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43kD Proteinin Vitro
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Molecular and Cellular Neuroscience 8, 430–438 (1997) Article No. CN970597
Clustering of GABAA Receptors by Rapsyn/43kD Protein in Vitro Shi-Hong Yang, Paul F. Armson, Jeon Cha, and William D. Phillips1 Institute for Biomedical Research, Department of Physiology, University of Sydney, Sydney, New South Wales 2006, Australia
Rapsyn, a 43-kDa protein on the cytoplasmic face of the postsynaptic membrane, is essential for clustering acetylcholine receptors (AChR) at the neuromuscular junction. When transfected into nonmuscle cells (QT-6), rapsyn forms discrete membrane domains and can cluster AChR into these same domains. Here we examined whether rapsyn can cluster other ion channels as well. When expressed in QT-6 cells, the GABAA receptor (human a1, b1, and g2 subunits) and the skeletal muscle sodium channel were each diffusely scattered across the cell surface. Rapsyn, when co-expressed, clustered the GABAA receptor as effectively as it clustered AChR in previous studies. Rapsyn did not cluster co-transfected sodium channel, confirming that it does not cluster ion channels indiscriminately. Rapsyn mRNA was detected at low levels in the brain by polymerase chain reaction amplification of reverse-transcribed RNA, raising the possibility of a broader role for rapsyn.
INTRODUCTION The clustering of neurotransmitter receptors in the postsynaptic portion of the cell membrane is a characteristic feature of a variety of chemical synapses in both the central and the peripheral nervous systems (Fertuck and Salpeter, 1976; Marshall, 1981; Triller et al., 1985; Killisch et al., 1991; Craig et al., 1994). Recently, the intracellular domains of the receptors, and associated cytoplasmic proteins, have been implicated in the spatially distinct clustering of glycine receptors and NMDA receptors (Kirsch et al., 1993; Kornau et al., 1995; Ehlers et al., 1996). However, the best studied example of postsynaptic receptor clustering is at the skeletal neuromuscular 1 To whom correspondence should be addressed at Institute for Biomedical Research and Department of Physiology (F13), University of Sydney, Sydney, New South Wales 2006, Australia. Fax: 612 9351-2058. E-mail: [email protected].
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junction where acetylcholine receptor (AChR) clustering contributes to the high efficacy of this synapse (Mathews and Salpeter, 1983). During synapse formation, the postsynaptic AChR cluster is thought to be induced to form by substances such as agrin, secreted by the nerve terminal (Magill-Solc and McMahan, 1990). Agrin activates the skeletal muscle transmembrane tyrosine kinase, MuSK, which leads to AChR clustering in the postsynaptic portion of the muscle cell membrane (Glass et al., 1996). While extracellular proteins such as agrin serve as signals to induce localized AChR clustering, the task of marshalling receptors into a high-density patch falls to intracellular proteins that are concentrated in the postsynaptic membrane. One of these, rapsyn/43kD (Frail et al., 1988), is a peripheral membrane protein that associates with the inner face of the plasma membrane with the aid of an amino-terminal myristate group (Musil et al., 1988; Phillips et al., 1991b). Rapsyn is thought to bind to AChR, immobilizing it in discrete membrane domains (Froehner et al., 1990; Phillips et al., 1991a). In turn, rapsyn may interact with the dystrophin-associated protein complex (Ervasti and Campbell, 1991) by binding to dystroglycan (Apel et al., 1995). Utrophin, which is closely co-localized with clustered AChR (Ohlendieck et al., 1991), can also bind to the dystrophinassociated protein complex (Matsumura et al., 1992; Kramarcy, et al., 1994) and probably helps to stabilize large AChR clusters (Phillips et al., 1993; Campanelli et al., 1994; Gee et al., 1994; Apel et al., 1995). An 87-kDa carboxy-terminal homologue of dystrophin may also play a role in formation and/or stabilization of the postsynaptic AChR clusters (Wagner et al., 1993). Thus rapsyn is thought to connect AChR to the membrane skeleton at sites of clustering. Considerable evidence supports the view that rapsyn 1044-7431/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
Rapsyn Clusters GABAA Receptors
serves as a linchpin in clustering AChR at the synapse. Rapsyn and AChR can be chemically cross-linked, suggesting a close one-to-one interaction at the membrane face (Burden et al., 1983). AChR clustered by rapsyn in heterologous cell types displays increased resistance to extraction with the non-ionic detergent, Triton X-100, suggesting a linkage to the cytoskeleton (Phillips et al., 1993). Finally, in rapsyn-deficient mice, neither AChR, nor dystroglycan, nor utrophin assembled into discrete clusters in the postsynaptic membrane (Gautam et al., 1995). Studies outlined above in which rapsyn and AChR were co-transfected into fibroblasts support the hypothesis that rapsyn helps to form a specialized postsynaptic membrane domain where it binds to and immobilizes AChR. Here we have employed the fibroblast expression system to explore the generality of this interaction and report that rapsyn can cluster a GABAA receptor (a member of the same receptor super family) but not a voltage-gated sodium channel.
RESULTS Co-expression of Rapsyn and GABAA Receptors The quail fibroblast cell line QT-6 was first transfected with GABAA receptor (combination of human a1, b1, and g2 subunits; Pritchett et al., 1988, 1989) alone. Cells stained prior to fixation with a monoclonal antibody specific for an extracellular epitope on the a1 subunit revealed a diffuse, fairly uniform pattern across the cell surface (Fig. 1B). Following transfection, only a minority of cells in each culture expressed the transfected gene (Phillips et al., 1991b). Hence, neighboring cells show only a low-level, nonspecific fluorescence (arrowheads in Fig. 1B) typical of that seen in cells transfected with rapsyn only (Fig. 1D). Of a total of 846 anti-GABAA receptor-positive cells from 5 transfections with the GABAA receptor plasmids, only 9 cells (0.9 6 0.9%; mean 6 standard deviation) displayed a nonuniform (patchy) distribution of cell surface staining. To localize rapsyn within transfected cells, it was necessary to fix and permeabilize the cells prior to immunostaining. Cells transfected with rapsyn alone displayed discrete rapsyn membrane patches (Fig. 1C; arrows) as previously reported (Phillips et al., 1991a). In cells co-transfected with GABAA receptor plus rapsyn, discrete two-dimensional patches of antiGABAA receptor staining replaced the diffuse cell surface anti-GABAA receptor staining seen with GABAA receptor alone (compare Figs. 1F and 1B). Anti-GABAA receptor-stained patches were found to co-localize pre-
431 cisely with anti-rapsyn stained membrane domains (arrows in Figs. 1F and 1E). Of those cells expressing rapsyn, 93 6 2% displayed rapsyn membrane domains. GABAA receptor staining was co-localized with rapsyn domains in 56 6 5% of rapsyn-positive cells. This was similar to results for AChR and rapsyn/43k protein co-clustering reported in previous studies (Phillips et al., 1991b) and reproduced for comparison in Fig. 2. The possibility that rapsyn might cluster all types of ion channels via some nonspecific interaction was tested by co-transfecting the adult skeletal muscle sodium channel (Na channel; George et al., 1992). These channels normally become restricted to the postsynaptic infoldings, adjoining the rapsyn/AChR-rich membrane domains at the neuromuscular junction. Cells transfected with Na channel and stained, after permeabilization, with antibody against intracellular epitopes of the Na channel displayed a combination of diffuse cellsurface and perinuclear, intracellular staining (Fig. 3B). Of 485 anti-Na-channel-positive cells from a total of three transfections with Na channel plasmid alone, only 11 cells (1.9 6 1.6%) showed a nonuniform (patchy) distribution of cell surface staining. Cells co-transfected with rapsyn and Na channel expression plasmids formed anti-rapsyn-stained patches on the cell surface as above (Fig. 3E) but anti-Na-channel staining did not become noticeably concentrated in the rapsyn-rich patches (Figs. 3E and 3F). Of 1576 rapsyn-positive cells examined from a total of 7 transfection experiments, 96 6 4% formed rapsyn-rich membrane domains but only 3 cells revealed any suggestion of co-clustering of Na channel with rapsyn (Fig. 2). This result confirms previous reports that rapsyn does not cluster ion channels nonselectively (see Discussion).
Detection of Rapsyn Messenger RNA in the Brain In previous studies, nuclease protection assays have detected rapsyn in skeletal muscle, heart, and kidney but not in brain or liver RNA, suggesting that rapsyn is not expressed in neurons (Musil et al., 1989). In view of its ability to cluster GABAA receptors we reexamined the possibility that rapsyn may be expressed in the brain using a more sensitive technique, namely, reverse transcription followed by polymerase chain reaction (PCR). Using oligonucleotide primers corresponding to nucleotides 1–24 (primer 98: sense) and nucleotides 730–754 (primer 97: antisense) of the original rapsyn cDNA, the expected 754-bp band was amplified from total RNA prepared from the mouse muscle cell line C2 after 30 cycles of amplification (data not shown). The same band was detected in three independent preparations of brain
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FIG. 1. GABAA receptor co-clustered with rapsyn in transfected QT-6 cells. (A and B) Cells were transfected with expression plasmids for GABAA receptor (a1, b1, and g2 subunits together) and stained with anti-GABAA receptor and anti-rapsyn antibodies prior to fixation. Three transfected cells reveal dispersed cell surface anti-GABAA receptor (FITC) staining (B). As expected, no rapsyn (TRITC) staining was seen in these cells (A). Small arrowheads indicate neighboring nontransfected cells in (B). (C and D) Cells transfected with rapsyn expression plasmid reveal anti-rapsyn (TRITC) stained membrane patches (C). No anti-GABAA receptor (FITC) staining was seen in these cells (D). (E and F) Cells transfected with both GABAA receptor and rapsyn plasmids reveal anti-rapsyn (TRITC) stained membrane patches (E) and colocalized anti-GABAA receptor staining (arrows in E and F). Bar is 20 µm for A–D and 10 µm for E and F.
total RNA when the number of thermal cycles was increased from 30 to 45. Figure 4 (top) shows results for RNA from different regions of the brainstem and for liver. Medulla (lane 1), pons (lane 2), inferior colliculus (lane 3), and superior colliculus (lane 4) all revealed the 754-bp rapsyn band while liver (lane 5) was negative. Primers specific for b-actin were used to confirm RNA integrity for each of these cDNA preparations (Fig. 4, lanes 6–10). The 754-bp rapsyn band was also detected after 45 cycles in telencephalon, cerebellum, and diencephalon (data not shown). False-positive results in RT-PCR can arise from contamination either with genomic DNA or with plasmids bearing the cDNAs. It seems unlikely that contamina-
tion was responsible for our observations for the following reasons: (1) annealing sites for primers 98 and 97 are separated by more than 2 kb of intronic DNA (Gautam et al., 1995) and no such large genomic band was seen (Fig. 4, lanes 1–5); (2) the 754-bp rapsyn band was not detected if the enzyme was left out of the reverse transcription incubation (data not shown); and (3) a rapsyn band of the correct size (1383 bp) was specifically amplified using an antisense primer that anneals to a 38 noncoding region that is absent from the rapsyn expression plasmids used within the laboratory (Fig. 4, lane 11), consistent with the presence of full-length rapsyn mRNA in the brain. The identity of the 754 and 1383-bp bands with
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DISCUSSION
FIG. 2. Frequency of clustering of ion channels with rapsyn on transfected cells. Open bars represent the percentage of rapsyn positive cells that displayed anti-rapsyn stained membrane patches. Shaded bars indicate the percentage of rapsyn-positive cells where anti-AChR, anti-GABAA receptor, or anti-Na-channel staining was coclustered with anti-rapsyn staining. AChR* results are reproduced from Phillips et al. (1991) for comparison. Error bars represent the standard deviation for six (GABAA receptor and Na channel) or three (AChR; Phillips et al., 1991b) independent transfection experiments. A minimum of 104 rapsyn-positive cells were counted for each transfection experiment.
rapsyn was confirmed in two ways. The original rapsyn cDNA (Frail et al., 1988) contains a HincII restriction site at nucleotide 585 (see Fig. 4 map). When digested with HincII, the 754-bp PCR product yielded the expected 585- and 169-bp fragments (Fig. 4, lane 13, note the weak residual 754-bp band indicating that the digestion reaction did not go to completion). The 1383-bp PCR product also yielded expected fragments of 585 and 789 bp (Fig. 4, lane 14). As a further test of whether the 754 and 1383-bp PCR products really represented rapsyn mRNA, they were reamplified using a rapsyn-specific sense primer internal to the primers used in the first round of amplification (Fig. 4 map; primer 89). When 1 µl of the 1383-bp PCR product was reamplified for 10 cycles with primers 89 and 97, the predicted 313-bp band was specifically amplified (Fig. 4, lane 15) confirming the identity of the band. A similar result was obtained when the 754-bp PCR product was used as template (data not shown). While the different brain regions displayed bands of differing intensities (e.g., Fig. 4, lanes 1–4), results from reverse transcription-PCR are not intrinsically quantitative and no attempt was made to determine the relative amounts of rapsyn mRNA.
