Evidence for two types of non-NMDA receptors in rat cerebellar purkinje cells maintained in slice cultures

Pergamon

0028-3908(94)00165-O

Neuropharmacology Vol. 34, No. 3, pp. 335-346, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0028-3908/95 $9.50 + 0.00

Evidence for Two Types of Non-NMDA Receptors in Rat Cerebellar Purkinje Cells Maintained in Slice Cultures A. RENARD,

F. CREPEL

and E. AUDINAT

Laboratoire de Neurobiologie et Neuropharmacologie du Developpement CNRS ERS 0100, Bbtiment 440, Universitk Paris-&d, 91405 Orsay Cedex, France (Accepted 15 December 1994) Summary-Pharmacological properties of non-MNDA receptors were investigated in Purkinje cells grown in rat cerebellar slice cultures and recorded in the whole-cell configuration of the patch-clamp technique. Dose-response curves for AMPA and domoate suggest that AMPA, in the concentration range tested, activated only AMPA receptors whereas, domoate activated two types of receptors, probably AMPA and kainate receptors, with ECSovalues of 8 and 0.5 PM, respectively. The Scatchard analysis of the dose-response relationship for domoate also suggest that both kainate and AMPA receptors were activated by domoate with approximate affinities of 5 and 0.07 PM-‘, respectively. The non-competitive non-NMDA receptors antagonist, GYKI 52466, reduced the amplitude of both AMPA- and domoate-activated currents, with a greater potency in reducing currents evoked by AMPA (IC,, = 10 PM) than those induced by domoate (IC,, = 105 PM). These results suggest that, in addition to AMPA receptors, Purkinje cells express kainate receptors and that these two types of non-NMDA receptors can be distinguished from each other on the basis of several pharmacological properties, including affinity for AMPA, domoate and GYKI 52466. Keywords-Patch-clamp,

AMPA receptors, kainate receptors, GYKI 52466, slice cultures.

The receptor-channels gated by the excitatory amino acid glutamate have been classified into three groups named for thei:r preferential agonist, a-amino3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA). Among the non-NMDA receptors, AMPA receptors are ubiquitously expressed in neurones of the central nervous system where they mediate the majority of fast synaptic transmission. Although these AMPA channels can also be gated by kainate, they are distinct from kainate receptors which have a lhigh affinity for kainate but little or no affinity for AMPA. Molecular cloning studies have identified different glutamate receptor subunits which can also be grouped in several sub-families on the basis of their structural homologies and properties of recombinantly expresse:d receptors. AMPA preferring receptors are assembled from subunits GluRl, 2, 3 and 4 (Hollman et al., 1989; Boulter et al., 1990; Keinanen et al., 1990; Nakanishi et al., 1990; Sakimura et al., 1990) whereas kainate receptors are assembled from subunits GluR5, 6, 7, KAl and KA2 (Bettler et al., 1990; Egebjerg et al., 1991; Werner et al., 1991; Bettler et al., 1992; Herb et al., 1992; Sakimura et al., 1992; Sommer et al., 1992).

The functional distinction between AMPA and kainate receptors remains unclear and the role of kainate receptors is still largely unknown. Most of the native non-NMDA receptors which have been characterized have functional properties resembling those of AMPA recombinant channels formed from GluRl4 subunits. To date, only three examples of native kainate receptors have been characterized functionally. The first has been described in acutely dissociated neurones of the dorsal root ganglia and has certain features of GluR5 subunits (Huettner, 1990). The second example has been observed in neurones of dissociated hippocampal cells cultures and most likely corresponds to the expression of GluR6 subunits (Lerma et al., 1993). More recently, Patneau and colleagues (Patneau et al., 1994) have reported rapidly desensitizing responses to kainate in glial cells of the oligodendrocyte lineage expressing high level of GluR6 and KA2 mRNAs. Subunits of both AMPA and kainate receptors appear to .be present in cerebellar Purkinje cells. Indeed, in situ hybridization, single-cell polymerase chain reaction and immunocytochemical analysis indicate that these neurones express GluRl, GluR2 , GluR3 AMPA receptor subunits (Keinanen et al., 1990; 335

