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

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1 Keywords: Kainate, Glutamate, Patch clamp 7717 Journal of Physiology (1998), 510.2, pp High-affinity kainate-type ion channels in rat cerebellar granule cells Karen E. Pemberton, Scott M. Belcher, James A. Ripellino and James R. Howe Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520_8066, USA (Received 17 December 1997; accepted after revision 23 March 1998) 1. 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 ìò) was consistent with activation of kainate-type channels. ConA enhanced the apparent potency of the kainate receptor ligand SYM 2081 by 100-fold. 2. In ConA-treated granule cells, currents evoked by 10 ìò kainate were not significantly reduced by the AMPA receptor antagonist GYKI 53655, nor were these currents significantly reduced by the co-application of 100 ìò AMPA. Currents activated by low concentrations of kainate in the presence of AMPA were completely inhibited by 10 ìò LaÅ. 3. 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. 4. 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 mvï 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, ãnoise, thatwereonaverage about1ps. 5. To compare further the properties of recombinant kainate channels with the native kainatetype channels in granule cells, we determined EC50 and ãnoise 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 ãnoise values determined for SYM 2081 closer to the values typically found for native kainate-type channels in granule cells. 6. The results demonstrate that cerebellar granule cells in culture express functional kainatetype 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-ª_aspartate (NMDA), á-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 & M ulle, 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, K ohler, Sprengel & Seeburg, 1991; K ohler, 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

2 402 K. E. Pemberton, S. M. Belcher, J. A. Ripellino and J. R. Howe J. Physiol (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 & Mellstr om, 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 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 kainatetype subunits GluR5, GluR6 and KA2. METHODS Cerebellar cultures Rat pups (aged 5 15 days) were anaesthetized with ether and decapitated, and primary cultures of cerebellar granule cells were prepared as described previously (Cull-Candy et al. 1988). Cells were dissociated without enzymes and plated onto acid-washed coverslips coated with poly-l-lysine (100 ìg ml for 1 h). The resulting cultures were kept in Dulbecco s modified Eagle s medium containing 25 ìò KCl and 10 % fetal bovine serum. Patch-clamp recording Whole-cell patch-clamp recordings were made from granule cells maintained in culture for 2 6 days. Granule cells were identified by their small size and capacitance (2 5 pf). Patch pipettes had initial resistances of 8 15 MÙ. Currents were recorded with an EPC 9 amplifier (HEKA, Lambrecht, Germany) controlled with software (Pulse, HEKA) run on a Macintosh Quadra 800 computer. Series resistance compensation was not employed. The series resistance (R) and the cell capacitance (C) were determined electronically by subtracting the capacitive currents at the onset and offset of a 10 mv voltage step. The cancellation of the whole-cell transients was virtually complete in all cases. Analog signals were low-pass filtered at 10 khz (Bessel-type, 3 db) and were stored on videotape with a VR10b digital data recorder (Instrutech, Great Neck, NY, USA) and a VCR at a sampling rate of 94 khz. The pipette solution contained (mò): CsCl, 150; CaClµ, 0 5; NaµEGTA, 5; and K-Hepes, 10. Spermine (100 ìò, Sigma) was included in the pipette solution in most experiments. The extracellular solution contained (mò): NaCl, 150; KCl, 2 5; CaClµ, 1; and Na-Hepes, 10. The ph of the pipette and extracellular solutions was adjusted to 7 2 with dilute HCl. dl-2-amino-5- phosphonovaleric acid (dl-apv, 100 ìò; RBI) was included in the extracellular solution to block NMDA receptors. Stock solutions of kainate (Sigma), AMPA (RBI), dl-apv, SYM 2081 ((2S,4R)-4- methylglutamate; Symphony Pharmaceuticals), and GYKI (1(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy- 3,4-dihydro-5H-2,3-benzodiazepine; Eli Lilly) were prepared and stored at 20 C. Frozen aliquots of the stock solutions were diluted to final concentration in extracellular solution on the day they were used. Agonists and antagonists were applied by local superfusion. Unless otherwise noted, coverslips of cells were preexposed to ìò ConA (Sigma) for 20 min to remove desensitization of kainate channels. All recordings were performed at room temperature (20 22 C). Single-cell RT-PCR and analysis of GluR5 and GluR6 editing Whole-cell patch-clamp recordings were established on granule cells in short-term primary culture and the cytoplasm was extracted from the cell by applying suction to the back of the recording pipette. The extracted cytoplasm was expelled into a siliconized 0 5 ml thin-walled microcentrifuge tube and the tubes were quickly immersed in a dry ice ethanol bath. First-strand cdna was synthesized from the cytoplasmic contents of single cells (in 6 7 ìl of pipette solution) with Superscript II reverse transcriptase (Life Technologies) using oligo(dt) and random hexamer primers (10 pmol) according to manufacturer s recommendations. The entire volume of the single-cell reverse transcription reactions was used for PCR amplification, consisting of one cycle of 94 C for 2 min; thirty cycles of: 94 C for 30 s, 58 C or 60 C for 30 s, 72 C for 30 s; and one cycle of 72 C for 10 min. The reaction volume was 50 ìl and contained 10 pmol of each primer pair. The primers used for PCR amplification of GluR5 and GluR6 are given in Table 1of Belcher & Howe (1997). Controls included mock cdna synthesis (no reverse transcriptase), samples treated with RNase A, and mock RT-PCR reactions in which template was excluded. For all samples analysed, no detectable product was generated from any of the negative control reactions.