Previous studies have shown that rapsyn formed discrete membrane domains when transfected into QT-6 fibroblasts or other nonmuscle cells. Co-expressed AChR became co-clustered in these domains (Froehner et al., 1990; Phillips et al., 1991a; Brennan et al., 1992). This clustering of AChR was not simply due to a nonspecific interaction since rapsyn did not co-cluster concanavilin A- and wheat germ agglutinin-binding glycoproteins, N-cadherin, CD8, glucose transporters, trk A, potassium channels, sodium channels, or the GLUR1 glutamate receptor subunit (Froehner et al., 1990; Maimone and Merlie, 1993; Yu and Hall, 1994; Gillespie et al., 1996; Phillips and Merlie, unpublished observations; present study). However, the present work shows that rapsyn can cluster the GABAA receptor (human a1, b1, and g2 subunits). This is to our knowledge the first report that rapsyn can interact with an ion channel other than the AChR. The range of ion channels that rapsyn is capable of clustering remains to be determined. Fibroblasts were transfected with a combination of the a1, b1, and g2 subunits. This combination was co-transfected because functional studies have shown that it produced larger whole cell conductances (presumably because the subunits assembled to form more cell surface channels) than for single subunits or binary combinations (Angelotti and Macdonald, 1993). However, the commercial anti-GABAA receptor antibody available to us was specific only for the a1 subunit of the receptor. Thus we were not able to test whether rapsyn might cluster the GABAA receptor complex via interaction with the a1, b1, or g2 subunits or all three. It will be interesting to examine the ability of rapsyn to cluster other isoforms of the GABAA receptor and indeed other members of the ligand-gated ion channel family. The means by which rapsyn immobilizes AChR into membrane domains remains to be fully defined but it seems likely that it involves a direct interaction between rapsyn and the AChR. Early chemical cross-linking experiments with Torpedo membranes suggested a close association between rapsyn and the b-subunit (Burden et al., 1983). Studies in Xenopus oocytes found that functional ACh-gated ion channels could be formed by co-expression of neuronal AChR b-subunits with muscle-type a, g, and d subunits, but that the ability of rapsyn to cluster these hybrid AChRs depended upon which b-subunit was injected (Wheeler et al., 1994). In contrast to these results, Maimone and Merlie (1993) found that each of the AChR subunits (a, b, g, and d) transfected individually into QT-6 cells were expressed
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FIG. 3. Na channel did not co-cluster with rapsyn in transfected QT-6 cells. Cells were transfected with expression plasmid for Na channel alone (A, B), rapsyn alone (C, D), or rapsyn plus Na channel (E, F). Cultures double-labeled after fixation and permeabilization were examined for TRITC anti-rapsyn staining (A, C, E) and FITC anti-Na channel staining (B, D, F). Cells transfected with Na channel alone showed a combination of intracellular and diffuse cell-surface staining (B). Examination of the TRITC optical channel confirmed the absence of antibody cross-reactivity and optical cross-bleed (A). Rapsyn-transfected cells stained with biotin-anti-rapsyn and TRITC-extravidin (Sigma) revealed rapsyn-rich membrane patches (arrowheads in C). A background level of FITC fluorescence was observed in both transfected and nontransfected cells examined after staining with anti-Na channel using this sandwich staining technique (B, D, and F). Cells transfected with both rapsyn and Na channel plasmids revealed similar anti-rapsyn (TRITC) stained membrane patches (arrowheads in E) but anti-Na channel (FITC) staining did not become clustered with rapsyn in these patches (arrowheads in F). Bar is 10 µm.
(at a low level) on the cell surface and could be clustered by rapsyn. Their results support the idea that each of the muscle AChR subunits possesses a binding site for rapsyn, but it is not known whether all these sites are exposed in the fully assembled AChR. Alignments of the short M1–M2 and long M3–M4 intracellular loops that might offer sites for rapsyn binding showed no more than a few amino acids conserved among AChR and GABAA receptor subunits (Schofield et al., 1987). Maimone and Merlie (1993) have suggested that rapsyn might recognize an amphipathic a-helix predicted for all the AChR subunits (Finer-
Moore and Stroud, 1984). However, the GABAA receptors do not share this predicted structure (Schofield et al., 1987). Identification of the site(s) to which rapsyn binds on the AChR and GABAA receptors must await studies with site-directed mutations of the receptor subunits. Despite its essential role in clustering AChR at the skeletal neuromuscular synapse, rapsyn has not previously been detected in the brain. Using an antibody binding assay, La Rochelle and Froehner (1986) detected rapsyn protein in the electric organ and skeletal muscle of the Torpedo ray but were unable to detect expression
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in the brain, heart, liver, or pancreas. Using RNase protection assays, Musil and co-workers surveyed expression across a range of mouse tissues and immortalized cell lines (Musil et al., 1989). Highest expression was found in the muscle cell line C2 with about 3-fold less rapsyn mRNA in skeletal muscle tissue. Smaller amounts of rapsyn mRNA were detected in heart and kidney (15 to 40-fold less than in C2 cells). They found no rapsyn band with total RNA from brain, liver, spleen, or uterus, concluding that if rapsyn was present in these tissues the concentration must have been less than 1⁄40 that found in C2 muscle cells. Quantitation of PCR results must be approached with great caution and we have not attempted to estimate the amount of rapsyn mRNA present in mouse brain. However, it would seem that rapsyn is expressed at a much lower level in brain than in C2 muscle cells. While prominent rapsyn bands were present with C2 cell RNA after just 30 cycles of amplification, detection of rapsyn in brain required 45 cycles to produce a robust band. The apparent low levels of rapsyn mRNA in brain might arise from a basal level of transcription throughout the heterogeneous collection of neurons, glia, and other cells that make up the brain. Alternatively, it might represent selective expression of rapsyn by a small subset of these cells. We are currently investigating the localization of rapsyn in an attempt to distinguish between these two possibilities.
EXPERIMENTAL METHODS FIG. 4. Reverse transcription followed by polymerase chain reaction detected rapsyn messenger RNA expression in the brainstem. The horizontal bar represents a map of the rapsyn cDNA, showing the location of start (ATG) and stop (TGA) codons, oligonucleotide primer annealing sites (arrows), and a HincII restriction site used in the analysis. Total RNA was reverse transcribed and amplified by polymerase chain reaction as described under Experimental Methods. (Top) Agarose gel electrophoresis of the products of 45 cycles of amplification of cDNA from the medulla (lane 1), pons (lane 2), inferior colliculus (lane 3), superior colliculus (lane 4), and liver (lane 5). Rapsyn primers 98 and 97 (see map at top of figure), yielded the expected 754-bp band for each of the brainstem regions. (Middle lanes 6–10) The products of amplification of the same cDNAs with primers specific for b-actin, to confirm RNA integrity (30 cycles of amplification; expected band 591 bp). (Bottom) Amplification of brainstem cDNA (lane 11) but not liver cDNA (lane 12) for 45 cycles with rapsyn primers 98 and A07 (see map) also yielded the expected band (1383 bp). Digestion of either the 754-bp product (lane 13) or the 1383-bp product (lane 14) with HincII in each case produced the expected fragments (754-bp product yielded 585 1 169 bp; 1383-bp product yielded 798 1 589 bp, note that digestions were incomplete). Lane 15 shows the product of reamplification of the brainstem 1383-bp product for 10 cycles with rapsyn primers 89 and 97, yielding the expected 313-bp band. Numbers at left show markers in base pairs.