A. Renard et al.

336

Pelligrini-Giampietro et al., 1991; Lambolez et al., 1992; Petralia and Wenthold, 1992; Martin et al., 1993) as well as GluR5 and KAl kainate receptor subunits (Bettler et al., 1990; Werner et al., 1991). We have used whole-cell patch-clamp recordings to investigate the expression of distinct non-NMDA receptors in Purkinje cells of cerebellar slice cultures. Since specific antagonists that distinguish kainate from AMPA receptors have not yet been described, the presence of these two types of receptors has been inferred, initially, from the differential potency of various agonists (Hollman et al., 1989; Dawson et al., 1990; Keinanen et al., 1990; Nakanishi et al., 1990; Bettler et al., 1992). We have thus compared the responses induced in Purkinje cells by AMPA and by domoate. AMPA is relatively selective for AMPA receptors, although at millimolar concentrations it can also activate some recombinant kainate receptors (Herb et al., 1992; Sommer et al., 1992), whereas domoate is a mixed agonist acting on both AMPA and kainate receptors with slightly different affinities (Honor& et al., 1982; Huettner, 1990; Egebjerg et al., 1991; Sommer et al., 1992; Johansen et al., 1993). Domoate was preferred to kainate because the desensitization of domoate responses at kainate receptors is less pronounced than that evoked by kainate itself (Huettner, 1990; Egebjerg et al., 1991; Herb et al., 1992; Sommer et al., 1992; Lerma et al., 1993). In addition, we have tested the effects of GYKI 52466, a non-competitive antagonist of non-NMDA receptors (Tarnawa et al., 1990; Donevan and Rogawski, 1993; Zorumski et aE., 1993). Results obtained in abduncens motoneurones suggest that this compound may differentially affect the responses mediated through the activation of AMPA and those due to the activation of kainate receptors (Ouardouz and Durand, 199 1). METHODS

Cerebellar slice cultures Parasagittal cerebellar slices 350 pm thick were obtained from Shermann rate neonates and were cultured following the “roller tube” technique as previously described (Gahwiler, 1984). The cultures were fed once weekly with a medium consisting of horse serum (25%), basal medium (Eagle, 50%) and Hank’s balanced salt solution (25%) supplemented with glucose to a final concentration of 6.5 g . 1-l. Electrophysiology and drug application For electrophysiological recordings, 15- to 45-dayold cultures were transferred to a recording chamber mounted on the stage of an inverted microscope and superfused at room temperature (18-22”C), at a rate of 0.5-l ml. min-‘, with a solution containing: NaCl 140 mM, CaCl, 1 mM, MgCl, 1 mM, KC1 2.5 mM, HEPES 10 mM (pH 7.3) and glucose 10 mM. Whole-cell recordings of the patch-clamp technique (Hamill et al.,

1981) were obtained with an RK 300 amplifier (Biologic) using pipettes with a tip resistance of l-3 MR when filled with the following internal solution: CsCl 140 mM, CaCl, 0.5 mM, MgCl, 3 mM, EGTA 5 mM and HEPES 10 mM (pH 7.2). The signal was filtered at 1 kHz and fed into a data acquisition system interfaced with an MS-DOS computer for on- and off-line analysis and reproduction using the Acquis 1 software developed by G. Sadoc. During drug application, the membrane potential was usually maintained at -60 mV. However, when high concentrations of agonist were used, recordings were often performed at -20 or +20 mV in order to reduce the amplitude of the agonist induced current and, therefore, to minimize the error due to the voltage drop through the uncompensated series resistance. In these cases, the linearity of the current/voltage relationship was first assessed with low concentrations of the tested agonist. Agonists and antagonists of the glutamate receptors were diluted in the external solution described above and were always supplemented with 0.5 PM tetrodotoxin (TTX) and sometimes with 20pM bicuculline. The drug-containing solutions were applied by local perfusion from a linear array of 7 tubes (i.d. 200pm) mounted on a hydrolic micromanipulator and positioned 5&100pm from the soma of the recorded neurone. AMPA and domoate were purchased from Tocris Neuramin (Bristol, U.K.). GYKI 52466 was generously provided by Dr I. Tarnawa (Institute for Drug Research, Hungary). All other drugs were obtained from Sigma. Data analysis A modification of the Scatchard analysis was introduced to analyze the experimental dose-response curve for domoate which could not be fitted properly with the logistical function used for the analysis of the AMPA responses (see Results section). The graphic representation of Scatchard is described by the equation: v/[A] = n&-v&