3 J. Physiol Kainate-type channels in granule cells 403 The extent of RNA editing at the QÏR site of GluR5 and GluR6 was determined using a protocol based on differential digestion of RT-PCR products with the restriction endonuclease BbvI (Belcher & Howe, 1997). The GluR5 and GluR6 RT-PCR products amplified from single granule cells (10 ìl ofa50ìl reaction) were digested for 4 6hat37 Cwith4 6UofBbvI (New England Biolabs, Beverly, MA, USA). The digestion products were fractionated on polyacrylamide gels and the molar ratios of edited and unedited products were determined by densitometric analysis. To determine the extent of editing at the two GluR6 sites in the first transmembrane segment (TMI), as well as the QÏR site, the entire population of GluR6 RT-PCR products generated from single granule cells was directly sequenced with 5'_end-labelled primers. The proportions of unedited and edited transcripts were then estimated by comparing the signal intensities of the dda and ddg lanes on the sequencing gel. To assess the sensitivity of comparisons based on the intensity of sequencing terminations, known amounts of the cdnas encoding the Q and R versions of GluR6 were mixed at ratios of 1 : 10, 1 : 1 and 10 : 1. Aliquots of the samples containing 1 ng of DNA were amplified by PCR and analysed by DNA sequencing. Not only were dda and ddg terminations detected in each sample, but densitometric estimates of the percentages of Q- and R-encoding products agreed within 5 % of the known percentages for each dilution. Immunoprecipitation analysis To compare further the properties of the native kainate-type channels in granule cells with those of recombinant kainate-type channels, experiments were also performed on HEK 293 cell lines that stably express either GluR6(R) or GluR6(R) and KA2 in a tetracycline-responsive manner (Howe, Skyrabin, Belcher, Zerillo & Schmauss, 1995; Howe, 1996). Immunoprecipitation analysis was performed to characterize the heteromeric interactions between GluR6 and KA2 in the GluR6(R) KA2-expressing cells. For this analysis, the cells were grown in Eagle s minimal essential medium with Earle s salts in the presence of G418 (0 5 mg ml ; Life Technologies), hygromycin B (200 U ml ; Calbiochem) and tetracycline (2 ìg ml ; Sigma). The expression of GluR6 and KA2 was induced by removing tetracycline from the media. Plates of % confluent cells were harvested and GluR subunit proteins solubilized as described previously (Ripellino, Neve & Howe, 1998). The extraction conditions used result in the solubilization of % of the GluR6 and KA2 proteins. Detergent extracts ( ìg protein) were precleared by incubating with immobilized protein A (Repligen, Needham, MA, USA) for 30 min at 4 C, separated by centrifugation, and GluR subunit-specific antibodies were added to the supernatants. Polyclonal anti- GluR6Ï7 and anti-ka2 antisera were purchased from Upstate Biotechnologies Inc. (Lake Placid, NY, USA) and used at a concentration of 2 ìg ml, which was sufficient to immunoprecipitate all of the specific antigen. Immunoprecipitations, Western blots, and densitometric comparisons were performed as described in Ripellino et al. (1998). Analysis of patch-clamp results For the analysis of agonist-evoked currents, signals were replayed from videotape and digitized at the original sampling rate of 94 khz. The record was low-pass filtered with a digital Gaussian filter at 2 khz ( 3 db) and compressed by a factor of 10 to generate a new file where the sampling rate was 9 4 khz. Concentration response data were generated by measuring the steady-state amplitude of agonist-evoked currents (from the digitized records) at a membrane potential, Vm, of 80 mv. The concentration response data were fitted with Hill-type equations: I = Imax Ï(1 + (EC50Ï [A]) n H), (1) where I is the steady-state current at a given concentration of agonist, [A]; and Imax, EC50, and nh are parameters to be estimated from the fit. A formally equivalent equation was used to estimate IC50 values for GYKI 53655, where the current values at each inhibitor concentration were normalized to the control amplitude. In some cases, agonist concentration response data were fitted with equations consisting of the sum of two Hill-type components. Current voltage (I V ) relationships for agonist-evoked currents were obtained by performing 500 ms voltage ramps ( 80mV to +80mV) during steady-state currents (responses to four ramps were averaged for each curve). The I V curves for kainate-type currents measured in the presence of AMPA were obtained by subtracting the I V curve during application of 100 ìò AMPA from the corresponding curve during the concurrent application of AMPA and kainate or SYM Difference currents were sampled at 1 khz. The small size of the kainate-evoked currents in most granule cells meant that errors as small as 2 3 pa in the subtraction procedure used to obtain the difference currents could result in significant errors in reversal potential, Vrev, determinations. These errors in turn could lead to significant errors in estimates of channel rectification from measurements of the ratio of the current amplitudes. Rather than adjust the I V curves such that Vrev was zero (K ohler et al. 1993), the I V curves were fitted with fourthorder polynomial functions and these fits used to generate slope conductance vs. potential (G V) relationships for each cell. The slopeconductancesdonotdependonvrev, and the ratio of the slope conductances at +40 and 40 mv (+40 mvï 40 mv ratio) was calculated for each cell and used as an index of rectification. To minimize unequal weighting of the data due to the increased current variance at large driving force, the standard deviation (ó) was calculated for contiguous ten point sections and the squared deviation for each data point was weighted during the least-squares minimization by its appropriate 1Ïó value. The first derivative of the polynomial fit was evaluated at +40 mv and 40 mv to calculate the +40 mvï 40 mv rectification ratio. The net one-sided spectral density, G(f), for agonist-evoked current noise was obtained as described in detail in Howe (1996). For kainate-type currents evoked in the presence of AMPA, the background spectrum was taken as the spectrum during application of 100 ìò AMPA. Net spectra were fitted with one Lorentzian, or the sum of two Lorentzian components, and the current variance was calculated from the fit as: Var I = (ðï2)óg(0)i fci, (2) i where G(0)i and fci are the zero-frequency asymptote and halfpower frequency of the ith component, respectively. The apparent single-channel current was taken to be: inoise = VarIÏI, where I is the mean steady-state agonist-evoked current at Vm = 80 mv; and the apparent single-channel conductance, ãnoise = iï(vm Vrev), was calculated assuming the reversal potential, Vrev, was 0 mv. The results of noise analysis of whole-cell currents recorded when ô=rc was greater than 200 ìs were discarded to minimize distortionoftheresultsbyrc filtering. Mean (± s.e.m.) values are given for the various parameters determined unless indicated otherwise.

4 404 K. E. Pemberton, S. M. Belcher, J. A. Ripellino and J. R. Howe J. Physiol RESULTS Pretreatment with ConA reveals a high-affinity kainate current The whole-cell patch-clamp technique was used to determine if kainate-type channels are expressed in rat cerebellar granule cells in primary culture. All the results reported here were obtained in the presence of the competitive NMDA-receptor antagonist dl-apv (100 ìò) to prevent activation of NMDA receptors. While kainate can also activate AMPA-type channels, pre-incubation of cells with the lectin concanavalin A (ConA) facilitates the detection of kainate-type currents by selectively reducing fast desensitization of these channels (see, for example: Huettner, 1990; Partin, Patneau, Winters, Mayer & Buonanno, 1993; Wong & Mayer, 1993). We therefore compared the potency of Figure 1. Pretreatment of cerebellar granule cells with ConA reveals high-affinity kainate-type currents Aa, whole-cell currents evoked at 80 mv by increasing concentrations of kainate in a cerebellar granule cell not exposed to ConA. Note the lack of a response to 5 ìò kainate. Breaks in the record are indicated by the diagonal bars. Ab, concentration response curve for steady-state currents evoked by kainate from thesamegranulecellasinaa. The Hill-type fit to the data gave an EC50 of 46 ìò. B, whole-cell currents evoked at 80 mv by 1 and 10 ìò kainate in a granule cell pre-exposed to ConA. Kainate applications are denoted by the bar above the traces. C, concentration response curve for kainate in a granule cell that was pretreated with ConA (different cell from that in B). The smooth line shows the two-component Hill-type fittotheresultswhichgaveec50 values of 5 and 56 ìò for the high- and low-affinity components, respectively.

5 J. Physiol Kainate-type channels in granule cells 405 kainate in granule cells that were or were not exposed to ConA. In the absence of exposure to ConA, applications of 10 ìò kainate rarely gave measurable whole-cell currents in granule cells, and responses were not seen at kainate concentrations below 10 ìò (Fig. 1A). The currents evoked by higher concentrations of kainate showed little, if any, desensitization, and concentration response data were obtained by measuring the steady-state currents evoked at 80 mv. Figure 1Ab shows a typical concentration responsecurveobtainedfromagranulecellthatwasnot exposed to ConA. The Hill-type fit to the results gave an EC50 value of 46 ìò. Similar measurements from six granule cells gave mean values for the EC50 and Hill coefficient (nh)of55 ± 5ìÒand1 7 ± 0 2, respectively. Whereas 10 ìò kainate rarely evoked whole-cell currents in untreated cells, concentrations of kainate of 10 ìò or less evoked measurable currents in the majority of granule cells (78 out of 105) in cultures pre-exposed to ConA (10 25 ìò for 20 min). The whole-cell currents evoked in a ConAtreated cell by 1 and 10 ìò kainate are shown in Fig. 1B. The whole-cell currents evoked at 80 mv by 10 ìò kainate in ConA-treated cells ranged from 4 to 85 pa. Inspection of frequency histograms of the current amplitudes indicated that the values were log-normally distributed. A Gaussian fit to the log-amplitude results gave a mean whole-cell current value of 14 pa (anti-log(mean ± s.d.): 5 47 pa, n = 59). The capacitance values measured for the cells were typically very similar (2 3 pf) and expressing the data as current densities (pa pf ) did not substantially reduce cellto-cell variability nor alter the log-normal nature of the distribution. To better assess the effect of ConA treatment on the apparent affinity of kainate for GluR channels in granule cells, concentration response data were obtained over a wide range of kainate concentrations. Figure 1C shows the results obtained from a ConA-treated granule cell with kainate concentrations ranging from 0 2 to 500 ìò. The results were fitted with the sum of two Hill-type components with respective EC50 values of 5 and 56 ìò. Two similar components were detected in each of six cells tested (10 12 concentrations per cell; 0 1 or 0 2 ìò to 0 5 or 1 mò kainate). The mean EC50 and nh values were 3 9 ± 0 7 ìò and 1 8 ± 0 4, respectively, for the high-affinity component, and 120 ± 30 ìò and 2 3 ± 0 4, respectively, for the lowaffinity component. Studies with the AMPA receptor antagonist GYKI Currents through AMPA-type channels are selectively reduced by 2,3-benzodiazepines (Donevan & Rogawski, 1993; Zorumski, Yamada, Price & Olney, 1993), and GYKI is one of the most selective of such non- Figure 2. Kainate-evoked currents in granule cells not pre-exposed to ConA are blocked by the AMPAreceptor antagonist GYKI A, whole-cell currents evoked by 200 ìò kainate in a granule cell held at 80mV in the presence of increasing concentrations of GYKI The cell was not pretreated with ConA. Portions of the continuous record are displayed so that the onsets of the applications (bar above the traces) are aligned. Kainate and GYKI were applied simultaneously; the time-dependent fade of the currents reflects the slow kinetics of the GYKI inhibition. Concentrations of GYKI are shown to the right of each trace. B, concentration response curve for GYKI inhibition of steady-state currents evoked by 200 ìò kainate (same cell as in A).Thefittotheresults (smooth curve) gave an IC50 of 8 2 ìò.