Cell Culture and Transfection Quail QT-6 fibroblasts were maintained as previously described (Blount and Merlie, 1988). Cells were transfected by a modification of the calcium phosphate technique of Chen and Okayama (1987; see also Phillips et al., 1991b). A total of 25 µg of plasmid DNA was used for each transfection. GABAA receptor transfections consisted of 2 µg each of Cytomegalovirus (CMV) expression plasmids encoding the a1, b1, and g2 subunits of the human GABAA receptor (Pritchett et al., 1988, 1989) plus or minus 10 µg of a rapsyn expression plasmid. The rapsyn plasmid (Phillips et al., 1991a) employed the Rous sarcoma virus long terminal repeat promoter. pBluescript (Stratagene, La Jolla, CA) was added to keep the DNA concentration constant between transfections. For sodium channel transfections, 4 µg of a CMV expression plasmid encoding the adult human skeletal muscle voltage-gated sodium channel (pRc/ CMV-hSkM1) plus or minus 4 µg of rapsyn expression plasmid was combined with pBluescript to make a total of 25 µg of plasmid DNA. Sodium channels expressed
436 from pRc/CMV-hSkM1 on heterologous cells have previously been characterized in voltage clamp studies (Chahine et al., 1994a, b). One to two days after transfection the cells were trypsin treated and plated onto glass coverslips in a 24-well tray at a density of 2 3 105 cells per well. Coverslips were incubated at 37°C for 1 more day in growth medium, rinsed in PBS, and then incubated at room temperature for 20 min in fixative containing 1% paraformaldehyde, 100 mM L-lysine, 10 mM sodium m-periodate, and 0.1% saponin in PBS. Coverslips were then rinsed once in PBS. Cells were permeabilized for 10 min in 1% Triton X-100 in PBS and washed 3 times in PBS. To reduce nonspecific antibody binding, coverslips were preblocked overnight in 1% bovine serum albumin in PBS at 4°C.
Immunofluorescent Staining Coverslips were incubated with antibodies by inverting them (cell side down) on a 20-µl drop of the diluted antibody on a sheet of Parafilm in a humidified chamber. Unless otherwise indicated, all antibodies were diluted in PBS containing 1% BSA. For anti-GABAA receptor staining, coverslips were incubated for 1.5 h at room temperature with a monoclonal antibody (bd 24) directed against an extracellular epitope on the a1 subunit (Boehringer Mannheim, Germany) (Ewert et al., 1990) diluted 1:5 and/or affinity purified rabbit antirapsyn antibody 5943p (Phillips et al., 1991b) diluted 1:50. After washing for 30 min in three changes of PBS, coverslips were incubated for 1.5 h with affinity-purified FITC-conjugated goat anti-mouse Ig (1:100) and TRITCgoat anti-rabbit Ig (1:100). After being washed as described above, the coverslips were rinsed briefly in distilled water and mounted for fluorescence microscopy using an antifading mount. Coverslips were examined on a Bio-rad MR 600 laser scanning microscope using a 100X oil objective. To visualize cell-surface anti-GABAA receptor staining, coverslips were removed directly from culture, were rinsed once with complete culture medium (Blount and Merlie, 1988), and were inverted cell-side down onto a 20-µl drop of complete medium containing anti-GABAA receptor (bd 24; diluted 1:5) and/or antirapsyn (1:50). After an incubation of 30 min on ice, coverslips were washed two times with complete medium on ice and then once in PBS over a total of 30 min. Cells were then fixed and stained with secondary antibodies as outlined above but without permeabilization. To visualize the relatively weak anti-Na-channel signal, an amplification step was employed (LaRochelle et al., 1989). Cells were fixed, permeabilized, and preb-
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locked as described above. Coverslips were then incubated overnight at 4°C with affinity-purified rabbit anti-Na-channel antibody directed against an intracellular loop segment conserved among sodium channels (Upstate Biotechnology, NY) (Dugandzija-Novakovic et al., 1995) diluted 1:90 in 1% BSA/PBS. After being washed as described above, coverslips were incubated in FITC-conjugated sheep anti-rabbit Ig (1:500) for 1.5 h at room temperature (secondary incubation). After being washed again, coverslips were incubated in FITCconjugated donkey anti-sheep IgG (1:500) for 1.5 h (tertiary incubation) prior to washing. To permit doublelabeling for Na channel and rapsyn and avoid antibody cross-reactivity, anti-rapsyn monoclonal antibody Mab 1234 (Peng and Froehner, 1985) was directly coupled to sulfosuccinimidobiotin as previously described (LaRochelle and Froehner, 1986). Biotinylated anti-rapsyn (1:66) was included in the secondary incubation and was visualized by adding TRITC-extravidin (Sigma Chemical Co., St. Louis, MO, diluted 1:50) to the tertiary incubation. To test for antibody specificity and possible optical bleed-through, in all experiments parallel coverslips were processed with deletion of one or another of the primary antibodies. Affinity-purified secondary antibodies were obtained from Sigma Chemical Co., Boehringer Manheim, or Silenius Laboratories (Hawthorn, VIC, Australia).
Detection of Rapsyn and Actin mRNA RNA was prepared from adult mouse brain regions and liver by a modification of the acid–phenol method of Chomczynski and Sacchi (1987). Reverse transcription was primed with random hexanucleotides and PCR was carried out with 1 µg RNA according to Perkin– Elmer Cetus (Cetus Corp.). Rapsyn primers were constructed corresponding to nucleotides 1–24 (primer 98) and 442–465 (primer 89) of the coding sequence and nucleotides 731–754 (primer 97) and 1360–1383 (Primer A07) of the antisense strand. Actin primers corresponded to nucleotides 46–67 of the coding strand and 614–636 of the antisense strand of mouse b-actin (Alonso et al., 1986). After amplification for the indicated number of cycles, the PCR products were separated by electrophoresis on a 3% agarose gel and were visualized by ethidium bromide fluorescence.
ACKNOWLEDGMENTS Expression plasmids for GABAA receptors and Na channel were kind gifts from Dr. Peter Schofield and Dr. Alfred George, respectively.
Rapsyn Clusters GABAA Receptors
Monoclonal anti-rapsyn/43k protein, Mab 1234, was generously provided by Dr. Stanley Froehner. The authors also thank Helen Karuso for valuable help and advice on reverse transcription-PCR, the University of Sydney Electron Microscope Unit for support with confocal microscopy, and Drs. Nicholas Lavidis, Peter Noakes, Vladimir Balcar, and Betsy Apel for critically reading the manuscript.
