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Kainate Receptor Subunits Expressed in Single Cultured Hippocampal Neurons: Molecular and Functional Variants by RNA Editing

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Kainate Receptor Subunits Expressed in Single Cultured Hippocampal Neurons: Molecular and Functional Variants by RNA Editing View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Neuron, Vol. 14, 1009-1017, May, 1995, Copyright © 1995 by Cell Press Kainate Receptor Subunits Expressed in Single Cultured Hippocampal Neurons: Molecular and Functional Variants by RNA Editing Diego Ruano,* Bertrand Lambolez,* the DNA and RNA sequences occurs, originating molecu- Jean Rossier,* Ana V. Paternain,t lar variants of the same subunit. In three glutamate recep- and Juan Lermat tor subunits, the AMPA receptor subunit GluR2 (GluR-B; *lnstitut Alfred Fessard Kein&nen et al., 1990; Boulter et al., 1990; Nakanishi et Centre National de la Recherche Scientifique al., 1990) and the kainate receptor subunits GluR5 (Bettler 91198 Gif-sur-Yvette Cedex et al., 1990) and GluR6 (Egebjerg et al., 1991), a single France n ucleotide exchange as a result of RNA editing determines tDepartamento de Plasticidad Neural the presence of a glutamine (Q) or arginine (R) residue in Instituto Cajal the Q/R site located in the second transmem brane domain Consejo Superior de Investigaciones Cientificas (Sommer et al., 1991). Recently, two additional sites gen- 28002 Madrid erated by editing, named IN and Y/C, located in the first Spain transmembrane domain have also been described in the GluR6 subunit (KShler et al., 1993). These three residues are critical determinants of calcium permeability and recti- Summary fication properties of kainate receptors as seen in homo- meric GluR6 channels expressed in host cells (KShler et To determine the kainate receptor subunits that are al., 1993; Egebjerg and Heinemann, 1993). In particular, found in native kainate receptors, we have applied a the editing of the Q/R site in the GluR6 subunit seems to multiplex PCR of cDNAs reverse transcribed from govern the rectification properties in homomeric GluR6 mRNA harvested from single cultured hippocam- channels (Herb et al., 1992; KShler et al., 1993). The uned- pal neurons after electrophysiological recording. We ited version encodes a glutamine and exhibits strong in- found that all the cells showing rapidly desensitizing ward rectification, whereas the edited version encodes an currents in response to kainate express the GluR6 sub- arginine at this position and does not rectify (Egebjerg et unit mRNA, and that some of them also express the al., 1993). However, calcium permeability seems to be GluR5 subunit mRNA. No GluR7, KA-1, or KA-2 subunit controlled by the Q/R site whenever both the IN and mRNAs were detected. Analysis of the editing sites of Y/C sites are edited (K6hler et al., 1993). the GluR6 mRNA demonstrated that the three editing Native kainate receptor channels have been character- sites present in these subunits are edited to a different ized only recently, and little is known about their subunit extent. Predominant expression of the unedited vari- composition (see Feldmeyer and Cull-Candy, 1994). Rap- ant (Q) was observed, but edited and unedited variants idly desensitizing responses upon kainate application may coexist in the same cell. In addition, we show that were first described in the peripheral nervous system the Q/R site from the GluR6 subunit controls functional (Huettner, 1990) and more recently in hippocampal neu- properties of native kainate receptors. rons (Lerma et al., 1993) and glia (Patneau et al., 1994). The subunit composition of these kainate receptors in cul- Introduction tured hippocampal neurons is unknown. Recently, a com- bination of whole-cell patch clamp recording and analysis The non-NMDA ionotropic glutamate receptors have been by polymerase chain reaction (PCR) in single identified classified into two different groups according to their neurons has been used to address the question of the binding affinity for the agonists a-amino-3-hydroxy-5- molecular composition of native glutamate receptors methylisoxazole-4-propionate (AMPA) and kainate (Mon- (Lambolez et al., 1992; Audinat et al. 1994; Bochet et al., aghan et al., 1989; Watkins et al., 1990). Five subunits, 1994; Jonas et al., 1994). This method was modified to named GluR5 (Bettler et al., 1990), GluR6 (Egebjerg et characterize, from a molecular point of view, native gluta- al., 1991), GluR7 (Bettler et al., 1992), KA-1 (Werner et mate receptors of the kainate type. Following a multiplex al., 1991), and KA-2 (Herb et al., 1992), have been demon- PCR (mPCR) protocol, all the kainate receptor subunits strated to generate glutamate receptors with high affinity were independently tested from the same cell. We have for kainate in expression systems (Bettler et al., 1992; Herb also analyzed RNA editing in the PCR-generated GluR6 et al., 1992; Lomeli et al., 1992; Werner et al., 