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Cloning and Tissue Distribution of a Novel P2X Receptor from Rat Brain
JOBNAME: BBRC 223#2 PAGE: 1 SESS: 13 OUTPUT: Thu Jun 20 08:56:17 1996 /xypage/worksmart/tsp000/72170k/44
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
223, 456–460 (1996)
0915
Cloning and Tissue Distribution of a Novel P2X Receptor from Rat Brain Florentina Soto,1 Miguel Garcia-Guzman, Christine Karschin, and Walter Stühmer Department of Molecular Biology of Neuronal Signals, Max-Planck Institute for Experimental Medicine, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany Received April 30, 1996 We have isolated the cDNA for a novel member (P2X6) of the ATP-gated ion channel family. The rat P2X6 nucleotide sequence encodes a 379 amino acid protein that conserves all the structural features of previously cloned P2X receptors, including the two putative transmembrane domains predicted by hydrophobicity plots. In situ hybridization analysis of rat brain sections showed a wide pattern of mRNA expression that is virtually identical to that already described for P2X4. Injection of P2X6 cRNA in Xenopus oocytes did not give rise to ATP-activated channels. Coexpression of P2X6 with P2X4 subunits produced currents which were not discernibly different from those of P2X4 expressed alone. © 1996 Academic Press, Inc.
ATP is not only the main energy source in the organism, but also acts as an extracellular messenger in a variety of cells and tissues (1, 2). The binding of ATP to P2X receptors opens, in the millisecond range, ionic channels that are non-selective for monovalent cations and permeable to Ca2+. Molecular cloning techniques led to the identification of 4 different P2X receptors: P2X1-P2X4 (3–11). Functional expression of the recombinant P2X receptors in either Xenopus oocytes or in transfected cells revealed several differences between them. This diversity in responses can be further increased by the ability of some subunits to form heteromultimeric complexes (6). In situ hybridization and tissue distribution analysis revealed that one or more of the cloned P2X receptors can be found in every tissue. The restricted expression pattern of P2X3, that appears to be only in sensory ganglia (5), contrasts with the wide expression pattern of P2X4 (7–10), while P2X1 and P2X2 have a more restrained distribution (12). Up to now, three P2X receptors have been detected in rat brain neurons: P2X1, P2X2 (12) and P2X4 (7–10). We have isolated two additional members, P2X5 (manuscript in preparation) and P2X6, of the purinergic receptor family using PCR-based cloning and low-stringency hybridization techniques. Here we report the cloning and tissue distribution of P2X6, a novel P2X receptor widely expressed in both the central nervous system and in peripheral tissues. MATERIALS AND METHODS cDNA cloning of a novel P2X channel. The P2X6 cDNA was initially detected by PCR in rat brain cDNA using degenerate primers previously described (10). The resulting 750 bp PCR fragment was isolated, 32P-labelled and used to screen 1×106 phages of a commercial rat brain cDNA library (Clontech) under low stringency hybridization conditions (13). None of the phages isolated contained the sequence encoding the N-terminal domain of the protein. A rapid amplification of cDNA ends (RACE) was performed using the Marathon cDNA amplification kit (Clontech). The primers used for amplification were an antisense primer specific for the P2X6 sequence (59-AGCCACTCCCACAGCCGTTTCTC-39) and a sense primer complementary to the anchor sequence (supplied with the kit). Electrophysiology. The full-length P2X6 clone was inserted into the pSGEM vector (10). Capped RNA synthesis and oocyte isolation, handling and two electrode voltage-clamp measurements were performed as previously described (14). The superfusing solution contained 115 mM NaCl, 2.8 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, pH47.2. Tissue expression analysis by PCR. Total RNA prepared by the single-step method (15) from various tissues was reverse transcribed and the resulting cDNAs were analyzed by PCR with specific primers for the P2X6 receptor and, as an internal control of the reaction, with specific primers for the transferrin receptor (TFR) (10). The sense and antisense primers used to detect P2X6 expression were 59-GAAAGGCTCTGACTGCTCTC-39 and 59-AGGAATGGATAGGCTGACAC-39, re-
1
Corresponding author. Fax:+49-551-3899644. e-mail:[email protected]. 456
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