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438 Mathews, B. J., and Salpeter, M. M. (1983). Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions. J. Neurosci. 3: 644–657. Matsumura, K., Ervasti, J. M., Ohlendieck, K., Kahl, S. D., and Campbell, K. P. (1992). Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature 360: 588–591. Musil, L. S., Carr, C., Cohen, J. B., and Merlie, J. P. (1988). Acetylcholine receptor-associated 43K protein contains covalently bound myristate. J. Cell Biol. 107: 1113–1121. Musil, L. S., Frail, D. E., and Merlie, J. P. (1989). The mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells. J. Cell Biol. 108: 1833–1840. Ohlendieck, K., Ervasti, J. M., Matsumura, K., Kahl, S. D., Leveille, C. J., and Campbell, K. P. (1991). Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle. Neuron 7: 499–508. Peng, H. B., and Froehner, S. C. (1985). Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells. J. Cell Biol. 100: 1698–1705. Phillips, W. D., Kopta, C., Blount, P., Gardner, P. D., Steinbach, J. H., and Merlie, J. P. (1991a). ACh receptor-rich membrane domains organized in fibroblasts by recombinant 43-kilodalton protein. Science 251: 568–570. Phillips, W. D., Maimone, M., and Merlie, J. P. (1991b). Mutagenesis of the 43-kD postsynaptic protein defines domains involved in plasma membrane targeting and AChR clustering. J. Cell Biol. 115: 1713– 1723. Phillips, W. D., Noakes, P. G., Roberds, S. L., Campbell, K. P., and
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Molecular and Cellular Neuroscience 8, 430–438 (1997) Article No. CN970597
Clustering of GABAA Receptors by Rapsyn/43kD Protein in Vitro Shi-Hong Yang, Paul F. Armson, Jeon Cha, and William D. Phillips1 Institute for Biomedical Research, Department of Physiology, University of Sydney, Sydney, New South Wales 2006, Australia
Rapsyn, a 43-kDa protein on the cytoplasmic face of the postsynaptic membrane, is essential for clustering acetylcholine receptors (AChR) at the neuromuscular junction. When transfected into nonmuscle cells (QT-6), rapsyn forms discrete membrane domains and can cluster AChR into these same domains. Here we examined whether rapsyn can cluster other ion channels as well. When expressed in QT-6 cells, the GABAA receptor (human a1, b1, and g2 subunits) and the skeletal muscle sodium channel were each diffusely scattered across the cell surface. Rapsyn, when co-expressed, clustered the GABAA receptor as effectively as it clustered AChR in previous studies. Rapsyn did not cluster co-transfected sodium channel, confirming that it does not cluster ion channels indiscriminately. Rapsyn mRNA was detected at low levels in the brain by polymerase chain reaction amplification of reverse-transcribed RNA, raising the possibility of a broader role for rapsyn.
INTRODUCTION The clustering of neurotransmitter receptors in the postsynaptic portion of the cell membrane is a characteristic feature of a variety of chemical synapses in both the central and the peripheral nervous systems (Fertuck and Salpeter, 1976; Marshall, 1981; Triller et al., 1985; Killisch et al., 1991; Craig et al., 1994). Recently, the intracellular domains of the receptors, and associated cytoplasmic proteins, have been implicated in the spatially distinct clustering of glycine receptors and NMDA receptors (Kirsch et al., 1993; Kornau et al., 1995; Ehlers et al., 1996). However, the best studied example of postsynaptic receptor clustering is at the skeletal neuromuscular 1 To whom correspondence should be addressed at Institute for Biomedical Research and Department of Physiology (F13), University of Sydney, Sydney, New South Wales 2006, Australia. Fax: 612 9351-2058. E-mail: [email protected].
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junction where acetylcholine receptor (AChR) clustering contributes to the high efficacy of this synapse (Mathews and Salpeter, 1983). During synapse formation, the postsynaptic AChR cluster is thought to be induced to form by substances such as agrin, secreted by the nerve terminal (Magill-Solc and McMahan, 1990). Agrin activates the skeletal muscle transmembrane tyrosine kinase, MuSK, which leads to AChR clustering in the postsynaptic portion of the muscle cell membrane (Glass et al., 1996). While extracellular proteins such as agrin serve as signals to induce localized AChR clustering, the task of marshalling receptors into a high-density patch falls to intracellular proteins that are concentrated in the postsynaptic membrane. One of these, rapsyn/43kD (Frail et al., 1988), is a peripheral membrane protein that associates with the inner face of the plasma membrane with the aid of an amino-terminal myristate group (Musil et al., 1988; Phillips et al., 1991b). Rapsyn is thought to bind to AChR, immobilizing it in discrete membrane domains (Froehner et al., 1990; Phillips et al., 1991a). In turn, rapsyn may interact with the dystrophin-associated protein complex (Ervasti and Campbell, 1991) by binding to dystroglycan (Apel et al., 1995). Utrophin, which is closely co-localized with clustered AChR (Ohlendieck et al., 1991), can also bind to the dystrophinassociated protein complex (Matsumura et al., 1992; Kramarcy, et al., 1994) and probably helps to stabilize large AChR clusters (Phillips et al., 1993; Campanelli et al., 1994; Gee et al., 1994; Apel et al., 1995). An 87-kDa carboxy-terminal homologue of dystrophin may also play a role in formation and/or stabilization of the postsynaptic AChR clusters (Wagner et al., 1993). Thus rapsyn is thought to connect AChR to the membrane skeleton at sites of clustering. Considerable evidence supports the view that rapsyn 1044-7431/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
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serves as a linchpin in clustering AChR at the synapse. Rapsyn and AChR can be chemically cross-linked, suggesting a close one-to-one interaction at the membrane face (Burden et al., 1983). AChR clustered by rapsyn in heterologous cell types displays increased resistance to extraction with the non-ionic detergent, Triton X-100, suggesting a linkage to the cytoskeleton (Phillips et al., 1993). Finally, in rapsyn-deficient mice, neither AChR, nor dystroglycan, nor utrophin assembled into discrete clusters in the postsynaptic membrane (Gautam et al., 1995). Studies outlined above in which rapsyn and AChR were co-transfected into fibroblasts support the hypothesis that rapsyn helps to form a specialized postsynaptic membrane domain where it binds to and immobilizes AChR. Here we have employed the fibroblast expression system to explore the generality of this interaction and report that rapsyn can cluster a GABAA receptor (a member of the same receptor super family) but not a voltage-gated sodium channel.