6 406 K. E. Pemberton, S. M. Belcher, J. A. Ripellino and J. R. Howe J. Physiol competitive AMPA receptor antagonists currently available (Paternain et al. 1995; Wilding & Huettner, 1995; Bleakman et al. 1996). If the kainate-evoked currents in granule cells not exposed to ConA result primarily from non-desensitizing activation of AMPA-type channels, then these currents should be inhibited by GYKI Figure 2A shows whole-cell currents evoked in a granule cell (not exposed to ConA) with 200 ìò kainate in the absence and presence of GYKI Inhibition of the currents is apparent with 1 ìò GYKI and is virtually complete at a GYKI concentration of 100 ìò. Kainate and GYKI were applied simultaneously and the fade in the currents reflects the slow kinetics of 2,3-benzodiazepine inhibition (Donevan & Rogawski, 1993). The concentration response curve for the steady-state inhibition produced by GYKI in the same granule cell is shown in Fig. 2B. The fit to the results gave an IC50 value of 8 2 ìò, which was close to the mean value obtained (8 1 ± 2 3 ìò, n = 5 cells). Whereas 100 ìò GYKI produced near-maximal inhibition of kainate-evoked currents in cells not preexposed to ConA, this concentration of the AMPA receptor blocker had little effect on currents evoked by 10 ìò kainate in ConA-treated granule cells (Fig. 3A), and the mean percentage change in the amplitude of these currents was not significantly different from zero ( 12 ± 6 %, n = 9 cells). In four of the cells, the currents evoked by 100 ìò kainate were also measured in the absence and presence of 100 ìò GYKI The results from these cells are shown in Fig. 3B. While the co-application of GYKI had little effect on the currents evoked by 10 ìò kainate, the currents evoked by 100 ìò kainate were reduced significantly (72 ± 3 %). Additionally, the currents evoked by 100 ìò kainate in the presence of GYKI were similar in size to the currents evoked in the same cells by 10 ìò kainate (with or without the antagonist), suggesting that the residual currents seen in the presence of 100 ìò GYKI were due to activation of kainate-type channels. Concentration response data obtained from one cell in the absence and presence of 100 ìò GYKI are shown in Fig. 3C. The results in the absence of the antagonist are fitted well with the sum of two Hill-type components with respective EC50 values of 1 9 and 96 ìò. The lower-affinity component is absent in the presence of 100 ìò GYKI 53655, Figure 3. High-affinity kainate-type currents in ConA-treated granule cells are resistant to block by GYKI A, whole-cell currents evoked at 80 mv by 10 ìò kainate in the absence (a) and presence(b) of 100 ìò GYKI in a ConA-treated granule cell. B, bargraph showing the mean steady-state amplitude of whole-cell currents evoked in 4 ConA-treated granule cells by 10 ìò kainate (left) and 100 ìò kainate (right) in both the absence (control, 5) and presence of 100 ìò GYKI (4). Error bars indicate s.e.m. The currents elicited by 10 ìò kainate were little affected by the non-competitive AMPA receptor antagonist, whereas the currents evoked by 100 ìò kainate were reduced in size to amplitudes similar to those seen with 10 ìò kainate. C, kainate concentration response data from a granule cell pretreated with ConA in the absence (control, 0) andpresence(1) of 100 ìò GYKI The two-component fit to the results obtained in the absence of GYKI gave EC50 values of 1 9 and 96 ìò. GYKI blocks the low-affinity, but not the high-affinity, component.

7 J. Physiol Kainate-type channels in granule cells 407 and the data can be fitted by a single Hill-type component with an EC50 of 1 6 ìò. Similar results were obtained from five ConA-treated granule cells; the single-component fits to the results obtained in the presence of 100 ìò GYKI gave a mean EC50 value for kainate of 2 4 ± 0 8 ìò (nh = 1 8 ± 0 4). AMPA does not inhibit the high-affinity kainate currents If, as the above results suggest, the currents evoked in ConA-treated granule cells by kainate concentrations of 10ìÒandbelowareprimarilytheresultofactivationof kainate-type channels, then concentrations of AMPA sufficient to occupy the majority of AMPA-type channels in the cells should have little effect on these currents. Applications of 100 ìò AMPA produced measurable currents in seventy-six out of eight-six granule cells examined. Pooled concentration response data for AMPA from eleven cells gave an EC50 of 12 ìò. The steady-state amplitude of currents evoked by 100 ìò AMPA was typically smaller (û 50 %) than the AMPA receptor component of kainate-evoked currents in the same cells. This result suggests that 100 ìò AMPA produced substantial steady-state desensitization of AMPA-type channels, although the speed of our solution changes was insufficient to detect it. Thus, 100 ìò AMPA would be expected to interfere with kainate activation of AMPA-type channels both by competing for occupancy of the receptors and by promoting receptor desensitization (Lerma et al. 1993). In ConA-treated granule cells that gave responses to 10 ìò kainate alone, twenty-three out of twenty-eight cells also showed responses to 10 ìò kainate when kainate was coapplied with 100 ìò AMPA (Fig. 4A). In each of these cells, the amplitude of the steady-state currents evoked by 10 ìò kainate was little affected by the presence of AMPA. The ratio of the steady-state currents evoked in the same cell by 10 ìò kainate alone and by 10 ìò kainate during the concurrent application of 100 ìò AMPA was 1 0 ± 0 1 (n = 23). However, the whole-cell currents evoked by 100 ìò kainate were reduced significantly in the presence of 100 ìò AMPA (mean reduction, 56 ± 5 %). As was the case for the results obtained with 100 ìò GYKI 53655, the amplitudeoftheresidualcurrentevokedby100ìòkainate was similar to the amplitude of the currents evoked in the same cell by 10 ìò kainate. The kainate EC50 values obtained in the presence of 100 ìò AMPA were similar to those obtained for the high-affinity Figure 4. Kainate-type currents in ConAtreated granule cells are insensitive to the concurrent application of AMPA and GYKI A, whole-cell currents evoked at 80 mv by 10 ìò kainate alone, and by 10 ìò kainate in the presence of 100 ìò AMPA, in a granule cell pretreated with ConA. Agonist applications are indicated by bars above the record. B, concentration response curve for currents evoked by kainate in the presence of 100 ìò AMPA in a ConA-treated granule cell (different cell from that in A). The Hill-type fit to the data gave an EC50 value of 3 2 ìò. C, bar-graph showing the mean amplitude of currents evoked by 100 ìò AMPA (left) andby10ìòkainateduringtheconcurrent application of 100 ìò AMPA (right). The respective currents were measured in each of 5 granule cells in the absence (control, 5) and presence of 100 ìò GYKI (4). Applications of GYKI that virtually abolished currents evoked by AMPA had no effect on the currents evoked by 10 ìò kainate. Error bars indicate the s.e.m.

8 408 K. E. Pemberton, S. M. Belcher, J. A. Ripellino and J. R. Howe J. Physiol component detected in two-component fits in the absence of competing ligands, as well as to the corresponding values measured in 100 ìò GYKI Concentration response data from one cell are shown in Fig. 4B. The mean EC50 obtained for kainate in the presence of 100 ìò AMPA was 3 1 ± 0 7 ìò, and the mean nh value was 1 6 ± 0 2 (n =8 cells, 4 6 kainate concentrations per cell). Figure 4C shows that 100 ìò GYKI had no effect on responses to 10 ìò kainate that were evoked in the presence of 100 ìò AMPA (mean change, 1 ± 13 %; n = 5 cells), whereas the currents evoked in the same cells by 100 ìò AMPA were completely inhibited (99 ± 1 %). High-affinity kainate currents are blocked by lanthanum Previous studies have shown that LaÅ inhibits kainatetype channels at concentrations that potentiate kainate activation of AMPA-type channels (Huettner, 1991; Wilding & Huettner, 1997). In our experiments, we found that currents evoked in ConA-treated granule cells by 10 ìò kainate were significantly reduced by 10 ìò LaÅ (Fig. 5A). On average, the amplitude of the steady-state current evoked by 10 ìò kainate was reduced by 84 ± 5 % (n =9 cells). In the same cells, 10 ìò LaÅ decreased the amplitude of the responses evoked by 100 ìò kainate by 42 ± 11 %. Applications of 10 ìò LaÅ had no significant effect on currents evoked by 100 ìò AMPA, but virtually abolished the currents elicited in the same cells by 10 ìò kainate in the presence of AMPA, reducing the kainate-activated currents by 91 ± 6 % (Fig. 5B, n = 6). In the presence of AMPA, LaÅ reduced responses to 100 ìò kainate by 67±14%. ConA reveals currents activated by the kainateselective ligand SYM 2081 The glutamate analogue SYM 2081 produces rapidly desensitizing activation of kainate-type channels at nanomolar concentrations, whereas the EC50 for SYM 2081 activation of AMPA-type channels has been reported to be about 300 ìò (Wilding & Huettner, 1997; Zhou et al. 1997). It was therefore of interest to compare the potency of SYM 2081 in cells that were and were not exposed to ConA. Figure 6A shows whole-cell currents evoked by increasing concentrations of SYM 2081 in a ConA-treated granule cell (Aa) and in a granule cell with intact kainate receptor desensitization (Ab). The concentration response data obtained from the two cells are plotted in Fig. 6B. The fit to the data from the cell not exposed to ConA (1)gaveanEC50 value of 216 ìò. The fit to the results from the ConAtreated cell (0) gaveanec50 value of 0 95ìÒ. Because SYM 2081 concentrations of 10 ìò and greater evoked measurable currents in some cells not exposed to ConA (suggesting activation of AMPA-type channels), the data from the ConA-treated cell were also fitted excluding the 10, 30 and 100 ìò points (dashed curve). The fit to the results for SYM 2081 concentrations of 3 ìò and less gave an EC50 value of 0 51 ìò. Analysis of concentration response data for SYM activated currents in five granule cells that were not pretreated with ConA gave mean EC50 and nh values of 260 ± 51 ìò and 1 6 ± 0 2, respectively. The mean EC50 and nh values obtained from eight ConA-treated granule cells were 3 3 ± 0 9 ìò and 1 1 ± 0 3, respectively, if data Figure 5. Kainate-type currents are blocked by lanthanum A, whole-cell currents evoked in a ConA-treated granule cell by kainate (10 and 100 ìò) in the absence (a) and presence (b) of 10 ìò LaÅ. Agonist applications are indicated by the bars above the traces. B, bar-graph showing the mean amplitude of the currents evoked by 10 ìò kainate (left) and 100 ìò kainate (right) in the absence (control, 5) and presence(4) of 10 ìò LaÅ. The respective currents were measured in 6 granule cells during the concurrent application of 100 ìò AMPA and were normalized to the size of the current evoked in each cell by 10 ìò kainate alone. Error bars indicate s.e.m.

9 J. Physiol Kainate-type channels in granule cells 409 for concentrations of ìò SYM 2081 were included; and the corresponding values if these values were excluded (as in Fig. 6B) were 1 7 ± 0 3 ìò and 1 3 ± 0 3, respectively (n = 8). Rectification behaviour of kainate-type currents in granule cells In ConA-treated granule cells, I V curves were measured for steady-state currents evoked by 10 ìò kainate under conditions where kainate activation of AMPA receptors was minimal (i.e. in the presence of either 100 ìò AMPA or 100 ìò GYKI 53655). Both inwardly and outwardly rectifying currents were observed. Current voltage curves from two different granule cells are shown in Fig. 7A and B. The slope conductance vs. potential (G V) relationships derived from the I V curves in Fig. 7A and B are shown in Fig.7C and D, respectively, and gave +40 mvï 40 mv ratios of 1 5 and 0 8, respectively. Values for the +40 mvï 40 mv ratio were measured for thirty-six granule cells. The mean +40 mvï 40 mv ratio was 1 3 ± 0 1, but the values varied substantially (ranging from 0 4 to 3 6). Current voltage curves were also obtained during steady-state responses to applications of 10 ìò SYM 2081 in five granule cells (in AMPA or GYKI 53655); the mean +40 mvï 40 mv ratio was 1 2 ± 0 1. Figure 6. Pretreatment of granule cells with ConA reveals activation by SYM 2081 of highaffinity kainate-type currents A, whole-cell currents evoked at 80 mv by increasing concentrations of SYM2081 in 2 granule cells that either were (a) or were not (b) pretreated with ConA. B, concentration response data from the complete set of results obtained for the cells in Aa and Ab: 0, data from a cell exposed to ConA; 1, results from a granule cell not pretreated with ConA. Continuous lines are one-component Hill-type fits to each set of data. The fit to the results obtained from the cell pretreated with ConA gave an EC50 value of 0 95 ìò, whereas the corresponding value obtained from the cell not exposed to ConA was 216 ìò. The dashed line indicates the Hill-type fit that was obtained for the results from the ConA-treated granule cell when the data for SYM 2081 concentrations of 10 ìò and above were excluded (EC50 = 0 51 ìò).

