Glutamate, the major excitatory neurotransmitter in the central nervous system, activates three different receptors that directly gate ion channels, namely receptors for AMPA (alpha-amino-3-hydroxy-5-methyl isoxozole propionic acid), NMDA (N-methyl-D-aspartate), and kainate, a structural analogue of glutamate. The contribution of AMPA and NMDA receptors to synaptic transmission and plasticity is well established. Recent work on the physiological function of kainate receptors has focused on the hippocampus, where repetitive activation of the mossy-fibre pathway generates a slow, kainate-receptor-mediated excitatory postsynaptic current (EPSC). Here we show that high-intensity single-shock stimulation (of duration 200 microseconds) of primary afferent sensory fibres produces a fast, kainate-receptor-mediated EPSC in the superficial dorsal horn of the spinal cord. Activation of low-threshold afferent fibres generates typical AMPA-receptor-mediated EPSCs only, indicating that kainate receptors may be restricted to synapses formed by high-threshold nociceptive (pain-sensing) and thermoreceptive primary afferent fibres. Consistent with this possibility, kainate-receptor-mediated EPSCs are blocked by the analgesic mu-opiate-receptor agonist Damgo and spinal blockade of both kainate and AMPA receptors produces antinociception. Thus, spinal kainate receptors contribute to transmission of somatosensory inputs from the periphery to the brain.
Small diameter dorsal root ganglion (DRG) neurons, which include cells that transmit nociceptive information into the spinal cord, are known to express functional kainate receptors. It is well established that exposure to kainate will depolarize C-fiber afferents arising from these cells. Although the role of kainate receptors on sensory afferents is unknown, it has been hypothesized that presynaptic kainate receptors may regulate glutamate release in the spinal cord. Here we show that kainate, applied at low micromolar concentrations in the presence of the AMPA-selective antagonist (RS)-4-(4-aminophenyl)-1,2-dihydro-1-methyl-2-propyl-carbamoyl-6,7-methylenedioxyphthalazine, suppressed spontaneous NMDA receptor-mediated EPSCs in cultures of spinal dorsal horn neurons. In addition, kainate suppressed EPSCs in dorsal horn neurons evoked by stimulation of synaptically coupled DRG cells in DRG-dorsal horn neuron cocultures. Interestingly, although the glutamate receptor subunit 5-selective kainate receptor agonist (RS)-2-␣-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) (2 M) was able to suppress DRG-dorsal horn synaptic transmission to a similar extent as kainate (10 M), it had no effect on excitatory transmission between dorsal horn neurons. Agonist applications revealed a striking difference between kainate receptors expressed by DRG and dorsal horn neurons. Whereas DRG cell kainate receptors were sensitive to both kainate and ATPA, most dorsal horn neurons responded only to kainate. Finally, in recordings from dorsal horn neurons in spinal slices, kainate and ATPA were able to suppress NMDA and AMPA receptormediated EPSCs evoked by dorsal root fiber stimulation. Together, these data suggest that kainate receptor agonists, acting at a presynaptic locus, can reduce glutamate release from primary afferent sensory synapses. Key words: kainate, presynaptic, ATPA, glutamate receptor subunit 5, glutamate, autoreceptor, excitatory synaptic transmission, NMDAGlutamate is the major excitatory transmitter at primary afferent synapses, at which it conveys sensory information to the CNS via postsynaptic AMPA, NMDA, and kainate (KA) receptors on spinal cord dorsal horn neurons (Yoshimura and Jessell, 1990;Li et al., 1999). In addition to postsynaptic receptors, many neurons express on their presynaptic terminals ionotropic receptors that are thought to regulate transmitter release , including receptors for heterologous transmitters as well as autoreceptors for the transmitter(s) released by the terminal itself.Much recent effort has focused on kainate receptors as possible presynaptic regulators of transmission. In the hippocampus, for example, presynaptic kainate receptor activation appears to reduce release of both glutamate (Chittajallu et al., 1996;Kamiya and Ozawa, 1998) and GABA (Clarke et al., 1997; RodriguezMoreno et al., 1997) (but see Frerking et al., 1999). At primary afferent synapses in the spinal cord, in addition to the postsynaptic kainate receptors that contribute to EPSCs evoked by highth...
