RESUMO
N-Methyl-d-aspartate receptors (NMDARs) are ionotropic ligand-gated glutamate receptors that mediate fast excitatory synaptic transmission in the central nervous system (CNS). Several neurological disorders may involve NMDAR hypofunction, which has driven therapeutic interest in positive allosteric modulators (PAMs) of NMDAR function. Here we describe modest changes to the tetrahydroisoquinoline scaffold of GluN2C/GluN2D-selective PAMs that expands activity to include GluN2A- and GluN2B-containing recombinant and synaptic NMDARs. These new analogues are distinct from GluN2C/GluN2D-selective compounds like (+)-(3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ) by virtue of their subunit selectivity, molecular determinants of action, and allosteric regulation of agonist potency. The (S)-enantiomers of two analogues (EU1180-55, EU1180-154) showed activity at NMDARs containing all subunits (GluN2A, GluN2B, GluN2C, GluN2D), whereas the (R)-enantiomers were primarily active at GluN2C- and GluN2D-containing NMDARs. Determination of the actions of enantiomers on triheteromeric receptors confirms their unique pharmacology, with greater activity of (S) enantiomers at GluN2A/GluN2D and GluN2B/GluN2D subunit combinations than (R) enantiomers. Evaluation of the (S)-EU1180-55 and EU1180-154 response of chimeric kainate/NMDA receptors revealed structural determinants of action within the pore-forming region and associated linkers. Scanning mutagenesis identified structural determinants within the GluN1 pre-M1 and M1 regions that alter the activity of (S)-EU1180-55 but not (R)-EU1180-55. By contrast, mutations in pre-M1 and M1 regions of GluN2D perturb the actions of only the (R)-EU1180-55 but not the (S) enantiomer. Molecular modeling supports the idea that the (S) and (R) enantiomers interact distinctly with GluN1 and GluN2 pre-M1 regions, suggesting that two distinct sites exist for these NMDAR PAMs, each of which has different functional effects.
Assuntos
Receptores de N-Metil-D-Aspartato , Transmissão Sináptica , Regulação Alostérica , Modelos Moleculares , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
AMPA and NMDA receptors are ligand-gated ion channels that depolarize postsynaptic neurons when activated by the neurotransmitter L-glutamate. Changes in the distribution and activity of these receptors underlie learning and memory, but excessive change is associated with an array of neurological disorders, including cognitive impairment, developmental delay, and epilepsy. All of the ionotropic glutamate receptors (iGluRs) exhibit similar tetrameric architecture, transmembrane topology, and basic framework for activation; conformational changes induced by extracellular agonist binding deform and splay open the inner helix bundle crossing that occludes ion flux through the channel. NMDA receptors require agonist binding to all four subunits, whereas AMPA and closely related kainate receptors can open with less than complete occupancy. In addition to conventional activation by agonist binding, we recently identified two locations along the inner helix of the GluK2 kainate receptor subunit where cysteine (Cys) substitution yields channels that are opened by exposure to cadmium ions, independent of agonist site occupancy. Here, we generate AMPA and NMDA receptor subunits with homologous Cys substitutions and demonstrate similar activation of the mutant receptors by Cd. Coexpression of the auxiliary subunit stargazin enhanced Cd potency for activation of Cys-substituted GluA1 and altered occlusion upon treatment with sulfhydryl-reactive MTS reagents. Mutant NMDA receptors displayed voltage-dependent Mg block of currents activated by agonist and/or Cd as well as asymmetry between Cd effects on Cys-substituted GluN1 versus GluN2 subunits. In addition, Cd activation of each Cys-substituted iGluR was inhibited by protons. These results, together with our earlier work on GluK2, reveal a novel mechanism shared among the three different iGluR subtypes for prying open the gate that controls ion entry into the pore.