(1)

where n is the number of sites per receptor, K, is the association constant and v is the saturation fraction which corresponds to the fraction of protein which have bound the ligand (v = (PA)/[(PA) + (P)], with P representing the unbound protein and PA the protein bound by its agonist A). The dose-response curve for domoate was obtained by measuring the current induced by the agonist at the steady-state of the responses. In a simplified kinetic scheme, we can assume that an equilibrium has been reached between the different forms of the receptor, i.e. unbound closed receptors (R), bound receptors in an open state (RoA) and bound receptors in a desensitized state Rn A.The saturation fraction, v, can therefore be defined by (R,A+ R,A)/(R+ ReA+ RnA) and

Non-NMDA

receptors in Purkinje cells

NMDA receptors were activated in Purkinje cells by AMPA and low doses of domoate, they could not be distinguished on the basis of the voltage-dependence of their effects.

equation (1) is now equivalent to: (RoA + RnA) (RoA + RnA)K, (R+RoA+R,A)[P;i=nKa-(R+RoA+R,A)’ (2) We assume that the amplitude of the largest response evoked by a saturating concentration of domoate was proportional to the total number of receptors of the recorded cells, i.e. IR + R,A + R,A. Therefore, the amplitude of the current induced by a given concentration of domoate normalized to the amplitude of the maximal response was used to approximate the saturation fraction. Scatchard representation was thus obtained by plotting the ratio of the normalized response to the concentration as a function of the normalized current. An approximation of K, was obtained by determining the slope of the least square fit of this plot. Since the currents induced by domoate were measured at a steady-state we could not estimate the proportion of desensitized receptors which will have to be taken into account in the interpretation of the Scatchard analysis (see Discussion section’).

IRESULTS Whole-cell recordings were obtained from Purkinje cells grown 15545 days in vitro in cerebellar slice cultures. In a first set of experiments, we aimed to investigate the presence of kainate receptors in these neurones by analyzing the desensitization of the responses induced by the mixed agonist kainate. Recent reports have shown a differential modulation by cyclothiazide and concanavalin A of desensitization at AMPA and kainate receptors (Patneau et al., 1993; Wong, 1993). Unfortunately, the large size of Purkinje cells and the fast kinetics of desensitization at non-NMDA receptors made difficult an accurate study of this phenomenon in these neurones. In addition, in our hands, concanavalin A had none effects on non-NMDA receptors expressed by neurones maintained in slice cultures (n = 5). Therefore, we based our study on the comparison of the steady state responses induced by AMPA and by domoate (see Introduction section). Current-voltage relationships of AMPA induced responses

331

and domoate-

The selective activation of AMPA receptors of Purkinje cells by 2.5 ZcM AMPA induced responses characterized by a linear current-voltage relationship [Fig. l(A)] with a reversal potential near 0 mV (n = 4). When Purkinje cells were maintained at -60 mV, 0.1 PM domoate induced a slowly rising, non-desensitizing inward current (Fig. 1). The amplitude of the domoate currents varied linearly between -60 and +60 mV with a reversal potential close to 0 mV [n = 4; Fig. l(B)]. Thus, if two different types of non-

