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The α-Bungarotoxin-binding Nicotinic Acetylcholine Receptor from Rat Brain Contains Only the α7 Subunit

1997; Elsevier BV; Volume: 272; