild-Type GluK2 and the M867I Mutant Kainate Receptor

Abstract

GluK2 is a kainate receptor subunit that is alternatively spliced at the C-terminus. Previous studies implicated GluK2 in autism. In particular, the methionine-to-isoleucine replacement at amino acid residue 867 (M867I) that can only occur in the longest isoform of the human GluK2 (hGluK2), as the disease (autism) mutation, is thought to cause gain-of-function. However, the kinetic properties of the wild-type hGluK2 and the functional consequence of this gain-of-function mutation at the molecular level are not well understood. To investigate whether the M867I mutation affects the channel properties of the human GluK2 kainate receptor, we have systematically characterized the rate and the equilibrium constants pertinent to channel opening and channel desensitization for this mutant and the wild-type hGluK2 receptor, along with the wild-type rat GluK2 kainate receptor (rGluK2) as the control. Our results show that the M867I mutation does not affect either the rate or the equilibrium constants of the channel opening but does slow down the channel desensitization rate by ~1.6-fold at saturating glutamate concentrations. It is possible that a consequence of this mutation on the desensitization rate is linked to facilitating the receptor trafficking and membrane expression, given the close proximity of M867 to the forward trafficking motif in the C-terminal sequence. By comparing the data of the wild-type human and rat GluK2 receptors, we also find that the human GluK2 has a ~3-fold smaller channel-opening rate constant but an identical channel-closing rate constant, and thus a channel-opening probability of 0.85 vs. 0.96 for rGluK2. Furthermore, the intrinsic equilibrium dissociation constant K 1 for hGluK2, like the EC 50 value, is ~2-fold lower than rGluK2. Our results therefore suggest that the human GluK2 is relatively a slowly activating channel but more sensitive to glutamate, as compared to the rat ortholog, despite the fact that the human and rat forms share 99% sequence homology.GluK2 (previously known as GluR6) is a subunit of the glutamate ion channel receptor family of kainate subtype (1-3). Within this family, glutamate ion channel receptors are subdivided by their prototypical agonists into N-methyl-D-aspartate (NMDA) receptors, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptors, and kainate receptors (3). Within the kainate receptor subtype, there are five subunits, i.e., GluK1-5. GluK2, like GluK1 and 3, can form functional, homomeric channels, whereas GluK4-5 can only form † This work was supported in part by grants from the National Institutes of Health (R01 NS060812), Department of Defense (W81XWH-04-1-0106), and the Muscular Dystrophy Association. * To whom correspondence should be addressed. ; lniu@albany.edu. SUPPORTING INFORMATION AVAILABLEThe supporting information includes a figure showing Hill equation fit to the dose-response relationship, and a table showing the results of the nonlinear regression data for the best fit of n, K 1 and k op to the observed rate constan...

Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases

“…5; Table 4). Characterization of the M867I missense mutation revealed no detectable effect on GluK2 receptor gating (Han et al, 2010), but revealed a modest ∼1.6-fold slowing of the desensitization time course. Additional SNP association studies on various populations support a role of GRIK2 in autism (Shuang et al, 2004;Dutta et al, 2007;Kim et al, 2007;Holt et al, 2010;Casey et al, 2012;Griswold et al, 2012).…”

Channel-Opening Kinetic Mechanism of Wild-Type GluK1 Kainate Receptors and a C-Terminal Mutant

“…This conclusion is also consistent with results from our study of other kainate receptor channels. 17, 19, 21, 30 Furthermore, as is known for kainate receptors, channel activation via a subset of subunits instead of all four in a tetrameric complex can indeed occur. 31 Thus, we favor an interpretation of k op and k cl at n = 2 being the representative values of the channel-opening and channel-closing rate constants for the wild-type GluK1-2a, wild-type GluK1-2b and the mutant GluK1-2b receptors.…”

“…For a laser-pulse photolysis measurement of a channel-opening kinetic rate constant, we also used at least two known concentrations of free glutamate with the same cell before and after a laser flash to calibrate the concentration of photolytically released glutamate. 17, 18, 21 The current amplitudes obtained from flow measurements were compared with that of the laser measurement with reference to the dose-response relationship. The flow measurement also allowed us to monitor any potential damage to the receptors and/or the cell for successive laser flashes with the same cell.…”

“…To describe the relationship between k obs and agonist concentration, we introduced a general mechanism of channel opening. 17, 18, 21 $$\mathrm{A}+\mathrm{nL}\stackrel{{\mathrm{normalK}}_1}{\rightleftharpoons }{\mathrm{AL}}_{\mathrm{normaln}}\underset{{\mathrm{normalk}}_{\mathrm{cl}}}{\overset{{\mathrm{normalk}}_{\mathrm{op}}}{\rightleftharpoons }}{\overline{\mathrm{AL}}}_{\mathrm{normaln}}$$ Here, A stands for the active, unliganded form of the receptor, L the ligand or glutamate, AL n the closed-channel state with n ligand molecules bound, and $\overline{{\mathit{AL}}_n}$ the open-channel state. The number of glutamate molecules to bind to the receptor and to open its channel, n , can be from 1 to 4, assuming that a receptor is a tetrameric complex and each subunit has one glutamate binding site.…”

GABA and Glutamate Receptors of the Autistic Brain

“…Electrophysiological experiments have shown that Xenopus oocytes expressing the rat version of M867I-GluK2 elicit larger ion currents than oocytes expressing the wild type receptor, and the increment of current correlates with an enhanced density of the mutated receptors on the plasma membrane of the oocytes (Strutz-Seebohm et al, 2006). Except for a slower rate of desensitization, no major changes in the kinetic properties of the human or rat version of the M867I-GluK2 could account for the observed increment in the ion currents (Han et al, 2010); therefore, the potential link between the M867I and the autistic phenotype could be related mostly to alterations in the normal trafficking of GluK2 in and out of presynaptic and postsynaptic terminals. Interestingly, despite a 99% homology between rat and human GluK2, both receptors had important kinetic and potency differences, highlighting the importance of the validation of animal models with data obtained from the human brain (Halladay et al, 2009;Limon et al, 2011).…”