Dose-response

curves for domoate and AMPA

Figure 2(A) illustrates the response of a Purkinje cell to the application of 5 and 50,uM AMPA. The response to 50 PM AMPA was characterized by an initial peak of current that decayed to a lower steadystate level during the application of the agonist which reflects the desensitization of the receptors. Steady state responses to AMPA increased for agonist concentrations between 0.3 PM and 60 PM. This is illustrated by the concentration-response curve shown on Fig. 2(A) which could be described by the following equation: Z = Z,,,

. (l/l + W,o/H”)

(3)

where Z is the observed current, I,,,, the maximum current, [A] is the agonist concentration, EC,, is the concentration that evoked a half maximal response, and n is the Hill coefficient. The fit of the data shown in Fig. 2(A) was obtained with EC,, and n equal to 6.2 PM and 1.46, respectively. This value of n indicated that more than one molecule of agonist was required for channel gating, suggesting a possible cooperative action of several AMPA subunits. In all tested Purkinje cells (n = 15) held at a membrane potential of -60 mV, inward currents were also evoked by concentrations of domoate ranging from 0.05 to 100 PM [Fig. 2(B), inset]. In some Purkinje cells (n = 3) responses could be detected with a domoate concentration as low as 0.01 PM. The amplitude of the responses increased with agonist concentration up to 100pM. Under these experimental conditions, i.e. a slow system for drug application, no marked desensitization was detected at any tested concentration of domoate [Fig. 2(B), inset]. The dose-response curve for domoate varied on at least three orders of magnitude on the logarithmic scale of domoate concentrations between 0.01 and 100pM [Fig. 2(B)]. The asymmetry of the experimental curve precluded a proper fit of the data points with equation (3). This led us to perform a Scatchard analysis of the concentration-response relationship for domoate, with the assumptions described in the Methods section. This Scatchard plot of the responses induced by domoate was characterized by two components [Fig. 3(B)]. The slopes of the linear regression of the first and the second components were 5.14 and 0.066 PM-‘, respectively, which suggests that domoate binds two types of receptors on Purkinje cells, probably kainate and AMPA receptors, with an apparent dissociation constant of 0.19 and 15 PM, respectively. Moreover, this Scatchard plot suggests that the contribution of AMPA and kainate receptors to the maximal responses to domoate were 90% and lo%, respectively. When these values were introduced

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Fig. 1. Voltage dependence of the responses induced in Purkinje cells by (A) AMPA and (B) domoate. (A) Left, whole-cell currents induced in Purkinje cell by the application of 2.5 /.IM AMPA at various holding potentials indicated on the left of each recording. Right, plot of the AMPAevoked current as a function of the holding potential. (B) Left, for another Purkinje cell, whole-cell currents induced by 0.5 PM domoate at various holding potentials and, right, the corresponding plot of the current as a function of the holding potential. 338

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Fig. 2. Dose-response relations for AMPA and domoate in Purkinje cells. (A) Dose-response curve for AMPA. Each point represents the mean + SD of the steady state current amplitude of 611 cells plotted as a fraction of the maximal current. Inset, Currents activated by 5 or 50 PM AMPA in a Purkinje cell at a holding potential of -60 mV. Experimental points were fitted by equation (3) (see Results section) with C,,, and n equal to 6.2 PM and I .46, respectively. (B) Dose-response curve for domoate. Each point represents the mean f SD of the steady state current amplitude of 3-15 cells plotted as a fraction of the maximal current. Inset, Currents activated by 0.5 or 50 PM domoate in a Purkinje cell at a holding potential of -60 mV. Experimental points were fitted by the sum of two dose-response curves (solid line) (see in Results section). The dashed lines are dose-response curves for the individual high and low components. Domoate activates AMPA receptors with EC, and nH equal to 8.6 PM and 1.7, respectively. Domoate activates kainate receptors with EC, and trn equal to 0.48 PM and 0.5, respectively.