10 410 K. E. Pemberton, S. M. Belcher, J. A. Ripellino and J. R. Howe J. Physiol Noise analysis of kainate-type currents in granule cells Although the conductances of some types of recombinant kainate-type channels are too small to detect single-channel currents directly, previous studies have shown that differences in the apparent unitary conductance of these channels can be detected from spectral density analysis of agonist-evoked current noise (Howe, 1996; Swanson et al. 1996). To estimate the unitary conductance of the channels underlying the kainate-type currents in granule cells, and to compare their conductance with previous estimates of the conductance of native (Huettner, 1990; Sahara et al. 1997) and recombinant kainate-type channels, noise analysis of steady-state whole-cell current noise was performed. While the whole-cell currents were small, the resolution in most recordings was sufficient to detect an increase in current noise during applications of 10 ìò kainate. Noise spectra were obtained from currents activated by 10 ìò kainate in forty-three granule cells where the kainate applications were made in the presence of either 100 ìò GYKI or 100 ìò AMPA, thus ensuring that the current fluctuations arose primarily from the gating of kainate-type channels. Similar data were obtained from seven cells with 10 ìò SYM Figure 8A showsaportionofthecurrentevokedby10ìò kainate in the presence of 100 ìò AMPA. The AMPA application is associated with a clear increase in noise, and there is an additional increase in noise during kainate application. Figure 8B shows the power spectrum for the kainate-evoked noise from the same cell, where the background spectrum was taken during the steady-state AMPA-evoked current. The spectrum was fitted with the sum of two Lorentzian components with half-power frequencies, fc1 and fc2, of 45 and 202 Hz, respectively. The ãnoise value obtained from the fit was 1 0 ps. The whole-cell current evoked by 10 ìò SYM 2081 in another granule cell in the presence of 100 ìò GYKI is shown in Fig. 8C. The power spectrum obtained from the same cell is shown in Fig. 8D. The spectrum was fitted adequately by a single Lorentzian with a half-power frequency of 55 Hz. The fit gaveaãnoise value of 1 1 ps. Figure 7. Rectification of kainate-type channels in granule cells A and B, I V relationships for steady-state currents evoked by 10 ìò kainate in the presence of 100 ìò AMPA from 2 different cerebellar granule cells. Currents were measured during 500 ms voltage ramps from 80 to +80 mv. The results are the difference currents obtained by subtracting the currents during application of 100 ìò AMPA from the currents during the concurrent application of 100 ìò AMPA and 10 ìò kainate. The smooth curves are fourth-order polynomial fits to the results. C and D, the first derivativeofthepolynomialfitstotheresultsinaand B, respectively. The values of the function at +40 and 40 mv were used to determine the ratio of the slope conductances at these membrane potentials.

11 J. Physiol Kainate-type channels in granule cells 411 Of the forty-three cells from which kainate spectra were obtained in the presence of AMPA or GYKI 53655, the power spectra from twenty-nine cells were adequately fitted by a single Lorentzian. The mean half-power frequency obtained from these spectra was 42 ± 4 Hz. In the remaining fourteen cells, two Lorentzian components were apparent, although the higher-frequency component was not especially prominent. The mean half-power frequencies for the two Lorentzian components were 28 ± 3 and 290 ± 32 Hz, and the mean ratio of the zero frequency asymptotes, G(0)1ÏG(0)2, was 33±6. The ãnoise values determined from the two Lorentzian spectra were somewhat, but not significantly, larger than the corresponding values obtained from the single Lorentzian fits (1 1 ± 0 3 ps and 0 75 ± 0 14 ps, respectively). In the presence of 100 ìò AMPA or 100 ìò GYKI 53655, the mean ãnoise estimated from the current fluctuations during the application of 10 ìò SYM 2081 was 2 0 ± 0 6 ps (n = 7 cells). This value did not differ significantly from the value obtained with 10 ìò kainate in the same cells (1 2 ± 0 3 ps). In three cells, the SYM 2081 spectra were best fitted with two Lorentzian components, whereas only a single component was resolved in the spectra from the other four cells. The mean ãnoise values from the single- and two- Lorentzian fits did not differ significantly. Single-cell RT-PCR analysis of GluR5 and GluR6 editing Previous studies have shown that granule cells express mrna encoding GluR5 and GluR6 (Bettler et al. 1990; Egebjerg et al. 1991; Bahn et al. 1994). Both the rectification behaviour and apparent unitary conductance of channels containing GluR5 and GluR6 are influenced by RNA editing at the QÏR site common to both subunits (K ohler et al. 1993; Swanson et al. 1996). We have recently shown that bothqandrversionsofglur5and GluR6 are present in RNA from populations of acutely isolated granule cells (Belcher & Howe, 1997). It was therefore of interest to determine whether this reflected the presence of unedited and edited variants in individual cells. Because our population results indicated that editing changes during early development, the cells were obtained at postnatal (P) days which would maximize detection of both unedited and edited cdnas (GluR5, P15; GluR6, P5 P10). Figure 8. Apparent unitary conductance of kainate-type channels in granule cells Whole-cell currents evoked at 80 mv by 10 ìò kainate in the presence of 100 ìò AMPA (A), and by 10 ìò SYM 2081 in the presence of 100 ìò GYKI (C) in 2 different ConA-treated granule cells. Agonist applications are indicated by the bars above the traces. In C, SYM 2081 and GYKI were applied simultaneously. B and D show the power spectra obtained from spectral density analysis of the current noise evoked by 10 ìò kainate and 10 ìò SYM 2081 during the responses shown in A and C, respectively. The power spectrum for the kainate-evoked noise was fitted (continuous curve) with the sum of two Lorentzian components with respective half-power frequencies of 45 and 202 Hz (dashed curves). The fit gave a ãnoise value of 1 0pS. The single Lorentzian fit to the SYM2081 results gave a ãnoise value of 1 1 ps.