JOBNAME: BBRC 223#2 PAGE: 2 SESS: 13 OUTPUT: Thu Jun 20 08:56:17 1996 /xypage/worksmart/tsp000/72170k/44
Vol. 223, No. 2, 1996
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FIG. 1. Multiple alignment of the P2X amino acid sequences. Residues conserved in all four rat sequences are boxed. The putative transmembrane segments are underlined (M1 and M2). The conserved cysteine residues are marked (&sb9;). spectively. The specificity of the PCR amplification was assessed by Southern blot with appropriate [32P]-a-dCTP randomprimed probes for the TFR and P2X6 genes. The final high stringency wash was 0.1× SSC, 0.1% SDS at 65°C. To rule out the possibility of genomic DNA contamination of the cDNA, we performed a PCR under the same conditions on 0.5 mg of rat genomic DNA. No bands were amplified when specific primers for P2X6 were used. The integrity of the genomic DNA used as a template was assessed by amplification using specific primers for an intronless gene (Kv 1.4). In situ hybridization. Digoxigenin-labelled sense and antisense cRNA probes were generated by in vitro transcriptions with DIG-UTP (Boehringer Mannheim) using the 750 bp PCR fragment as a template. Horizontal and sagittal sections (10–16 mm) of adult rat brain were cut on a cryostat, fixed and dehydrated. Pretreatments, hybridization of sections and immunological detection were carried out as reported (16). Color development was stopped after 12–30 hrs. For controls, adjacent sections were hybridized with sense RNA or digested with RNase before hybridization.
RESULTS AND DISCUSSION We have isolated from rat brain, a full length cDNA coding for a new member of the P2X receptor family (P2X6). The deduced amino acid sequence shows 45, 38, 39, 45 and 48% identity with P2X1, P2X2, P2X3, P2X4 and P2X5, respectively (Fig. 1). The hydrophobicity plot of P2X6
FIG. 2. Analysis of P2X4 mRNA tissue expression by RT-PCR and Southern blot autoradiography using a P2X6-specific probe (P2X6 arrow) and a rat transferrin receptor probe (TFR arrow) as a control for RT-PCR efficiency. 457
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FIG. 3. In situ hybridization with DIG-labelled RNA probes shows the localization of P2X6 mRNA in adult rat brain in (A) hippocampus, (C) olfactory bulb, (D) cortex, (E) cerebellum, (F) facial nucleus of the brainstem, and (G) ependymal layer lining the ventricle. Control sections are shown only for (B) hippocampus and (H) ependymal layer. Scale bars represent 250 mm in A, B, G, H and 100 mm in C, D, E, F. 458
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predicts a topology similar to previously described members of the family. There are two possible transmembrane regions flanking a long extracellular loop. The short N- and C-terminal regions are thought to be intracellular. P2X6 contains residues that are conserved in all the recombinant P2X receptors, including ten cysteine residues in the putative extracellular loop which may form disulfide bridges (Fig. 1). The P2X6 has N-glycosylation consensus sites at amino acid positions Asn-157, Asn-187 and Asn-202. RT-PCR analysis reveals that transcripts for the P2X6 receptor are present in most of the tissues analyzed (Fig. 2). This distribution is clearly similar to that displayed by P2X4 (10), but is not as widespread. Interestingly, P2X6 was not detected in RNA isolated from thymus, blood vessels and vas deferens, where the only P2X receptors detected have been P2X1 (17) and P2X4 (10). The expression of P2X6 in secretory tissues such as pituitary and adrenal gland suggests that this receptor may play a role in regulating hormone secretion. The distribution in central nervous system was further analyzed by in situ hybridization of rat brain sections (Fig. 3). P2X6 mRNA was abundantly expressed throughout the brain with a distribution pattern matching the previously described expression of P2X4 receptor mRNA (10). The only exception was the especially strong labelling of the ependymal cell layer lining the ventricular surface with the P2X6 probe (G), where P2X4 mRNA has not been detected. Strong hybridization signals were found in hippocampus (A), olfactory bulb mitral and tufted cells (C), cortex (D), cerebellar Purkinje cells (E), deep cerebellar nuclei, and in most of the brainstem nuclei, such as the facial nucleus (F). The reticular thalamic nucleus was strongly labelled, whereas moderate staining was observed in other thalamic nuclei, hypothalamus, amygdala, inferior colliculus and substantia nigra. Low levels of transcript were detected in the caudate putamen and superior colliculus. No staining was observed