Assuntos
Cádmio/farmacologia , Cisteína , Receptores de AMPA/fisiologia , Receptores de Ácido Caínico/fisiologia , Receptores de N-Metil-D-Aspartato , Ácido Glutâmico , Receptores de N-Metil-D-Aspartato/fisiologiaRESUMO
Kainate receptors are ligand-gated ion channels that have two major roles in the central nervous system: they mediate a postsynaptic component of excitatory neurotransmission at some glutamatergic synapses and modulate transmitter release at both excitatory and inhibitory synapses. Accumulating evidence implicates kainate receptors in a variety of neuropathologies, including epilepsy, psychiatric disorders, developmental delay, and cognitive impairment. Here, to gain a deeper understanding of the conformational changes associated with agonist binding and channel opening, we generate a series of Cys substitutions in the GluK2 kainate receptor subunit, focusing on the M3 helices that line the ion pore and form the bundle-crossing gate at the extracellular mouth of the channel. Exposure to 50 µM Cd produces direct activation of homomeric mutant channels bearing Cys substitutions in (A657C), or adjacent to (L659C), the conserved SYTANLAAF motif. Activation by Cd is occluded by modification with 2-aminoethyl MTS (MTSEA), indicating that Cd binds directly and specifically to the substituted cysteines. Cd potency for the A657C mutation (EC50 = 10 µM) suggests that binding involves at least two coordinating residues, whereas weaker Cd potency for L659C (EC50 = 2 mM) implies that activation does not require tight coordination by multiple side chains for this substitution. Experiments with heteromeric and chimeric channels indicate that activation by Cd requires Cys substitution at only two of the four subunits within a tetrameric receptor and that activation is similar for substitution within subunits in either the A/C or B/D conformations. We develop simple kinetic models for the A657C substitution that reproduce several features of Cd activation as well as the low-affinity inhibition observed at higher Cd concentrations (5-20 mM). Together, these results demonstrate rapid and reversible channel activation, independent of agonist site occupancy, upon Cd binding to Cys side chains at two specific locations along the GluK2 inner helix.
Assuntos
Cádmio/farmacologia , Cisteína/química , Receptores de Ácido Caínico/metabolismo , Substituição de Aminoácidos , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Subunidades Proteicas , Receptores de Ácido Caínico/genética , Receptor de GluK2 CainatoRESUMO
Positive modulators of NMDA receptors are important candidates for therapeutic development to treat psychiatric disorders including autism and schizophrenia. Sulfated neurosteroids have been studied as positive allosteric modulators of NMDA receptors for years, but we understand little about the cellular fate of these compounds, an important consideration for drug development. Here we focus on a visualizable sulfated neurosteroid analogue, KK-169. As expected of a pregnenolone sulfate analogue, the compound strongly potentiates NMDA receptor function, is an antagonist of GABAA receptors, exhibits occlusion with pregnenolone sulfate potentiation, and requires receptor domains important for pregnenolone sulfate potentiation. KK-169 exhibits somewhat higher potency than the natural parent, pregnenolone sulfate. The analogue contains a side-chain alkyne group, which we exploited for retrospective click labeling of neurons. Although the anionic sulfate group is expected to hinder cell entry, we detected significant accumulation of KK-169 in neurons with even brief incubations. Adding a photolabile diazirine group revealed that the expected plasma membrane localization of KK-169 is likely lost during fixation. Overall, our studies reveal new facets of the structure-activity relationship of neurosteroids at NMDA receptors, and their intracellular distribution suggests that sulfated neurosteroids could have unappreciated targets in addition to plasma membrane receptors.
Assuntos
Membrana Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Regulação Alostérica , Animais , Membrana Celular/metabolismo , Células Cultivadas , Química Click , Citoplasma/metabolismo , Agonistas de Aminoácidos Excitatórios/química , Ácido Glutâmico/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Camundongos , Oócitos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Relação Estrutura-Atividade , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Xenopus laevisRESUMO
UNLABELLED: NMDA receptors are ligand-gated ion channels that underlie transmission at excitatory synapses and play an important role in regulating synaptic strength and stability. Functional NMDA receptors require two copies of the GluN1 subunit coassembled with GluN2 (and/or GluN3) subunits into a heteromeric tetramer. A diverse array of allosteric modulators can upregulate or downregulate NMDA receptor activity. These modulators include both synthetic compounds and endogenous modulators, such as cis-unsaturated fatty acids, 24(S)-hydroxycholesterol, and various neurosteroids. To evaluate the structural requirements for the formation and allosteric modulation of NMDA receptor pores, we have replaced portions of the rat GluN1, GluN2A, and GluN2B subunits with homologous segments from the rat GluK2 kainate receptor subunit. Our results with these chimeric constructs show that the NMDA receptor transmembrane domain is sufficient to account for most pore properties, but that regulation by some allosteric modulators requires additional cytoplasmic or extracellular domains. SIGNIFICANCE STATEMENT: Glutamate receptors mediate excitatory synaptic transmission by forming cation channels through the membrane that open upon glutamate binding. Although many compounds have been identified that regulate glutamate receptor activity, in most cases the detailed mechanisms that underlie modulation are poorly understood. To identify what parts of the receptor are essential for pore formation and sensitivity to allosteric modulators, we generated chimeric subunits that combined segments from NMDA and kainate receptors, subtypes with distinct pharmacological profiles. Surprisingly, our results identify separate domain requirements for allosteric potentiation of NMDA receptor pores by pregnenolone sulfate, 24(S)-hydroxycholesterol, and docosahexaenoic acid, three endogenous modulators derived from membrane constituents. Understanding where and how these compounds act on NMDA receptors should aid in designing better therapeutic agents.