RESULTS Co-expression of Rapsyn and GABAA Receptors The quail fibroblast cell line QT-6 was first transfected with GABAA receptor (combination of human a1, b1, and g2 subunits; Pritchett et al., 1988, 1989) alone. Cells stained prior to fixation with a monoclonal antibody specific for an extracellular epitope on the a1 subunit revealed a diffuse, fairly uniform pattern across the cell surface (Fig. 1B). Following transfection, only a minority of cells in each culture expressed the transfected gene (Phillips et al., 1991b). Hence, neighboring cells show only a low-level, nonspecific fluorescence (arrowheads in Fig. 1B) typical of that seen in cells transfected with rapsyn only (Fig. 1D). Of a total of 846 anti-GABAA receptor-positive cells from 5 transfections with the GABAA receptor plasmids, only 9 cells (0.9 6 0.9%; mean 6 standard deviation) displayed a nonuniform (patchy) distribution of cell surface staining. To localize rapsyn within transfected cells, it was necessary to fix and permeabilize the cells prior to immunostaining. Cells transfected with rapsyn alone displayed discrete rapsyn membrane patches (Fig. 1C; arrows) as previously reported (Phillips et al., 1991a). In cells co-transfected with GABAA receptor plus rapsyn, discrete two-dimensional patches of antiGABAA receptor staining replaced the diffuse cell surface anti-GABAA receptor staining seen with GABAA receptor alone (compare Figs. 1F and 1B). Anti-GABAA receptor-stained patches were found to co-localize pre-
431 cisely with anti-rapsyn stained membrane domains (arrows in Figs. 1F and 1E). Of those cells expressing rapsyn, 93 6 2% displayed rapsyn membrane domains. GABAA receptor staining was co-localized with rapsyn domains in 56 6 5% of rapsyn-positive cells. This was similar to results for AChR and rapsyn/43k protein co-clustering reported in previous studies (Phillips et al., 1991b) and reproduced for comparison in Fig. 2. The possibility that rapsyn might cluster all types of ion channels via some nonspecific interaction was tested by co-transfecting the adult skeletal muscle sodium channel (Na channel; George et al., 1992). These channels normally become restricted to the postsynaptic infoldings, adjoining the rapsyn/AChR-rich membrane domains at the neuromuscular junction. Cells transfected with Na channel and stained, after permeabilization, with antibody against intracellular epitopes of the Na channel displayed a combination of diffuse cellsurface and perinuclear, intracellular staining (Fig. 3B). Of 485 anti-Na-channel-positive cells from a total of three transfections with Na channel plasmid alone, only 11 cells (1.9 6 1.6%) showed a nonuniform (patchy) distribution of cell surface staining. Cells co-transfected with rapsyn and Na channel expression plasmids formed anti-rapsyn-stained patches on the cell surface as above (Fig. 3E) but anti-Na-channel staining did not become noticeably concentrated in the rapsyn-rich patches (Figs. 3E and 3F). Of 1576 rapsyn-positive cells examined from a total of 7 transfection experiments, 96 6 4% formed rapsyn-rich membrane domains but only 3 cells revealed any suggestion of co-clustering of Na channel with rapsyn (Fig. 2). This result confirms previous reports that rapsyn does not cluster ion channels nonselectively (see Discussion).
Detection of Rapsyn Messenger RNA in the Brain In previous studies, nuclease protection assays have detected rapsyn in skeletal muscle, heart, and kidney but not in brain or liver RNA, suggesting that rapsyn is not expressed in neurons (Musil et al., 1989). In view of its ability to cluster GABAA receptors we reexamined the possibility that rapsyn may be expressed in the brain using a more sensitive technique, namely, reverse transcription followed by polymerase chain reaction (PCR). Using oligonucleotide primers corresponding to nucleotides 1–24 (primer 98: sense) and nucleotides 730–754 (primer 97: antisense) of the original rapsyn cDNA, the expected 754-bp band was amplified from total RNA prepared from the mouse muscle cell line C2 after 30 cycles of amplification (data not shown). The same band was detected in three independent preparations of brain
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FIG. 1. GABAA receptor co-clustered with rapsyn in transfected QT-6 cells. (A and B) Cells were transfected with expression plasmids for GABAA receptor (a1, b1, and g2 subunits together) and stained with anti-GABAA receptor and anti-rapsyn antibodies prior to fixation. Three transfected cells reveal dispersed cell surface anti-GABAA receptor (FITC) staining (B). As expected, no rapsyn (TRITC) staining was seen in these cells (A). Small arrowheads indicate neighboring nontransfected cells in (B). (C and D) Cells transfected with rapsyn expression plasmid reveal anti-rapsyn (TRITC) stained membrane patches (C). No anti-GABAA receptor (FITC) staining was seen in these cells (D). (E and F) Cells transfected with both GABAA receptor and rapsyn plasmids reveal anti-rapsyn (TRITC) stained membrane patches (E) and colocalized anti-GABAA receptor staining (arrows in E and F). Bar is 20 µm for A–D and 10 µm for E and F.
total RNA when the number of thermal cycles was increased from 30 to 45. Figure 4 (top) shows results for RNA from different regions of the brainstem and for liver. Medulla (lane 1), pons (lane 2), inferior colliculus (lane 3), and superior colliculus (lane 4) all revealed the 754-bp rapsyn band while liver (lane 5) was negative. Primers specific for b-actin were used to confirm RNA integrity for each of these cDNA preparations (Fig. 4, lanes 6–10). The 754-bp rapsyn band was also detected after 45 cycles in telencephalon, cerebellum, and diencephalon (data not shown). False-positive results in RT-PCR can arise from contamination either with genomic DNA or with plasmids bearing the cDNAs. It seems unlikely that contamina-
tion was responsible for our observations for the following reasons: (1) annealing sites for primers 98 and 97 are separated by more than 2 kb of intronic DNA (Gautam et al., 1995) and no such large genomic band was seen (Fig. 4, lanes 1–5); (2) the 754-bp rapsyn band was not detected if the enzyme was left out of the reverse transcription incubation (data not shown); and (3) a rapsyn band of the correct size (1383 bp) was specifically amplified using an antisense primer that anneals to a 38 noncoding region that is absent from the rapsyn expression plasmids used within the laboratory (Fig. 4, lane 11), consistent with the presence of full-length rapsyn mRNA in the brain. The identity of the 754 and 1383-bp bands with
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DISCUSSION
FIG. 2. Frequency of clustering of ion channels with rapsyn on transfected cells. Open bars represent the percentage of rapsyn positive cells that displayed anti-rapsyn stained membrane patches. Shaded bars indicate the percentage of rapsyn-positive cells where anti-AChR, anti-GABAA receptor, or anti-Na-channel staining was coclustered with anti-rapsyn staining. AChR* results are reproduced from Phillips et al. (1991) for comparison. Error bars represent the standard deviation for six (GABAA receptor and Na channel) or three (AChR; Phillips et al., 1991b) independent transfection experiments. A minimum of 104 rapsyn-positive cells were counted for each transfection experiment.
rapsyn was confirmed in two ways. The original rapsyn cDNA (Frail et al., 1988) contains a HincII restriction site at nucleotide 585 (see Fig. 4 map). When digested with HincII, the 754-bp PCR product yielded the expected 585- and 169-bp fragments (Fig. 4, lane 13, note the weak residual 754-bp band indicating that the digestion reaction did not go to completion). The 1383-bp PCR product also yielded expected fragments of 585 and 789 bp (Fig. 4, lane 14). As a further test of whether the 754 and 1383-bp PCR products really represented rapsyn mRNA, they were reamplified using a rapsyn-specific sense primer internal to the primers used in the first round of amplification (Fig. 4 map; primer 89). When 1 µl of the 1383-bp PCR product was reamplified for 10 cycles with primers 89 and 97, the predicted 313-bp band was specifically amplified (Fig. 4, lane 15) confirming the identity of the band. A similar result was obtained when the 754-bp PCR product was used as template (data not shown). While the different brain regions displayed bands of differing intensities (e.g., Fig. 4, lanes 1–4), results from reverse transcription-PCR are not intrinsically quantitative and no attempt was made to determine the relative amounts of rapsyn mRNA.
