High-affinity kainate-type ion channels in rat cerebellar granule cells.

Patch-clamp recordings were made from rat cerebellar granule cells in primary culture. In cells pre-exposed to concanavalin A (ConA) to remove kainate receptor desensitization, concentration-response data for kainate showed two components. The EC50 value for the high-affinity component (4 μM) was consistent with activation of kainate-type channels. ConA enhanced the apparent potency of the kainate receptor ligand SYM 2081 by 100-fold. In ConA-treated granule cells, currents evoked by 10 μM kainate were not significantly reduced by the AMPA receptor antagonist GYKI 53655, nor were these currents significantly reduced by the co-application of 100 μM AMPA. Currents activated by low concentrations of kainate in the presence of AMPA were completely inhibited by 10 μM La3+. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that granule cells express both unedited (Q) and edited (R) versions of GluR5, with the majority of the GluR5 transcripts being unedited. In contrast, GluR6(R) was detected in seven cells and GluR6(Q) was detected in one granule cell. Whole-cell current-voltage curves for kainate-type currents in granule cells were measured and the ratio of the slope conductances at +40 mV and −40 mV was used as an index of rectification. The mean +40 mV/-40 mV ratio determined from thirty-six granule cells was 1.3 ± 0.1. Spectral density analysis of kainate-evoked whole-cell current noise gave values for the apparent single-channel conductance, γnoise, that were on average about 1 pS. To compare further the properties of recombinant kainate channels with the native kainate-type channels in granule cells, we determined EC50 and γnoise values for SYM 2081 in stable cell lines expressing either GluR6(R) or GluR6(R) and KA2. Co-expression of KA2 with GluR6(R) shifts the EC50 and γnoise values determined for SYM 2081 closer to the values typically found for native kainate-type channels in granule cells. The results demonstrate that cerebellar granule cells in culture express functional kainate-type channels and that in most cells these channels show properties that are similar to those determined for heteromeric channels formed from GluR6(R) and KA2. However, the results also suggest that different granule cells express different repertoires of kainate-type channels with different, and perhaps variable, subunit composition. The ionotropic glutamate receptors (GluRs) can be divided into three subtypes based on their selectivity for agonists: N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate. Considerable evidence indicates that GluRs are oligomeric assemblies of individual subunit proteins, and five different gene products have been identified which are known to form kainate-type channels (GluR5, GluR6, GluR7, KA1 and KA2; Hollmann & Heinemann, 1994; Bettler & Mulle, 1995). Some kainate receptor subunits can form homomeric channels (GluR5 and GluR6), whereas others (KA1 and KA2) do not appear to form functional homomeric channels, but their coassembly with either GluR5 or GluR6 results in channels with unique properties (Herb, Burnashev, Werner, Sakmann, Wisden & Seeburg, 1992; Howe, 1996; Swanson, Feldmeyer, Kaneda & Cull-Candy, 1996). Additional functional diversity of kainate-type channels can result from RNA editing (Sommer, Kohler, Sprengel & Seeburg, 1991; Kohler, Burnashev, Sakmann & Seeburg, 1993; Swanson et al. 1996). In comparison to NMDA- and AMPA-type channels, relatively little is known about the properties and role of kainate-type channels in central neurons, despite the widespread expression of kainate receptor subunits in brain (Wisden & Seeburg, 1993; Bahn, Volk & Wisden, 1994). Native kainate-type channels were first identified in dorsal root ganglion neurons (Huettner, 1990), and subsequent studies have characterized the properties of kainate-type channels in cultured hippocampal neurons (Lerma, Paternain, Naranjo & Mellstrom, 1993; Paternain, Morales & Lerma, 1995; Wilding & Huettner, 1997). Recent evidence supports the involvement of kainate-type channels in synaptic transmission in the hippocampus (Castillo, Malenka & Nicoll, 1997; Clarke et al. 1997; Rodriguez-Moreno, Herreras & Lerma, 1997; Vignes & Collingridge, 1997). Kainate-type channels have also been identified in rat trigeminal ganglion neurons, and the biophysical characteristics of these channels, along with reverse transcriptase-polymerase chain reaction (RT-PCR) studies, suggest that they may be heteromeric assemblies containing KA2 and the edited (R) version of GluR5 (Sahara, Noro, Iida, Soma & Nakamura, 1997). The properties of GluR channels in cultured cerebellar granule cell neurons have been the focus of extensive investigation (Cull-Candy, Howe & Ogden, 1988; Howe, Cull-Candy & Colquhoun, 1991; Wyllie, Traynelis & Cull-Candy, 1993). In situ hybridization studies have shown that granule cells express mRNAs encoding the kainate-type subunits GluR5, GluR6 and KA2 (Bettler et al. 1990; Egebjerg, Bettler, Hermans-Borgmeyer & Heinemann, 1991; Herb et al. 1992; Bahn et al. 1994), suggesting that granule cells express kainate-type channels. However, the EC50 values determined previously for kainate in granule cells (and outside-out patches from these neurons) are more consistent with kainate activation of AMPA-type channels (Traynelis & Cull-Candy, 1991; Wyllie et al. 1993). Because many kainate-type channels are known to exhibit rapid and complete desensitization (Herb et al. 1992; Lerma et al. 1993), kainate-type responses may have been missed in earlier studies, where the agonist applications were relatively slow and kainate receptor desensitization was intact. In the present study, we have isolated and characterized kainate-type channels in cerebellar granule cells by removing kainate receptor desensitization with concanavalin A (ConA) and applying kainate, or the kainate receptor ligand SYM 2081 (Zhou et al. 1997), in the presence of AMPA or the AMPA receptor antagonist GYKI 53655. Our results indicate that granule cells express channels with a high affinity for kainate which show properties consistent with the known properties of channels formed from the kainate-type subunits GluR5, GluR6 and KA2.