A. Renard et al.

340 in a logistic dose-response I=

L,,

equation curves:

consisting

of the sum of two

- W/l + W,,,/L’W3 + (W 1 + (EC,, /[Al);)

(4)

Fl and F2 were the fractions of each type of non-NMDA receptors, a reasonable fit was obtained [Fig. 2(B)], which allowed in turn to determine that domoate activates kainate and AMPA receptors with ECSoequal 0.5 and 8.6 PM, respectively. In addition, the Hill coefficients were respectively equal to 0.5 and 1.5 for kainate receptors and AMPA receptors suggesting that at least two molecules of domoate were required for gating channel coupled to AMPA receptors whereas only one molecule of domoate seems sufficient to activate kainate receptors. where

Eflects of GYKI responses

52466 on domoate and AMPA -induced

The effect of the non-competitive antagonist, GYKI 52466, was tested on the responses of Purkinje cells to AMPA. As illustrated in Fig. 4(A), 60 ,uM GYKI 52466 reduced the amplitude of the AMPA current to < 10% of control. This inhibition of AMPA currents by GYKI 52466 was voltage independent (not shown). As expected for a non-competitive antagonist, the percentage of inhibition of the AMPA-induced currents by GYKI 52466 was identical for responses induced by 100 PM or by 1 PM AMPA, 98% f 1.4% (n = 5) and 99% f 1% (n = 7), respectively. GYKI 52466 also reduced the amplitude of domoateevoked currents and, at all concentrations of agonist tested, this inhibition was not dependent on the potential at which the membrane was held [Fig. 4(C)], suggesting that GYKI 52466 do not act as an open-channel blocker on the non-NMDA receptors of Purkinje cells. However, GYKI 52466 was less efficient in blocking the responses to domoate than those to AMPA. Moreover, in all tested cells the percentage of inhibition of the domoate-activated currents was dependent on the agonist concentration [Fig. 4(B)]. 60 PM GYKI 52466 reduces the average amplitude of the responses to

50 PM and 0.1 PM domoate by 70% rt 7.6% (n = 7) and 33.7% + 4.1% (n = 7), respectively. These results cannot be explained by a competitive inhibition of the domoate responses by GYKI 52466, rather, they suggest that GYKI 52466 inhibits two types of domoate gated receptor-channels with different affinities. Accordingly, we have studied the effect of increasing concentration of GYKI 52466 on the responses induced by AMPA and by low doses of domoate in order to determine the affinity of GYKI 52466 for the receptors activated by both agonists. Figure 5 shows the concentration-dependent block by GYKI 52466 (0.03-600 PM) of the steady-state currents evoked by 10pM AMPA (closed square) and by 0.1 ,uM domoate (closed triangle). We have not tested doses of GYKI 52466 >600 PM because of its limited solubility in saline solution. The AMPA responses were inhibited by GYKI 52466 with an approximate IC,, of 10pM. In contrast, the currents evoked by 0.1 ,UM domoate were not significantly affected by concentrations of GYKI 52466 < 10 PM and the IC,, obtained in this case was 105 PM. This indicated that the affinity of GYKI 52466 for the receptors gated by 0.1 PM domoate was lower than the affinity for receptors gated by AMPA. The Hill coefficients for GYKI 52466 inhibition of currents induced by 10 PM AMPA and by 0.1 ,uM domoate were 0.80 and 1 respectively, suggesting that this antagonist interacts with only one site on AMPA and kainate receptors. Finally, we analyzed the effect of GYKI 52466 on the entire dose-response curve for domoate. For each neurone, six different concentrations of domoate were applied in the presence of 6 PM GYKI 52466. In addition, the responses to 10 PM domoate applied with and without the antagonist were also measured to normalize the data to the dose-response curve for domoate obtained in the absence of GYKI 5246. Figure 3 (inset) shows the response of a Purkinje cell to 10 PM domoate in the absence of the antagonist (left panel) and the time-dependent blocking effect of 6 PM GYKI 52466 on this response (right panel). All the responses were measured when the currents had reached a steady-state (usually 5 set after the start of the