in any fiber pathways, in adjacent rat brain sections hybridized with sense RNA probe (H), or in control sections digested with RNase before hybridization (B). P2X4 and P2X6 seem to be the predominant forms of P2X receptors in the brain, since the other forms described, P2X1 and P2X2, are not so highly expressed or widely distributed (12). To characterize the functional properties of the recombinant P2X6 receptor, the cRNA coding for the protein was injected in Xenopus oocytes. Upon application of ATP (up to 500 mM), no inward current was detected. No responses were seen when other nucleotides such as ADP, GTP, UTP, AMP, or the nucleoside adenosine (100 mM) were used. Since it is possible that P2X6 is neither an agonist-binding subunit nor capable of creating a homomeric functional channel, we attempted to coexpress P2X6 with P2X4, the P2X receptor that has a very similar distribution in the brain. When both cRNAs were coinjected into Xenopus oocytes, the agonist-induced currents (ATP, 100 mM) were identical in pharmacology and kinetics to those of the control oocytes injected only with P2X4. We did not observe a dominant negative action of P2X6 on the P2X4 expression level (as determined by current amplitude). It is conceivable that combinations of P2X6 with other members of the family are required to produce a functional channel when expressed in Xenopus oocytes. Furthermore, the channel properties of P2X6 might depend on the expression system used. ACKNOWLEDGMENTS We thank Katja Antonnen for excellent technical assistance and Drs. Michael Hollmann and Anant Parekh for critically reading the manuscript. We are grateful to Drs. G. Collo, R. A. North, E. Kawashima, Merlo-Pich, S. Neidhart, A. Surprenant and G. Buell for kindly sharing unpublished data on P2X6.
REFERENCES 1. 2. 3. 4. 5. 6.
Burnstock, G. (1990) Ann. N.Y. Acad. Sci. 603, 1–17. Dubyak, G. R., and El-Moatassim, C. (1993) Am. J. Physiol. 265, C577–C606. Valera, S., Hussy, N., Evans, R. J., Adami, N., North, R. A., and Surprenant, A. (1994) Nature 371, 516–519. Brake, A. J., Wagenbach, M. J., and Julius, D. (1994) Nature 371, 519–523. Chen, C. C., Akopian, A. N., Sivilotti, L., Colquhoun, D., Burnstock, G., and Wood, J. N. (1995) Nature 377, 428–431. Lewis, C., Neidhart, S., Holy, C., North, R. A., Buell, G., and Surprenant, A. (1995) Nature 377, 432–435. 459
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Bo, X., Zhang, Y., Nassar, M., Burnstock, G., and Schoepfer, R. (1995) FEBS Lett. 375, 129–133. Buell, G., Lewis, C., Collo, G., North, R. A., and Surprenant, A. (1996) EMBO J. 15, 55–62. Seguela, P., Haghighi, A., Soghomonian, J., and Cooper, E. (1996) J. Neurosci. 16, 448–455. Soto, F., Garcia-Guzman, M., Gomez-Hernandez, J. M., Hollmann, M., Karschin, C., and Stühmer, W. (1996) Proc. Natl. Acad. Sci. USA. 93, 3684–3688. Wang, C. Z., Namba, N., Gonoi, T., Iganaki, N., and Seino, S. (1996) Biochem. Biophys. Res. Commun. 220, 196–202. Kidd, E. J., Grahames, C. B. A., Simon, J., Michel, A. D., Barnard, E. A., and Humprey, P. P. A. (1995) Mol. Pharmacol. 48, 569–573. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Stühmer, W. (1992) Methods Enzymol. 207, 319–345. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159. Bartsch, S, Bartsch, U., Dorries, U., Faissner, A., Weller, A., Ekblom, P., and Schachner, M. (1992) J. Neurosci. 12, 736–749. Surprenant, A., Buell, G., and North, R. A. (1995) Trends Neurosci. 18, 224–229.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
223, 456–460 (1996)
0915
Cloning and Tissue Distribution of a Novel P2X Receptor from Rat Brain Florentina Soto,1 Miguel Garcia-Guzman, Christine Karschin, and Walter Stühmer Department of Molecular Biology of Neuronal Signals, Max-Planck Institute for Experimental Medicine, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany Received April 30, 1996 We have isolated the cDNA for a novel member (P2X6) of the ATP-gated ion channel family. The rat P2X6 nucleotide sequence encodes a 379 amino acid protein that conserves all the structural features of previously cloned P2X receptors, including the two putative transmembrane domains predicted by hydrophobicity plots. In situ hybridization analysis of rat brain sections showed a wide pattern of mRNA expression that is virtually identical to that already described for P2X4. Injection of P2X6 cRNA in Xenopus oocytes did not give rise to ATP-activated channels. Coexpression of P2X6 with P2X4 subunits produced currents which were not discernibly different from those of P2X4 expressed alone. © 1996 Academic Press, Inc.
ATP is not only the main energy source in the organism, but also acts as an extracellular messenger in a variety of cells and tissues (1, 2). The binding of ATP to P2X receptors opens, in the millisecond range, ionic channels that are non-selective for monovalent cations and permeable to Ca2+. Molecular cloning techniques led to the identification of 4 different P2X receptors: P2X1-P2X4 (3–11). Functional expression of the recombinant P2X receptors in either Xenopus oocytes or in transfected cells revealed several differences between them. This diversity in responses can be further increased by the ability of some subunits to form heteromultimeric complexes (6). In situ hybridization and tissue distribution analysis revealed that one or more of the cloned P2X receptors can be found in every tissue. The restricted expression pattern of P2X3, that appears to be only in sensory ganglia (5), contrasts with the wide expression pattern of P2X4 (7–10), while P2X1 and P2X2 have a more restrained distribution (12). Up to now, three P2X receptors have been detected in rat brain neurons: P2X1, P2X2 (12) and P2X4 (7–10). We have isolated two additional members, P2X5 (manuscript in preparation) and P2X6, of the purinergic receptor family using PCR-based cloning and low-stringency hybridization techniques. Here we report the cloning and tissue distribution of P2X6, a novel P2X receptor widely expressed in both the central nervous system and in peripheral tissues. MATERIALS AND METHODS cDNA cloning of a novel P2X channel. The P2X6 cDNA was initially detected by PCR in rat brain cDNA using degenerate primers previously described (10). The resulting 750 bp PCR fragment was isolated, 32P-labelled and used to screen 1×106 phages of a commercial rat brain cDNA library (Clontech) under low stringency hybridization conditions (13). None of the phages isolated contained the sequence encoding the N-terminal domain of the protein. A rapid amplification of cDNA ends (RACE) was performed using the Marathon cDNA amplification kit (Clontech). The primers used for amplification were an antisense primer specific for the P2X6 sequence (59-AGCCACTCCCACAGCCGTTTCTC-39) and a sense primer complementary to the anchor sequence (supplied with the kit). Electrophysiology. The full-length P2X6 clone was inserted into the pSGEM vector (10). Capped RNA synthesis and oocyte isolation, handling and two electrode voltage-clamp measurements were performed as previously described (14). The superfusing solution contained 115 mM NaCl, 2.8 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, pH47.2. Tissue expression analysis by PCR. Total RNA prepared by the single-step method (15) from various tissues was reverse transcribed and the resulting cDNAs were analyzed by PCR with specific primers for the P2X6 receptor and, as an internal control of the reaction, with specific primers for the transferrin receptor (TFR) (10). The sense and antisense primers used to detect P2X6 expression were 59-GAAAGGCTCTGACTGCTCTC-39 and 59-AGGAATGGATAGGCTGACAC-39, re-
1
Corresponding author. Fax:+49-551-3899644. e-mail:[email protected]. 456
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
JOBNAME: BBRC 223#2 PAGE: 2 SESS: 13 OUTPUT: Thu Jun 20 08:56:17 1996 /xypage/worksmart/tsp000/72170k/44