We have used whole-cell recordings and rapid agonist applications to characterize the physiological properties of kainate receptors expressed by rat hippocampal neurons in dissociated cell culture. Activation of NMDA and AMPA receptors was prevented by inclusion of the noncompetitive antagonists MK-801 (2 M) and GYKI 53655 (100 M), respectively. In the presence of these inhibitors, both kainate (EC 50 ϭ 23 M) and glutamate (EC 50 ϭ 310 M) evoked desensitizing currents. Maximal peak currents for kainate with GYKI 53655 were 15 Ϯ 3% as large as in control solutions without GYKI. In contrast to currents mediated by AMPA receptors, kainate currents recorded in GYKI were blocked potently by lanthanum (IC 50 ϭ 2 M) and were desensitized by 1 M 2S,4R-4-methylglutamate (SYM 2081). Coapplication of either 5 M AMPA or 500 M aspartate had little effect on responses to kainate, although AMPA alone elicited current at 1 mM. In most cells, the currents evoked by kainate, glutamate, and SYM 2081 varied linearly with membrane potential and reversed near 0 mV. Kainate elicited substantial current at steady state (ϳ30% of peak), whereas responses to glutamate and SYM 2081 desensitized almost completely within 0.2-2 sec. Inhibition produced by a 10 sec desensitizing prepulse was half-maximal at 0.22 M for SYM 2081 and 13 M for glutamate. Recovery from desensitization to kainate and glutamate was Ͼ80% complete within 60 sec but was three-to fourfold slower after exposure to SYM 2081. Exposure to Concanavalin A blocked desensitization of the currents but also reduced the peak current amplitudes. Collectively, these results confirm that kainate-preferring receptors underlie the currents evoked by kainate, glutamate, or SYM-2081 in the presence of GYKI 53655; they are not mediated by electrogenic transport or by AMPA-preferring receptors that are insensitive to GYKI. In contrast to previous work on embryonic hippocampal neurons, our results show that the properties of kainate receptors expressed by cells from older animals are distinct from those displayed by homomeric assemblies of the GluR6 subunit.
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.
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 Ϫ70 to ϩ40 mV and is noncompetitive, consistent with allosteric regulation of channel function.
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 ∼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.
Whole‐cell recordings from cultured rat hippocampal neurons, from freshly dissociated dorsal root ganglion (DRG) neurons and from human embryonic kidney (HEK) 293 cells expressing the glutamate receptor GluR6 subunit were used to study the modulation of kainate receptor channels by long chain fatty acids. In all three cell types, application of cis‐unsaturated fatty acids caused a dose‐dependent reduction in whole‐cell currents evoked by kainate. Docosahexaenoic acid (DHA), arachidonic acid (AA), linolenic acid and linoleic acid all produced substantial inhibition at a concentration of 50 μm, whereas inhibition by linolenelaidic acid and linolelaidic acid was significantly weaker. Fully saturated fatty acids were essentially inactive. With continuous exposure to active fatty acids, the peak current elicited by kainate declined over a time course of several minutes to reach a steady‐state level less than 50 % of the initial amplitude. Recovery was slow in control solution, but was speeded up by exposure to bovine serum albumin (0.5 mg ml−1), a protein that binds fatty acids with submicromolar affinity. The inhibition in neurons was half‐maximal with 5–15 μm AA or DHA, but potency was at least 10‐fold greater at GluR6 in HEK 293 cells. Inhibition by AA or DHA was unaffected by extracellular nordihydroguaiaretic acid (10 μm), indomethacin (10 μm), 17‐octadecynoic acid (30 μm) or 1‐(5‐isoquinolinylsulphonyl)‐2‐methylpiperazine dihydrochloride (H‐7; 10 μm). Furthermore, inclusion of H‐7 (100 μm), BAPTA (10 mm), AA (50 μm), antioxidants, or the protein kinase C inhibitor PKC19‐36 (20 μm) in the internal solution had little effect on whole‐cell currents and did not prevent inhibition of currents by extracellular application of AA or DHA. We conclude that the inhibition produced by cis‐unsaturated fatty acids does not require conversion to oxidized metabolites or activation of PKC. Instead, active compounds may interact directly with an extracellular, or intramembraneous, site on kainate receptors.
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