(Fig. 3 Opposite) Fig. 3. Effect of GYKI 52466 on the dose-response relations for domoate. (A) The dose-response relations for domoate obtained in the presence of 6 FM GYKI 52466 is represc,rted by the empty squares and has been normalized to the dose-response curve for domoate obtained without the antagonist, already shown in Fig. 2 and indicated here with the filled squares for comparison. Experimental points represent the mean f SD of the normalized values from 5 to 7 cells. (B) and (C) Scatchard analysis of the dose-response relations for domoate obtained without (B) or with 6 PM GYKI 52466 in the external bathing solutions (C). Scatchard plot is derived from normalized responses to domoate shown in (A) and represents the ratio of the normalized responses over the agonist concentration (bound/free) as a function of the normalized responses (bound). The biphasic curve corresponds to the sum of two linear regressions according to the equation v/(A) = nK, - v& where v is the fraction saturation, n is the sites number per receptor, and K, is the apparent association constant and represent the slope of each curve. The normalized response was used to approximate the saturation fraction of the receptors (see text for further details). The two association constants for the high and the low affinity sites were equal to 5.14 and 0.066 PM-’ respectively in absence of GYKI 52466. In presence of 6 PM GYKI 52466, domoate binds both AMPA and kainate receptors with apparent affinity equal to 4.7 and O.l2pM-i, respectively.

Non-NMDA

receptors in Purkinje cells

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Fig. 4. Inhibition by GYKI 52466 of the AMPA and domoate-induced responses. (A) Effects of the non competitive antagonist GYKI 52466 on the AMPA-induced currents. Comparison of the elfects of 60 PM GYKI 52466 on the whole-cell currents induced by 100 or 1 PM AMPA. Note that the percentage of inhibition by GYKI 52466 is close to 100% at both concentrations of agonist. (B) Comparison of the effects of 60 PM GYKI 52466 on the currents induced by 50 or 0.1 PM domoate. Note that the blocking action of GYKI 52466 is less pronounced for the lowest concentration of agonist. (C) Left, inhibition by 60 PM GYKI 52466 of the whole-cell currents evoked by 0.5 PM domoate at various holding potentials and, right the correspondings current-voltage relations for the responses obtained in the absence (m) and presence (0) of GYKI 52466. 342

Non-NMDA

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in Purkinje

343

cells

1.2

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-0.2 GYKI pM Fig. 5. Concentration-response relationships for block of steady state AMPA and domoate currents by GYKI 52466. Plot of the fractional block of the responses induced by 0.1 PM domoate (A) and 10 PM AMPA (m) as a function of GYKI 52466 concentration. Data points represent the mean f SD of the fractional block values from 9 experiments with 0.1 PM domoate and 6 experiments with 10pM AMPA. The curves represent the non-linear fit of the data points according to the logistical function of the noncompetitive inhibition model where [GYKI] is the concentration of GYKI 52466, IC, is the 50% blocking l/l + (IC,,/[GYKl])“” concentration, and nH is the slope factor (0.9-l for these two curves).

the antagonist only for concentrations of domoate > 1 ,uM but not for lower concentrations (Fig. 3). This observation was confirmed by the comparison of the Scatchard analysis obtained in the absence and in the presence of 6pM GYKI 52466 [Fig. 3(B) and (C)l.

In the presence of 6pM GYKI 52466 [Fig. 3(C)], the Scatchard plot was still characterized by two components. However, the second component, corresponding to the low affinity site for domoate, was much more reduced than the component representing the high