Assuntos
Domínios Proteicos/fisiologia , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Ácido Araquidônico/farmacologia , Cálcio/metabolismo , Dimerização , Ácidos Docosa-Hexaenoicos/farmacologia , Relação Dose-Resposta a Droga , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Hidroxicolesteróis/farmacologia , Ácido Caínico/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Modelos Moleculares , N-Metilaspartato/farmacologia , Técnicas de Patch-Clamp , Pregnenolona , Domínios Proteicos/genética , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/genética , Ratos , Receptores de N-Metil-D-Aspartato/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Ionotropic glutamate receptors comprise two conformationally different A/C and B/D subunit pairs. Closed channels exhibit fourfold radial symmetry in the transmembrane domain (TMD) but transition to twofold dimer-of-dimers symmetry for extracellular ligand binding and N-terminal domains. Here, to evaluate symmetry in open pores we analysed interaction between the Q/R editing site near the pore loop apex and the transmembrane M3 helix of kainate receptor subunit GluK2. Chimeric subunits that combined the GluK2 TMD with extracellular segments from NMDA receptors, which are obligate heteromers, yielded channels made up of A/C and B/D subunit pairs with distinct substitutions along M3 and/or Q/R site editing status, in an otherwise identical homotetrameric TMD. Our results indicate that Q/R site interaction with M3 occurs within individual subunits and is essentially the same for both A/C and B/D subunit conformations, suggesting that fourfold pore symmetry persists in the open state.
Assuntos
DNA Complementar/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Sequência de Aminoácidos , Ácidos Docosa-Hexaenoicos/metabolismo , Eletrofisiologia , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptores Ionotrópicos de Glutamato/química , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
RNA editing at the Q/R site near the apex of the pore loop of AMPA and kainate receptors controls a diverse array of channel properties, including ion selectivity and unitary conductance and susceptibility to inhibition by polyamines and cis-unsaturated fatty acids, as well as subunit assembly into tetramers and regulation by auxiliary subunits. How these different aspects of channel function are all determined by a single amino acid substitution remains poorly understood; however, several lines of evidence suggest that interaction between the pore helix (M2) and adjacent segments of the transmembrane inner (M3) and outer (M1) helices may be involved. In the present study, we have used double mutant cycle analysis to test for energetic coupling between the Q/R site residue and amino acid side chains along the M3 helix. Our results demonstrate interaction with several M3 locations and particularly strong coupling to substitution for L614 at the level of the central cavity. In this location, replacement with smaller side chains completely and selectively reverses the effect of fatty acids on gating of edited channels, converting strong inhibition of wild-type GluK2(R) to nearly 10-fold potentiation of GluK2(R) L614A.
Assuntos
Mutação , Receptores de Ácido Caínico/metabolismo , Termodinâmica , Potenciais de Ação , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Ácidos Graxos/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/genética , Receptor de GluK2 CainatoRESUMO
RNA editing of kainate receptor subunits at the Q/R site determines their susceptibility to inhibition by cis-unsaturated fatty acids as well as block by cytoplasmic polyamines. Channels comprised of unedited (Q) subunits are strongly blocked by polyamines, but insensitive to fatty acids, such as arachidonic acid (AA) and docosahexaenoic acid (DHA), whereas homomeric edited (R) channels resist polyamine block but are inhibited by AA and DHA. In the present study, we have analyzed fatty acid modulation of whole-cell currents mediated by homomeric recombinant GluK2 (formerly GluR6) channels with individual residues in the pore-loop, M1 and M3 transmembrane helices replaced by scanning mutagenesis. Our results define three abutting surfaces along the M1, M2, and M3 helices where gain-of-function substitutions render GluK2(Q) channels susceptible to fatty acid inhibition. In addition, we identify four locations in the M3 helix (F611, L614, S618, and T621) at the level of the central cavity where Arg substitution increases relative permeability to chloride and eliminates polyamine block. Remarkably, for two of these positions, L614R and S618R, exposure to fatty acids reduces the apparent chloride permeability and potentiates whole-cell currents approximately 5 and 2.5-fold, respectively. Together, our results suggest that AA and DHA alter the orientation of M3 in the open state, depending on contacts at the interface between M1, M2, and M3. Moreover, our results demonstrate the importance of side chains within the central cavity in determining ionic selectivity and block by cytoplasmic polyamines despite the inverted orientation of GluK2 as compared with potassium channels and other pore-loop family members.
Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Ativação do Canal Iônico , Mutagênese Sítio-Dirigida , Receptores de Ácido Caínico/metabolismo , Espermina/metabolismo , Alanina , Sequência de Aminoácidos , Arginina , Linhagem Celular , Cloretos/metabolismo , Humanos , Potenciais da Membrana , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Técnicas de Patch-Clamp , Permeabilidade , Conformação Proteica , Receptores de Ácido Caínico/antagonistas & inibidores , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/genética , Relação Estrutura-Atividade , Transfecção , Triptofano , Receptor de GluK2 CainatoRESUMO
Neuronal proteins of the BTB/kelch and PDZ domain families interact with different regions of the cytoplasmic C-terminal domain of the GluR6 kainate receptor subunit. The BTB/kelch protein KRIP6 binds within a 58 amino acid segment of GluR6 proximal to the plasma membrane. In contrast, PDZ domain proteins, such as PICK1 and PSD95, interact with the last 4 residues of the GluR6 C-terminus. KRIP6 reduces peak currents mediated by recombinant GluR6 receptors and by native kainate receptors in neurons, whereas PICK1 stabilizes kainate receptors at synapses. Thus, protein-protein interactions at the C-terminal domain of GluR6 are important for regulating kainate receptor physiology. Here, we show by co-clustering and co-immunoprecipitation that KRIP6 interacts with PICK1 in heterologous cells. In addition, we demonstrate a novel modulation of GluR6 receptors by PICK1 resulting in increased peak current and relative desensitization of GluR6-mediated currents, phenotypes opposite to those produced by KRIP6. Importantly, these effects cancel out when KRIP6 and PICK1 are co-expressed together with GluR6. KRIP6 and PICK1 strongly co-cluster and co-immunoprecipitate regardless of the presence of GluR6. Immunofluorescence analysis reveals that GluR6 can either join the KRIP6-PICK1 clusters or remain separate; however, co-expression of KRIP6 reduces the fraction of PICK1 that co-immunoprecipitates with GluR6. Taken together, these results indicate that, in addition to a previously demonstrated direct interaction with the GluR6 C-terminal domain, KRIP6 regulates kainate receptors by inhibiting PICK1 modulation via competition or a mutual blocking effect.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas do Citoesqueleto/fisiologia , Proteínas Nucleares/fisiologia , Receptores de Ácido Caínico/fisiologia , Animais , Western Blotting , Células COS , Linhagem Celular , Chlorocebus aethiops , Eletrofisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Imunoprecipitação , Ácido Caínico/farmacologia , Domínios PDZ/fisiologia , Técnicas de Patch-Clamp , Receptor de GluK2 CainatoRESUMO
RNA editing at the Q/R site in the GluR5 and GluR6 subunits of neuronal kainate receptors regulates channel inhibition by lipid-derived modulators including the cis-unsaturated fatty acids arachidonic acid and docosahexaenoic acid. Kainate receptor channels in which all of the subunits are in the edited (R) form exhibit strong inhibition by these compounds, whereas wild-type receptors that include a glutamine (Q) at the Q/R site in one or more subunits are resistant to inhibition. In the present study, we have performed an arginine scan of residues in the pore loop of the GluR6(Q) subunit. Amino acids within the range from -19 to +7 of the Q/R site of GluR6(Q) were individually mutated to arginine and the mutant cDNAs were expressed as homomeric channels in HEK 293 cells. All but one of the single arginine substitution mutants yielded functional channels. Only weak inhibition, typical of wild-type GluR6(Q) channels, was observed for substitutions +1 to +6 downstream of the Q/R site. However, arginine substitution at several locations upstream of the Q/R site resulted in homomeric channels exhibiting strong inhibition by fatty acids, which is characteristic of homomeric GluR6(R) channels. Based on homology with the pore loop of potassium channels, locations at which R substitution induces susceptibility to fatty acid inhibition face away from the cytoplasm toward the M1 and M3 helices and surrounding lipids.
