Receptor Occupancy and Channel-opening Kinetics

Pei, Weimin; Ritz, Mark; McCarthy, Michael; Huang, Zhen; Niu, Li

doi:10.1074/jbc.m611821200

Cited by 16 publications

(36 citation statements)

References 34 publications

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“…We favor, therefore, an interpretation of n = 2 as the minimal number of glutamate molecules bound to a receptor to open the channel. This conclusion was also consistent with the results from a study of an AMPA receptor mutant where k obs was collected up to saturation (34), and from studies of both AMPA and kainate receptor channels (17, 19, 22). Furthermore, as is known in kainate receptors, channel activation by activating only a subset of subunits, instead of all four, in a tetrameric complex can occur (35).…”

Section: Resultssupporting

confidence: 89%

“…To achieve a better estimate of n, K 1 and k op by nonlinear regression using eq 2, we decided to first fix the value of k cl so that we reduced one variable. This was possible because when L ≪ K 1 , eq 2 was reduced to k obs ≈ k cl , suggesting that (a) k obs at a low glutamate concentration would reflect k cl , and (b) the value of k cl was independent of whatever the n value might be as in eq 2 (22, 34). Based on this rationale, we identified k obs of 310 s −1 as k cl for the wild-type and mutant hGluK2 receptors.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2010

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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...

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

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

et al. 2010

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“…5 in Sun et al, 2002). The former was confirmed by determining the rate of channel closure for GluR1 L497Y (Pei et al, 2007), whereas kinetic modeling of the same mutant suggested that both entry into the desensitized state and the rate of ligand dissociation were decreased relative to GluR1 WT (Mitchell and Fleck, 2007). Although this modeling study did not incorporate slower channel closure, it seems probable that a combination of these three factors blocks desensitization and slows deactivation in the AMPA receptor leucine to tyrosine mutants.…”

Section: Discussionmentioning

confidence: 76%

A Nondesensitizing Kainate Receptor Point Mutant

et al. 2009

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Ionotropic glutamate receptor (iGluR) desensitization can be modulated by mutations that change the stability of a dimer formed by the agonist binding domain. Desensitization of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors can be blocked by a single point mutation (e.g., GluR2 L483Y) that stabilizes this dimer in an active conformation. In contrast, desensitization of kainate receptors can be slowed, but not blocked, by similar dimer interface mutations. Only covalent cross-linking via introduced disulfides has been previously shown to block kainate receptor desensitization completely. We have now identified an apparently nondesensitizing GluR6 point mutant (D776K) located at the apex of the ligand binding (S1S2) domain dimer interface. Asp776 is one of a cluster of four charged residues in this region that together mediate direct dimer interactions and contribute to the binding sites for one chloride and two sodium ions. Despite the localized ϩ4 change in the net charge of the S1S2 dimer, the D776K mutation actually increased the thermodynamic stability of the dimer. Unlike GluR6 wild type, the D776K mutant is insensitive to external cations but retains sensitivity to external anions. We therefore hypothesize that the unexpected phenotype of this charge reversal mutation results from the substitution of the sodium ions bound within the dimer interface by the introduced lysine NH 3 ϩ groups. The nondesensitizing D776K mutant provides insights into kainate receptor gating and represents a potentially useful new tool for dissecting kainate receptor function.

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“…I max is the maximum current amplitude, and I t is the current amplitude at time t . Our previous studies of AMPA receptors, including a mutant AMPA receptor, for their channel-opening kinetic mechanisms led us to conclude that binding of two glutamate molecules per receptor (i.e., n = 2) was sufficient to open the channel (38). Using the laser-pulse photolysis technique, we previously determined the k op of (8.0 ± 0.49) × 10 4 s −1 and the k cl of (2.6 ± 0.20) × 10 3 s −1 , respectively, for the channel-opening kinetic constants of the GluA2Q flip receptor (21).…”

Section: Homologous Competitive Binding Assaymentioning

confidence: 99%

Potent and Selective Inhibition of the Open-Channel Conformation of AMPA Receptors by an RNA Aptamer

et al. 2010

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Inhibitors of AMPA receptors are useful as biochemical probes for structure-function studies and as drug candidates for a number of neurological disorders and diseases. Here we report the identification of an RNA inhibitor or aptamer by an in vitro evolution approach. Using a laserpulse photolysis technique, we further characterized the mechanism of inhibition of this aptamer on the AMPA receptor channel-opening rate process in the microsecond-to-millisecond time domain. Our results show that the aptamer we isolated is a noncompetitive inhibitor that selectively inhibits the open-channel conformation of AMPA receptors with nanomolar affinity. The potency and the selectivity of this noncompetitive aptamer rival those of small molecule inhibitors. Our results therefore demonstrate the utility of this approach to develop water-soluble, highly potent and conformation-selective noncompetitive inhibitors of AMPA receptors.A protein is generally dynamic and adapts a specific conformation for function (1-2). Using molecular agents that bind selectively to a specific protein conformation among its conformational repertoire is thus a powerful means to exert a tighter molecular recognition to more effectively regulate the function of that protein, and to even engineer a new protein function. For instance, small chemical compounds have been found to stabilize a conformation for some apoptotic procaspases to induce autoproteolytic activation of these proenzymes (3). Catalytic antibodies have been created, based on transition-state structural analogs, to accelerate chemical reactions by stabilizing their rate-determining transition states along reaction pathways (4).Here we describe the discovery of RNA inhibitors or aptamers that selectively target the open-channel conformation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)1-subtype glutamate ion channel receptors. The open-channel conformation exists in the time span of microsecond to a few milliseconds (5), after the receptors bind to glutamate, the endogenous neurotransmitter; even in the millisecond time scale, the glutamate-bound receptors turn into the desensitized, channel-closed receptor form (6). Only through the transient, open-channel conformation of the postsynaptic receptors is a nerve impulse transmitted in the central nervous system. AMPA receptors are indispensible for † This work was funded in part by grants from Department of Defense (W81XWH-04-1-0106) (to L.N.), National Institutes of Health (R01 NS060812) (to L.N.), and Muscular Dystrophy Association (to L.N.) and a postdoctoral fellowship from Muscular Dystrophy Association (to Z.H.). * To whom correspondence should be addressed. lniu@albany.edu. .. SUPPORTING INFORMATION AVAILABLEThe supporting information includes a schematic diagram showing SELEX, additional data showing that AG1407 in full length inhibited GluA2Q flip receptor more strongly as glutamate concentration increased, and additional data showing that although weak, AG56 inhibited the open-channel conformation of Glu...

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Receptor Occupancy and Channel-opening Kinetics

Cited by 16 publications

References 34 publications

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

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

A Nondesensitizing Kainate Receptor Point Mutant

Potent and Selective Inhibition of the Open-Channel Conformation of AMPA Receptors by an RNA Aptamer

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