A. Renard

344

affinity site for domoate. The relationship could be fitted with two linear regressions with slopes equal to 0.12 and 4.7 PM-‘, respectively. DISCUSSION Our results suggest that Purkinje cells express two types of non-NMDA receptors, most likely AMPA and kainate receptors, which can be distinguished on the basis of several pharmacological properties. Domoate binds kainate and AMPA receptors of Purkinje cells with apparent dissociation constants of about 0.20 and 15 PM, respectively. By contrast, in the concentration range tested, AMPA activates only AMPA receptors with an ECSoof 6 PM. This latter value is similar to that found for recombinant heteromeric channels formed by the combination of GluRl, 2 and 3 (Nakanishi et al., 1990) the three AMPA-receptor subunits expressed by Purkinje cells (Lambolez et al., 1992). Finally, GYKI 52466 is a much more efficient antagonist of the responses mediated through the activation of AMPA receptors than those mediated via kainate receptors. The first indication that AMPA and kainate receptors are co-expressed in Purkinje cells was obtained by analyzing the dose-response curve for domoate. Because this curve could not be fitted by a form of a Michaelis-Menten equation but with the sum of two Michaelis-Menten equations (see Results section), the data were analyzed according to a modification of the Scatchard analysis (see Methods section). The Scatchard plot for domoate is characterized by two components, suggesting the presence of two binding sites with different affinities for the agonist. However, this analysis is based on the measurement of currents which reflect only the number of receptor-channels in an open state and assumes that, for a given class of receptors, the conductance of the channel does not change with the agonist concentration. Rather than reflecting the presence of two types of non-NMDA receptors, one might argue that the biphasic shape of the Scatchard plot for domoate could be explained solely by the properties of AMPA receptors. In particular even though domoate is usually considered as a non-desensitizing agonist at AMPA receptors, a desensitization with kinetics too fast to be detected in our experimental conditions cannot be totally dismissed (Patneau et al., 1993). If desensitization corresponds to an open state with lower levels of conductance, as has been suggested for AMPA receptors (Tang et al., 1989), then a fraction of the current could be carried by desensitized receptors which are supposed to have a higher affinity for the agonist than nondesensitized receptors (Trussell et al., 1988; Patneau et al. 1991; Randy et al., 1993). In this case, a Scatchard plot with two components could be obtained with the additional assumption that the proportion of desensitized channels varies as a function of the agonist concentration. However, in the present study, the high affinity component of the Scatchard graph for domoate corre-

et al.

sponds to the low concentrations of agonist. Therefore, this would imply that AMPA receptors are more desensitized by low concentrations than by high concentrations of domoate, which seems unlikely. On the other hand if desensitization corresponds to a closed state of the receptors, then the amplitude of the current induced by domoate represents an under-estimation of the number of bound receptors, proportional to the level of desensitization. On the Scatchard plot, this underestimation would affect both coordinates of each experimental points (see Methods section) leading to a modification of the slope of the plot but not of its biphasic character. Moreover, when fractions of total domoate-induced current carried by AMPA and kainate receptors determined from Scatchard plot were introduced in a logistic equation consisting of the sum of two dose-response curves, a good fit of the dose-response curve for domoate was obtained. Therefore, these results favor the hypothesis that both AMPA and kainate receptors are present in cerebellar Purkinje cells and indicates that, concentrations of domoate < 1 FM selectively activate kainate receptors but not AMPA receptors in these neurones. Because desensitization of the responses at both types of receptors cannot be totally excluded from consideration, the values of the dissociation constants determined by our analysis are amenable to revision. Nevertheless, the EC,, observed for the high affinity component of the domoate responses in Purkinje cells (EC,, = 0.5 PM) compares favorably the EC,, of 0.7 PM determined from the concentration-response curve for domoate at kainate receptors of dorsal root ganglion (Huettner, 1990). The hypothesis that Purkinje cells co-express two types of non-NMDA receptors is also supported by the differential effects of GYKI 52466 on AMPA and domoate induced responses. GYKI 52466 inhibits the currents evoked by AMPA in Purkinje cells with an IC,, of 10 PM. This inhibition does not depend on the concentration of AMPA. These observations are in agreement with previous reports indicating that GYKI 52466 blocks AMPA receptors of hippocampal neurones in a non-competitive manner with an IC,, of 7.5 PM (Donevan and Rogawski, 1993; Zorumski et al., 1993). As reported by Donevan and Rogawski (1993) in hippocampal neurones, GYKI 52466 also antagonizes in a voltage-independent manner the responses induced by AMPA and domoate in Purkinje cells. Furthermore, this blocking effect seems also to be mediated via a noncompetitive mechanism since, on the Scatchard plot, it does not modify significantly the apparent association constants of domoate either at its low or high affinity sites. However, the percentage of inhibition by GYKI 52466 is larger for the responses induced by high concentrations of domoate than for those evoked by lower concentrations of the agonist. The effect of GYKI 52466 on the dose-response curve for domoate is explained by its higher affinity for the receptors which are activated by AMPA and by high concentrations of domoate than