1991). How- fragments derived from these cells in correlation with their ever, only the GluR5 and GluR6 subunits can form func- functional properties. tional channels when expressed in vitro (Egebjerg et al., 1991; Sommer et al., 1992; K6hler et al., 1993). The map- Results ping of the mRNAs for these subunits in the central ner- vous system by in situ hybridization has revealed a differ- mPCR ential distribution, with GluR6 and KA-2 being the most The mPCR assay was designed to amplify the five different widely expressed (Wisden and Seeburg, 1993). kainate receptor subunits (GluR5-7 and KA-1 and KA-2) One source of potential heterogeneity in glutamate re- expressed in single cells. For this purpose, a first round ceptor subunits is RNA editing. Editing of RNA is a phe- of PCR mixing the four specific primer pairs for GluR5, nomenon in which a single nucleotide exchange between GluR6, GluR7, KA-1, and KA-2 (these subunits were ampli- Neuron 1010 Figure 1. Kainate Receptor Subunits Present A B in Whole Rat Brain and Purkinje Cells (A) cDNA reverse transcribed from RNA ex- tracted from whole brains of 21-day-old rats M RS R6 R7 KA M M RSR6 R7KA M was used (0.5 ng) as a template to amplify the high affinity kainate receptor subunits, using the mPCR protocol (see Experimental Proce- dures). The 208, 259, 421, and 512 bp bands 603 correspond to the GluR5, GluR6, GluR7, and KA-1/KA-2 PCR-generated fragments, respec- 310 tively. 234 (B). The same protocol was applied to RNA harvested from a cerebellar Purkinje cell. The molecular weight markers are as in (A). Note the absence of amplification of GluR6 and GluR7 subunits in cerebellar Purkinje cell. Two other Purkinje cells were analyzed with similar Whole brain cDNA Purkinje cell results. Lanes M in (A)and (B), FX174 Haelll molecular weight markers. fled using the same pair of primers) subunits was per- for GluR5, GluR6, GluR7, KA-1, and KA-2 subunits, re- formed. The PCR products were subsequently purified spectively (data not shown). The mPCR assay was then and used in a second round of PCR as a template to am- used to amplify specific kainate receptor subunits ex- plify the different subunits. In this second round, each sub- pressed in single Purkinje cells from rat cerebellar slice unit was amplified independently using its specific pair of cultures. As expected from in situ hybridization results in primers, resulting in four PCR products corresponding to Purkinje cells (Wisden and Seeburg, 1993), neither GluR6 GluR5, GluR6, GluR7, KA-1, and KA-2 cDNAs, respec- nor GluR7 were detected, but GluR5, KA-1, and KA-2 sub- tively. To evaluate the reliability of this assay, mPCR was unit mRNAs were observed (Figure 1B). Thus, all of the used to amplify the five different kainate receptor subunits kainate receptor,.subunits in whole rat brain, as well as using RNA purified from adult rat brain. Figure 1A is an specific subunits expressed at the single cell level, could ethidium bromide-stained gel demonstrating that all of the be amplified by the mPCR protocol. kainate receptor subunits were amplified when cDNA from whole rat brain was used as a template. Fragments of the Kainate Receptor Subunits Expressed by Single predicted size were obtained, and they were identified by Hippocampal Cells Southern blot analysis with specific probes and specific The electrophysiological properties of the glutamate re- restriction enzymes: Bbvl, Maell, Alul, EcoRI, and Kpnl ceptors of the kainate subtype in single cultured neurons Figure 2. Responses to Kainate and Southern A Cell #1 B Cell #18 Blot Analysis of Glutamate Receptor Subunits Kainate AMPA in Cultured Hippocampal Neurons Kainate AMPA (A and B) Whole-cell currents evoked by rapid ........... application of kainate (300 I~M) and S-AMPA (200 ~M) are illustrated for two cultured hippo- campal cells voltage-clamped at -60 mV, which were subjected to RT-mPCR analysis. The duration of agonist application is indicated I 5o 100 1lo by the bar above each trace. Note the lack of pA pA pA response to AMPA of the cell shown in (B). (C) The cDNA fragments obtained after RT- 400 ms 400 ms mPCR corresponding to the GluR5-7 and KA-1 and KA-2 subunits from 11 cultured hippocam- pal cells, responding to kainate as in (B), were resolved on four agarose gels, transfered onto Cells C , four nylon membranes, and hybridized with the GluR6-, GluR5-, GluR7-, and KA-1/KA-2-speci- 1 8 11 12 13 14 15 18 2425 2628 fic probes. All cells expressed the GluR6 sub- unit (first row), and in some of them GluR5 was also detected (second row). No signal was ob- GluR6 tained with GluR7 and KA-1/KA-2 probes in any cell (data not shown). Cell 1 did not respond to kainate with transient currents, but with re- sponses of the AMPA receptor-mediated type (A). No signal corresponding to the GluR5-6 GluR5 -0 (column 1) nor GluR7, KA-1, and KA-2 subunit mRNAs (data not shown) were detected in this and in two additional cells with similar electro- physiological characteristics.
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