12 412 K. E. Pemberton, S. M. Belcher, J. A. Ripellino and J. R. Howe J. Physiol Whole-cell recordings were obtained from granule cells in short-term primary culture, and the cytoplasmic contents were harvested and used as a template for RT-PCR amplification with GluR5- or GluR6-specific primers. The ratios of unedited and edited GluR5 products were determined from analysis of the restriction fragments obtained upon BbvI digestion (Belcher & Howe, 1997). The data obtained for GluR5 from three individual granule cells are shown in Fig. 9A. In each cell the majority of GluR5 transcripts are unedited, although some product arising from edited RNA is also detected. Sufficient GluR5-specific product for reliable analysis was amplified from eight granule cells (from P15 rats). The mean percentage of edited GluR5 transcripts was 24 ± 5 %. In addition to the QÏR site in the putative pore-forming region, GluR6 contains two sites in TMI that are also subject to RNA editing (K ohler et al. 1993). To analyse editing at the three sites in GluR6, in each of eight granule cells (from P5 P10 rats) the population of GluR6 RT-PCR products was directly sequenced with 5'-end-labelled primers. The proportions of unedited and edited transcripts were then estimated by comparing the signal intensities of the dda and ddg lanes on the sequencing gel. Two examples of this analysis for the QÏR site in GluR6 are shown in Fig. 9B. In each case there is a strong termination in the ddg lane and no detectable termination in the dda lane, suggesting that thevastmajorityofglur6mrnaineachgranulecellwas edited at the QÏR site. In seven of the eight cells analysed, the GluR6 products appeared to be completely edited at all three sites, whereas the reaction products from the remaining cell were edited at both TMI sites and unedited at the QÏR site. At each editing site analysed, specific terminations for both dda and ddg were never detected. Analysis of samples containing known amounts of the cdnas encoding GluR6(Q) and GluR6(R) indicated that we could have detected either cdna if it represented at least 10 % of the RT-PCR product from a given cell (see Methods). Kainate and SYM 2081 activation of GluR6(R) and GluR6(R)ÏKA2 channels In addition to GluR5 and GluR6, cerebellar granule cells also express KA2. Co-expression of KA2 with GluR6 results in channels with unique properties (Herb et al. 1992; Howe, 1996; Swanson et al. 1996), and there is evidence for heteromeric interactions between GluR6 and KA2 in cerebellum (Ripellino et al. 1998). Characterization of heteromeric GluR6ÏKA2 channels is therefore relevant to understanding Figure 9. RT-PCR analysis of GluR5 and GluR6 editing in single cerebellar granule cells A, GluR5 RT-PCR amplification products from 3 granule cells. The samples were separated on a 6 % polyacrylamide gel that was stained with ethidium bromide and photographed. The lane marked ( ) contains undigested RT-PCR products. Lanes 1 3 show RT-PCR products from each cell after digestion with BbvI. The sizes of the uncut GluR5 product (313 bp) and the edited (R, 187 bp) and unedited (Q, 106 bp) restriction fragments are indicated at the sides of each panel. The lane labelledmcontainsa1kbdnaladder(life Sciences) as a size standard. B, sequence analysis of GluR6-specific RT-PCR products obtained from 2 granule cells (GC-5-4 and GC-5-3). The position of the QÏR site is indicated. Note that for both cells a termination signal is only detected in the ddg lane.

13 J. Physiol Kainate-type channels in granule cells 413 the subunit composition of native kainate-type channels in granule cells. This characterization is complicated, however, by the possible expression of both homomeric and heteromeric channels in cells co-transfected with GluR6 and KA2. Clonal HEK 293 cells that stably express GluR6(R) and KA2 were therefore analysed to determine to what extent the cells express channels that are heteromeric assemblies containing both subunits. Western blots of GluR6 and KA2 protein in the GluR6(R) KA2 stable transfectants are shown in Fig. 10A. Equal amounts of protein from parallel cultures in which kainate subunit expression was suppressed with tetracycline Figure 10. GluR6 and KA2 protein expression and agonist affinity in HEK 293 cells stably expressing both subunits A, Western blots of total cellular protein from HEK 293 cells stably expressing the GluR6 and KA2 subunits. The immunoblots were probed with anti-glur6ï7 (left) or anti-ka2 (right) antibody. The cells were harvested before (NI) and 5 days after (I) inducing expression of GluR6 and KA2 by removing tetracycline from the media. Note that the background expression of KA2 in the presence of tetracycline is lower than the corresponding expression of GluR6, and that the subsequent induction of expression is greater. B, detergent extracts of total cellular protein from cells stably expressing GluR6 KA2 were immunoprecipitated with anti-ka2 (left) or anti-glur6ï7 (right) antibody. Aliquots of the detergent extracts (DE) and postimmunoprecipitate supernatants (S) and pellets (P) were subjected to SDS PAGE and immunoblotted with anti-glur6ï7 (left) or anti-ka2 (right) antibody. The positions of 120 and 85 kda molecular weight standards are shown. C, whole-cell currents (a) evoked at 80 mv by increasing concentrations of SYM 2081, and the resultant concentration response data (b), in a ConA-treated HEK 293 cell stably expressing the GluR6(R) and KA2 subunits. SYM 2081 applications are indicated by bars above the current traces. The fit to the results gave an EC50 value of 0 74 ìò.