Non-NMDA

receptors in Purkinje cells

for those activated by low concentrations of domoate. Indeed, the IC,, of GYKI 52466 for the responses induced by 0.1 PM domoate is at least ten times higher than for those evoked by AMPA (see above and Fig. 5) and by 50 FM domoate l(IC,, equal 12.9pM, not shown). This strongly suggests that GYKI 52466 discriminates between two types of non-NMDA receptors expressed by Purkinje cells and may therefore be a powerful tool to study native kainate receptors in isolation. Our results thus provide pharmacological evidence for the expression of functional kainate receptors in Purkinje cells. mRNAs encoding the GluR5 and KAl subunits of kainate receptors have been detected in these neurones by in situ hybridization (Bettler et al., 1990; Werner et al., 1991). The actual subunit composition of kainate receptors expressed by Purkinje cells is not known but one might expect that GluR5 and KAl subunits are combined in heteromeric structures since KAl subunits do not form functional channels in the homomeric configuration (Werner et al., 1991). Unlike the GluRS-KA2 combination (Herb et al., 1992), the properties of GluRS-KA.1 heteromeric channels have not yet been investigated in expression studies with cloned subunits. Kainate receptors of Purkinje cells appear to have features in common with those described in sensory neurones of the dorsal root ganglia (Huettner, 1990) which are likely to result from the expression of GluR5 (Bettler et al., 1990) and probably KA2 (Herb et al., 1992). In both cell types, the affinity for domoate is close to 0.5 PM and the current/voltage relation of the responses induced by ,domoate are linear (Huettner, 1990). A similar affinity for domoate has also been found in the case of cloned GluR5 homomeric channels but these receptors are characterized by an inwardly rectifying current/voltage relation (Sommer et al., 1992). As recently reported ‘by Partin and colleagues (1990) on recombinant glutamate receptors in Xenopus oocytes, cyclothiazide blocked desensitization of AMPA receptors whereas concanavalinA has much stronger effects on desensitization at kainate-but not AMPAreceptors. However, in our hands, concanavalinA did not significantly potentiate the steady state currents induced in Purkinje cells by low concentrations of domoate or kainate (not shown). This lack of sensitivity to ConcanavalinA coulsd simply be explained by the subunits composition of kainate receptors expressed in Purkinje cells. ConcanavalinA has not yet been tested on all the possible combinations of kainate subunits. The number of these combinations increases by the existence of several spliced and edited variants of each subunit (Kolher et al., 1993) and therefore we do not wether a high sensitivity to concanavalinA is actually the “hallmark” of all kainate receptors. The amount of current carried by kainate receptorchannels in response to a high concentration of a mixed agonist such as domoate represents a minor part of the total current flowing through non-NMDA channels. This is likely to be due to a smaller number of kainate

345

receptors as compared with AMPA receptors. However, the proportion of kainate receptors may be underestimated if the conductances of these receptor-channels are lower than that of AMPA receptors. Single-channel studies are now required to gather more information on the functional characteristics of native kainate receptors and to evaluate their physiological role in Purkinje cells. would like to thank Dr Corradeti and Dr Capiod for valuable comments and suggestions, Ryan Dammerman for his help on the manuscript and Nathalie Gibelin for excellent technical assistance. This work was supported by grants from Institut National de la Sante et de la Recherche Medicale (C.R.E. 930801). Acknowledgements-We

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