Previous studies have shown that rapsyn formed discrete membrane domains when transfected into QT-6 fibroblasts or other nonmuscle cells. Co-expressed AChR became co-clustered in these domains (Froehner et al., 1990; Phillips et al., 1991a; Brennan et al., 1992). This clustering of AChR was not simply due to a nonspecific interaction since rapsyn did not co-cluster concanavilin A- and wheat germ agglutinin-binding glycoproteins, N-cadherin, CD8, glucose transporters, trk A, potassium channels, sodium channels, or the GLUR1 glutamate receptor subunit (Froehner et al., 1990; Maimone and Merlie, 1993; Yu and Hall, 1994; Gillespie et al., 1996; Phillips and Merlie, unpublished observations; present study). However, the present work shows that rapsyn can cluster the GABAA receptor (human a1, b1, and g2 subunits). This is to our knowledge the first report that rapsyn can interact with an ion channel other than the AChR. The range of ion channels that rapsyn is capable of clustering remains to be determined. Fibroblasts were transfected with a combination of the a1, b1, and g2 subunits. This combination was co-transfected because functional studies have shown that it produced larger whole cell conductances (presumably because the subunits assembled to form more cell surface channels) than for single subunits or binary combinations (Angelotti and Macdonald, 1993). However, the commercial anti-GABAA receptor antibody available to us was specific only for the a1 subunit of the receptor. Thus we were not able to test whether rapsyn might cluster the GABAA receptor complex via interaction with the a1, b1, or g2 subunits or all three. It will be interesting to examine the ability of rapsyn to cluster other isoforms of the GABAA receptor and indeed other members of the ligand-gated ion channel family. The means by which rapsyn immobilizes AChR into membrane domains remains to be fully defined but it seems likely that it involves a direct interaction between rapsyn and the AChR. Early chemical cross-linking experiments with Torpedo membranes suggested a close association between rapsyn and the b-subunit (Burden et al., 1983). Studies in Xenopus oocytes found that functional ACh-gated ion channels could be formed by co-expression of neuronal AChR b-subunits with muscle-type a, g, and d subunits, but that the ability of rapsyn to cluster these hybrid AChRs depended upon which b-subunit was injected (Wheeler et al., 1994). In contrast to these results, Maimone and Merlie (1993) found that each of the AChR subunits (a, b, g, and d) transfected individually into QT-6 cells were expressed
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FIG. 3. Na channel did not co-cluster with rapsyn in transfected QT-6 cells. Cells were transfected with expression plasmid for Na channel alone (A, B), rapsyn alone (C, D), or rapsyn plus Na channel (E, F). Cultures double-labeled after fixation and permeabilization were examined for TRITC anti-rapsyn staining (A, C, E) and FITC anti-Na channel staining (B, D, F). Cells transfected with Na channel alone showed a combination of intracellular and diffuse cell-surface staining (B). Examination of the TRITC optical channel confirmed the absence of antibody cross-reactivity and optical cross-bleed (A). Rapsyn-transfected cells stained with biotin-anti-rapsyn and TRITC-extravidin (Sigma) revealed rapsyn-rich membrane patches (arrowheads in C). A background level of FITC fluorescence was observed in both transfected and nontransfected cells examined after staining with anti-Na channel using this sandwich staining technique (B, D, and F). Cells transfected with both rapsyn and Na channel plasmids revealed similar anti-rapsyn (TRITC) stained membrane patches (arrowheads in E) but anti-Na channel (FITC) staining did not become clustered with rapsyn in these patches (arrowheads in F). Bar is 10 µm.
(at a low level) on the cell surface and could be clustered by rapsyn. Their results support the idea that each of the muscle AChR subunits possesses a binding site for rapsyn, but it is not known whether all these sites are exposed in the fully assembled AChR. Alignments of the short M1–M2 and long M3–M4 intracellular loops that might offer sites for rapsyn binding showed no more than a few amino acids conserved among AChR and GABAA receptor subunits (Schofield et al., 1987). Maimone and Merlie (1993) have suggested that rapsyn might recognize an amphipathic a-helix predicted for all the AChR subunits (Finer-
Moore and Stroud, 1984). However, the GABAA receptors do not share this predicted structure (Schofield et al., 1987). Identification of the site(s) to which rapsyn binds on the AChR and GABAA receptors must await studies with site-directed mutations of the receptor subunits. Despite its essential role in clustering AChR at the skeletal neuromuscular synapse, rapsyn has not previously been detected in the brain. Using an antibody binding assay, La Rochelle and Froehner (1986) detected rapsyn protein in the electric organ and skeletal muscle of the Torpedo ray but were unable to detect expression
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in the brain, heart, liver, or pancreas. Using RNase protection assays, Musil and co-workers surveyed expression across a range of mouse tissues and immortalized cell lines (Musil et al., 1989). Highest expression was found in the muscle cell line C2 with about 3-fold less rapsyn mRNA in skeletal muscle tissue. Smaller amounts of rapsyn mRNA were detected in heart and kidney (15 to 40-fold less than in C2 cells). They found no rapsyn band with total RNA from brain, liver, spleen, or uterus, concluding that if rapsyn was present in these tissues the concentration must have been less than 1⁄40 that found in C2 muscle cells. Quantitation of PCR results must be approached with great caution and we have not attempted to estimate the amount of rapsyn mRNA present in mouse brain. However, it would seem that rapsyn is expressed at a much lower level in brain than in C2 muscle cells. While prominent rapsyn bands were present with C2 cell RNA after just 30 cycles of amplification, detection of rapsyn in brain required 45 cycles to produce a robust band. The apparent low levels of rapsyn mRNA in brain might arise from a basal level of transcription throughout the heterogeneous collection of neurons, glia, and other cells that make up the brain. Alternatively, it might represent selective expression of rapsyn by a small subset of these cells. We are currently investigating the localization of rapsyn in an attempt to distinguish between these two possibilities.
EXPERIMENTAL METHODS FIG. 4. Reverse transcription followed by polymerase chain reaction detected rapsyn messenger RNA expression in the brainstem. The horizontal bar represents a map of the rapsyn cDNA, showing the location of start (ATG) and stop (TGA) codons, oligonucleotide primer annealing sites (arrows), and a HincII restriction site used in the analysis. Total RNA was reverse transcribed and amplified by polymerase chain reaction as described under Experimental Methods. (Top) Agarose gel electrophoresis of the products of 45 cycles of amplification of cDNA from the medulla (lane 1), pons (lane 2), inferior colliculus (lane 3), superior colliculus (lane 4), and liver (lane 5). Rapsyn primers 98 and 97 (see map at top of figure), yielded the expected 754-bp band for each of the brainstem regions. (Middle lanes 6–10) The products of amplification of the same cDNAs with primers specific for b-actin, to confirm RNA integrity (30 cycles of amplification; expected band 591 bp). (Bottom) Amplification of brainstem cDNA (lane 11) but not liver cDNA (lane 12) for 45 cycles with rapsyn primers 98 and A07 (see map) also yielded the expected band (1383 bp). Digestion of either the 754-bp product (lane 13) or the 1383-bp product (lane 14) with HincII in each case produced the expected fragments (754-bp product yielded 585 1 169 bp; 1383-bp product yielded 798 1 589 bp, note that digestions were incomplete). Lane 15 shows the product of reamplification of the brainstem 1383-bp product for 10 cycles with rapsyn primers 89 and 97, yielding the expected 313-bp band. Numbers at left show markers in base pairs.