14 414 K. E. Pemberton, S. M. Belcher, J. A. Ripellino and J. R. Howe J. Physiol (not induced, NI), or in which expression was induced by removing tetracycline from the media (induced, I), were electrophoresed in adjacent lanes of the same gel. The background expression of GluR6 is substantial in the presence of tetracycline, whereas relatively little KA2 expression is seen in the non-induced cells and the induction of expression obtained with KA2 is consequently more robust (Fig. 10A). At 5 6 days post-induction, quantitative densitometricanalysisgaveameanglur6:ka2ratioof 1 5 ± 0 2 (n = 4) in the cell line studied here. At these postinduction times, it was possible to immunoprecipitate the vast majority of either subunit protein with antibody directed against the other. The results of one such immunoprecipitation experiment are shown in Fig. 10B. Detergent extracts of total cellular protein were immunoprecipitated with anti-ka2 (left) or anti-glur6ï7 (right) antibody, and aliquots of the detergent extracts and the postimmunoprecipitate supernatants and pellets were immunoblotted with anti-glur6ï7 (left) or anti-ka2 (right). The nearly complete depletion of immunoreactivity from the supernatant in both cases suggests that the vast majority of GluR6 and KA2 proteins are present in heteromeric assemblies. Similar results were obtained in three additional experiments. The co-immunoprecipitation results might be misleading if functional plasmalemmal receptors comprise only a small fraction of the total cellular receptor pool. We therefore determined the extent to which AMPA, which activates GluR6ÏKA2 channels but not GluR6 homomers (Egebjerg et al. 1991; Herb et al. 1992), would compete with kainate for activation of the functional channels present in the cells expressing both GluR6(R) and KA2. Previous results indicate that the EC50 value for kainate in cells expressing GluR6(R) alone is about 0 5 ìò, whereas the corresponding values for kainate and AMPA in GluR6(R) KA2-expressing cells are about 1 5 and 250 ìò, respectively (Howe, 1996). Therefore, 3 mò AMPA should effectively prevent activation of GluR6(R)ÏKA2 channels by 0 5 ìò kainate, but not interfere with kainate activation of any homomeric GluR6(R) channels present. As expected from previous work (Egebjerg et al. 1991), applications of 3 mò AMPA did not produce detectable currents in three cells expressing GluR6(R) alone and did not reduce currents evoked by 0 5 ìò kainate. In contrast, 3 mò AMPA produced currents in each of the six GluR6(R) KA2-expressing cells studied. Applications of 0 5 ìò kainate gave clear inward currents when applied alone in the same cells (52 1 ± 16 0 pa, at 80 mv), but the currents were markedly reduced (97 ± 3 %) when the applications were repeated in the presence of 3 mò AMPA. The results show that the expression of homomeric GluR6 channels is minimal in our stable GluR6(R) KA2-transfected cells. Kainate displays 3-fold lower affinity for GluR6ÏKA2 heteromers than GluR6 homomeric channels. It was therefore of interest to determine whether SYM 2081 also discriminates between these two types of recombinant channels. Whole-cell currents evoked at 80 mv by SYM 2081 in a HEK 293 cell stably expressing the GluR6(R) and KA2 subunits are shown in Fig. 10Ca. Thefit to the concentration response curve (Fig. 10Cb) gave an EC50 value of 0 74 ìò. The mean EC50 and nh values determined for SYM 2081 in five cells expressing both GluR6(R) and KA2 were 0 58 ± 0 12 ìò and 1 2 ± 0 04, respectively. The corresponding values determined in GluR6(R)-expressing cells were 0 22 ± 0 02 ìò and 1 6 ± 0 4, respectively (n = 4). Thus, heteromeric GluR6(R)ÏKA2 channels show about 3-fold lower affinity for SYM 2081 than homomeric GluR6(R) channels. In addition, the coexpression of KA2 results in a shallower dependence on agonist concentration, as evidenced by the lower nh value in the cells expressing GluR6(R)ÏKA2 heteromeric channels. Both of these results are similar to those reported previously for kainate (Howe, 1996). Agonist-specific differences in ãnoise have been reported for certain types of recombinant AMPA-type channels (Swanson, Kamboj & Cull-Candy, 1997). We therefore compared the characteristics of the current fluctuations seen with kainate and SYM 2081 in our stable GluR6(R)- and GluR6(R) KA2-expressing cells. The mean ãnoise value obtained for GluR6(R) channels with 10 ìò kainate was similar to values reported previously (0 25 ± 0 04 ps, n = 14). Estimates of ãnoise obtained in three cells with 10 ìò SYM 2081 gave a mean value of 0 20 ± 0 06 ps and were close to the values obtained in the same cells with 10 ìò kainate. The mean ãnoise values determined with 10 ìò kainate and 10 ìò SYM 2081 in six GluR6(R) KA2- expressing cells were 0 74 ± 0 10 and 0 54 ± 0 04 ps, respectively. Power spectra obtained with both agonists contained two Lorentzian components with similar halfpower frequencies. The ãnoise values we have obtained for the GluR6(R)ÏKA2 channels studied here are similar to those reported previously under conditions where the relative expression of the two subunits was unknown (Howe, 1996; Swanson et al. 1996). If GluR channels are pentameric, then the relative abundance of GluR6 and KA2 in the clonal line examined suggests that, on average, the channels have a GluR6 : KA2 stoichiometry of approximately 3:2. We have found no evidence for agonist-specific differences in ãnoise for either homomeric or heteromeric kainate-type channels. DISCUSSION Identification of kainate-type channels in granule cells One of our main findings is that exposure of granule cells to ConA reveals a component of steady-state currents that are activated by low concentrations of kainate. Several lines of evidence support the conclusion that the channels underlying these currents are of the kainate subtype.