Assuntos
Substituição de Aminoácidos , Ácidos Graxos/fisiologia , Ativação do Canal Iônico/fisiologia , Receptores de Ácido Caínico/fisiologia , Sequência de Aminoácidos , Aminoácidos/genética , Animais , Arginina/genética , Linhagem Celular , Membrana Celular/metabolismo , Concanavalina A/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Eletrofisiologia , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ácido Caínico/farmacologia , Potenciais da Membrana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Edição de RNA , Ratos , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/genética , Receptor de GluK2 CainatoRESUMO
Whereas many interacting proteins have been identified for AMPA and NMDA glutamate receptors, fewer are known to directly bind and regulate function of kainate receptors. Using a yeast two-hybrid screen for interacting partners of the C-terminal domain of GluR6a, we identified a novel neuronal protein of the BTB/kelch family, KRIP6. KRIP6 binds to the GluR6a C-terminal domain at a site distinct from the PDZ-binding motif and it co-immunoprecipitates with recombinant and endogenous GluR6. Co-expression of KRIP6 alters GluR6 mediated currents in a heterologous expression system reducing peak current amplitude and steady-state desensitization, without affecting surface levels of GluR6. Endogenous KRIP6 is widely expressed in brain and overexpression of KRIP6 reduces endogenous kainate receptor-mediated responses evoked in hippocampal neurons. Taken together, these results suggest that KRIP6 can directly regulate native kainate receptors and provide the first evidence for a BTB/kelch protein in direct functional regulation of a mammalian glutamate receptor.
Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de Ácido Caínico/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Potenciais Pós-Sinápticos Excitadores , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Ratos , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Receptor de GluK2 CainatoRESUMO
RNA editing within the pore loop controls the pharmacology and permeation properties of ion channels formed by neuronal AMPA and kainate receptor subunits. Genomic sequences for the glutamate receptor 2 (GluR2) subunit of AMPA receptors and the GluR5 and GluR6 subunits of kainate receptors all encode a neutral glutamine (Q) residue within the channel pore that can be converted by RNA editing to a positively charged arginine (R). Receptors comprised of unedited subunits are permeable to calcium and display inwardly rectifying current-voltage relationships, because of blocking of outward current by intracellular polyamines. In contrast, receptors that include edited subunits conduct less calcium, resist polyamine block, and have relatively linear current-voltage relationships. We showed previously that cis-unsaturated fatty acids, including arachidonic acid and docosahexanoic acid, exert a potent block of native kainate receptors as well as homomeric recombinant receptors formed by transfection of heterologous cells with cDNA for the GluR6(R) subunit. Here, we show that fatty acid blockade of recombinant homomeric and heteromeric kainate receptors is strongly dependent on editing at the Q/R site. Recombinant channels that include unedited subunits exhibit significantly weaker block than channels made up of fully edited subunits. Inhibition of fully edited channels is equivalent at voltages from -70 to +40 mV and is noncompetitive, consistent with allosteric regulation of channel function.
Assuntos
Arginina , Membrana Celular/metabolismo , Ácidos Graxos/farmacologia , Ácidos Graxos/fisiologia , Glutamina , Edição de RNA , Receptores de Ácido Caínico/antagonistas & inibidores , Receptores de Ácido Caínico/metabolismo , Animais , Arginina/genética , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glutamina/genética , Humanos , Ácido Caínico/farmacologia , Camundongos , Edição de RNA/genética , Receptores de Ácido Caínico/agonistas , Receptores de Ácido Caínico/genéticaRESUMO
Presynaptic kainate (KA) receptors regulate synaptic transmission at both excitatory and inhibitory synapses in the spinal cord dorsal horn. Previous work has demonstrated pharmacological differences between the KA receptors expressed by rat dorsal horn neurons and those expressed by the primary afferent sensory neurons that innervate the dorsal horn. Here, neurons isolated from KA receptor subunit-deficient mice were used to evaluate the contribution of glutamate receptor subunit 5 (GluR5) and GluR6 to the presynaptic control of transmitter release and to KA receptor-mediated whole-cell currents in these two cell populations [corrected]. Deletion of GluR6 produced a significant reduction in KA receptor-mediated current density in dorsal horn neurons, whereas GluR5 deletion caused no change in current density but removed sensitivity to GluR5-selective antagonists. Presynaptic modulation of inhibitory transmission between dorsal horn neurons was preserved in cells from either GluR5- or GluR6-deficient mice. In DRG neurons, in contrast, GluR5 deletion abolished KA receptor function, whereas deletion of GluR6 had little effect on peak current density but increased the rate and extent of desensitization. These results highlight fundamental differences in KA receptor physiology between the two cell types and suggest possible strategies for the pharmacological modulation of nociception.