Cell Culture and Transfection Quail QT-6 fibroblasts were maintained as previously described (Blount and Merlie, 1988). Cells were transfected by a modification of the calcium phosphate technique of Chen and Okayama (1987; see also Phillips et al., 1991b). A total of 25 µg of plasmid DNA was used for each transfection. GABAA receptor transfections consisted of 2 µg each of Cytomegalovirus (CMV) expression plasmids encoding the a1, b1, and g2 subunits of the human GABAA receptor (Pritchett et al., 1988, 1989) plus or minus 10 µg of a rapsyn expression plasmid. The rapsyn plasmid (Phillips et al., 1991a) employed the Rous sarcoma virus long terminal repeat promoter. pBluescript (Stratagene, La Jolla, CA) was added to keep the DNA concentration constant between transfections. For sodium channel transfections, 4 µg of a CMV expression plasmid encoding the adult human skeletal muscle voltage-gated sodium channel (pRc/ CMV-hSkM1) plus or minus 4 µg of rapsyn expression plasmid was combined with pBluescript to make a total of 25 µg of plasmid DNA. Sodium channels expressed
436 from pRc/CMV-hSkM1 on heterologous cells have previously been characterized in voltage clamp studies (Chahine et al., 1994a, b). One to two days after transfection the cells were trypsin treated and plated onto glass coverslips in a 24-well tray at a density of 2 3 105 cells per well. Coverslips were incubated at 37°C for 1 more day in growth medium, rinsed in PBS, and then incubated at room temperature for 20 min in fixative containing 1% paraformaldehyde, 100 mM L-lysine, 10 mM sodium m-periodate, and 0.1% saponin in PBS. Coverslips were then rinsed once in PBS. Cells were permeabilized for 10 min in 1% Triton X-100 in PBS and washed 3 times in PBS. To reduce nonspecific antibody binding, coverslips were preblocked overnight in 1% bovine serum albumin in PBS at 4°C.
Immunofluorescent Staining Coverslips were incubated with antibodies by inverting them (cell side down) on a 20-µl drop of the diluted antibody on a sheet of Parafilm in a humidified chamber. Unless otherwise indicated, all antibodies were diluted in PBS containing 1% BSA. For anti-GABAA receptor staining, coverslips were incubated for 1.5 h at room temperature with a monoclonal antibody (bd 24) directed against an extracellular epitope on the a1 subunit (Boehringer Mannheim, Germany) (Ewert et al., 1990) diluted 1:5 and/or affinity purified rabbit antirapsyn antibody 5943p (Phillips et al., 1991b) diluted 1:50. After washing for 30 min in three changes of PBS, coverslips were incubated for 1.5 h with affinity-purified FITC-conjugated goat anti-mouse Ig (1:100) and TRITCgoat anti-rabbit Ig (1:100). After being washed as described above, the coverslips were rinsed briefly in distilled water and mounted for fluorescence microscopy using an antifading mount. Coverslips were examined on a Bio-rad MR 600 laser scanning microscope using a 100X oil objective. To visualize cell-surface anti-GABAA receptor staining, coverslips were removed directly from culture, were rinsed once with complete culture medium (Blount and Merlie, 1988), and were inverted cell-side down onto a 20-µl drop of complete medium containing anti-GABAA receptor (bd 24; diluted 1:5) and/or antirapsyn (1:50). After an incubation of 30 min on ice, coverslips were washed two times with complete medium on ice and then once in PBS over a total of 30 min. Cells were then fixed and stained with secondary antibodies as outlined above but without permeabilization. To visualize the relatively weak anti-Na-channel signal, an amplification step was employed (LaRochelle et al., 1989). Cells were fixed, permeabilized, and preb-
Yang et al.
locked as described above. Coverslips were then incubated overnight at 4°C with affinity-purified rabbit anti-Na-channel antibody directed against an intracellular loop segment conserved among sodium channels (Upstate Biotechnology, NY) (Dugandzija-Novakovic et al., 1995) diluted 1:90 in 1% BSA/PBS. After being washed as described above, coverslips were incubated in FITC-conjugated sheep anti-rabbit Ig (1:500) for 1.5 h at room temperature (secondary incubation). After being washed again, coverslips were incubated in FITCconjugated donkey anti-sheep IgG (1:500) for 1.5 h (tertiary incubation) prior to washing. To permit doublelabeling for Na channel and rapsyn and avoid antibody cross-reactivity, anti-rapsyn monoclonal antibody Mab 1234 (Peng and Froehner, 1985) was directly coupled to sulfosuccinimidobiotin as previously described (LaRochelle and Froehner, 1986). Biotinylated anti-rapsyn (1:66) was included in the secondary incubation and was visualized by adding TRITC-extravidin (Sigma Chemical Co., St. Louis, MO, diluted 1:50) to the tertiary incubation. To test for antibody specificity and possible optical bleed-through, in all experiments parallel coverslips were processed with deletion of one or another of the primary antibodies. Affinity-purified secondary antibodies were obtained from Sigma Chemical Co., Boehringer Manheim, or Silenius Laboratories (Hawthorn, VIC, Australia).
Detection of Rapsyn and Actin mRNA RNA was prepared from adult mouse brain regions and liver by a modification of the acid–phenol method of Chomczynski and Sacchi (1987). Reverse transcription was primed with random hexanucleotides and PCR was carried out with 1 µg RNA according to Perkin– Elmer Cetus (Cetus Corp.). Rapsyn primers were constructed corresponding to nucleotides 1–24 (primer 98) and 442–465 (primer 89) of the coding sequence and nucleotides 731–754 (primer 97) and 1360–1383 (Primer A07) of the antisense strand. Actin primers corresponded to nucleotides 46–67 of the coding strand and 614–636 of the antisense strand of mouse b-actin (Alonso et al., 1986). After amplification for the indicated number of cycles, the PCR products were separated by electrophoresis on a 3% agarose gel and were visualized by ethidium bromide fluorescence.
ACKNOWLEDGMENTS Expression plasmids for GABAA receptors and Na channel were kind gifts from Dr. Peter Schofield and Dr. Alfred George, respectively.
Rapsyn Clusters GABAA Receptors
Monoclonal anti-rapsyn/43k protein, Mab 1234, was generously provided by Dr. Stanley Froehner. The authors also thank Helen Karuso for valuable help and advice on reverse transcription-PCR, the University of Sydney Electron Microscope Unit for support with confocal microscopy, and Drs. Nicholas Lavidis, Peter Noakes, Vladimir Balcar, and Betsy Apel for critically reading the manuscript.
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