Vol. 223, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Multiple alignment of the P2X amino acid sequences. Residues conserved in all four rat sequences are boxed. The putative transmembrane segments are underlined (M1 and M2). The conserved cysteine residues are marked (&sb9;). spectively. The specificity of the PCR amplification was assessed by Southern blot with appropriate [32P]-a-dCTP randomprimed probes for the TFR and P2X6 genes. The final high stringency wash was 0.1× SSC, 0.1% SDS at 65°C. To rule out the possibility of genomic DNA contamination of the cDNA, we performed a PCR under the same conditions on 0.5 mg of rat genomic DNA. No bands were amplified when specific primers for P2X6 were used. The integrity of the genomic DNA used as a template was assessed by amplification using specific primers for an intronless gene (Kv 1.4). In situ hybridization. Digoxigenin-labelled sense and antisense cRNA probes were generated by in vitro transcriptions with DIG-UTP (Boehringer Mannheim) using the 750 bp PCR fragment as a template. Horizontal and sagittal sections (10–16 mm) of adult rat brain were cut on a cryostat, fixed and dehydrated. Pretreatments, hybridization of sections and immunological detection were carried out as reported (16). Color development was stopped after 12–30 hrs. For controls, adjacent sections were hybridized with sense RNA or digested with RNase before hybridization.
RESULTS AND DISCUSSION We have isolated from rat brain, a full length cDNA coding for a new member of the P2X receptor family (P2X6). The deduced amino acid sequence shows 45, 38, 39, 45 and 48% identity with P2X1, P2X2, P2X3, P2X4 and P2X5, respectively (Fig. 1). The hydrophobicity plot of P2X6
FIG. 2. Analysis of P2X4 mRNA tissue expression by RT-PCR and Southern blot autoradiography using a P2X6-specific probe (P2X6 arrow) and a rat transferrin receptor probe (TFR arrow) as a control for RT-PCR efficiency. 457
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FIG. 3. In situ hybridization with DIG-labelled RNA probes shows the localization of P2X6 mRNA in adult rat brain in (A) hippocampus, (C) olfactory bulb, (D) cortex, (E) cerebellum, (F) facial nucleus of the brainstem, and (G) ependymal layer lining the ventricle. Control sections are shown only for (B) hippocampus and (H) ependymal layer. Scale bars represent 250 mm in A, B, G, H and 100 mm in C, D, E, F. 458
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predicts a topology similar to previously described members of the family. There are two possible transmembrane regions flanking a long extracellular loop. The short N- and C-terminal regions are thought to be intracellular. P2X6 contains residues that are conserved in all the recombinant P2X receptors, including ten cysteine residues in the putative extracellular loop which may form disulfide bridges (Fig. 1). The P2X6 has N-glycosylation consensus sites at amino acid positions Asn-157, Asn-187 and Asn-202. RT-PCR analysis reveals that transcripts for the P2X6 receptor are present in most of the tissues analyzed (Fig. 2). This distribution is clearly similar to that displayed by P2X4 (10), but is not as widespread. Interestingly, P2X6 was not detected in RNA isolated from thymus, blood vessels and vas deferens, where the only P2X receptors detected have been P2X1 (17) and P2X4 (10). The expression of P2X6 in secretory tissues such as pituitary and adrenal gland suggests that this receptor may play a role in regulating hormone secretion. The distribution in central nervous system was further analyzed by in situ hybridization of rat brain sections (Fig. 3). P2X6 mRNA was abundantly expressed throughout the brain with a distribution pattern matching the previously described expression of P2X4 receptor mRNA (10). The only exception was the especially strong labelling of the ependymal cell layer lining the ventricular surface with the P2X6 probe (G), where P2X4 mRNA has not been detected. Strong hybridization signals were found in hippocampus (A), olfactory bulb mitral and tufted cells (C), cortex (D), cerebellar Purkinje cells (E), deep cerebellar nuclei, and in most of the brainstem nuclei, such as the facial nucleus (F). The reticular thalamic nucleus was strongly labelled, whereas moderate staining was observed in other thalamic nuclei, hypothalamus, amygdala, inferior colliculus and substantia nigra. Low levels of transcript were detected in the caudate putamen and superior colliculus. No staining was observed in any fiber pathways, in adjacent rat brain sections hybridized with sense RNA probe (H), or in control sections digested with RNase before hybridization (B). P2X4 and P2X6 seem to be the predominant forms of P2X receptors in the brain, since the other forms described, P2X1 and P2X2, are not so highly expressed or widely distributed (12). To characterize the functional properties of the recombinant P2X6 receptor, the cRNA coding for the protein was injected in Xenopus oocytes. Upon application of ATP (up to 500 mM), no inward current was detected. No responses were seen when other nucleotides such as ADP, GTP, UTP, AMP, or the nucleoside adenosine (100 mM) were used. Since it is possible that P2X6 is neither an agonist-binding subunit nor capable of creating a homomeric functional channel, we attempted to coexpress P2X6 with P2X4, the P2X receptor that has a very similar distribution in the brain. When both cRNAs were coinjected into Xenopus oocytes, the agonist-induced currents (ATP, 100 mM) were identical in pharmacology and kinetics to those of the control oocytes injected only with P2X4. We did not observe a dominant negative action of P2X6 on the P2X4 expression level (as determined by current amplitude). It is conceivable that combinations of P2X6 with other members of the family are required to produce a functional channel when expressed in Xenopus oocytes. Furthermore, the channel properties of P2X6 might depend on the expression system used. ACKNOWLEDGMENTS We thank Katja Antonnen for excellent technical assistance and Drs. Michael Hollmann and Anant Parekh for critically reading the manuscript. We are grateful to Drs. G. Collo, R. A. North, E. Kawashima, Merlo-Pich, S. Neidhart, A. Surprenant and G. Buell for kindly sharing unpublished data on P2X6.
REFERENCES 1. 2. 3. 4. 5. 6.
Burnstock, G. (1990) Ann. N.Y. Acad. Sci. 603, 1–17. Dubyak, G. R., and El-Moatassim, C. (1993) Am. J. Physiol. 265, C577–C606. Valera, S., Hussy, N., Evans, R. J., Adami, N., North, R. A., and Surprenant, A. (1994) Nature 371, 516–519. Brake, A. J., Wagenbach, M. J., and Julius, D. (1994) Nature 371, 519–523. Chen, C. C., Akopian, A. N., Sivilotti, L., Colquhoun, D., Burnstock, G., and Wood, J. N. (1995) Nature 377, 428–431. Lewis, C., Neidhart, S., Holy, C., North, R. A., Buell, G., and Surprenant, A. (1995) Nature 377, 432–435. 459
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Bo, X., Zhang, Y., Nassar, M., Burnstock, G., and Schoepfer, R. (1995) FEBS Lett. 375, 129–133. Buell, G., Lewis, C., Collo, G., North, R. A., and Surprenant, A. (1996) EMBO J. 15, 55–62. Seguela, P., Haghighi, A., Soghomonian, J., and Cooper, E. (1996) J. Neurosci. 16, 448–455. Soto, F., Garcia-Guzman, M., Gomez-Hernandez, J. M., Hollmann, M., Karschin, C., and Stühmer, W. (1996) Proc. Natl. Acad. Sci. USA. 93, 3684–3688. Wang, C. Z., Namba, N., Gonoi, T., Iganaki, N., and Seino, S. (1996) Biochem. Biophys. Res. Commun. 220, 196–202. Kidd, E. J., Grahames, C. B. A., Simon, J., Michel, A. D., Barnard, E. A., and Humprey, P. P. A. (1995) Mol. Pharmacol. 48, 569–573. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Stühmer, W. (1992) Methods Enzymol. 207, 319–345. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156–159. Bartsch, S, Bartsch, U., Dorries, U., Faissner, A., Weller, A., Ekblom, P., and Schachner, M. (1992) J. Neurosci. 12, 736–749. Surprenant, A., Buell, G., and North, R. A. (1995) Trends Neurosci. 18, 224–229.
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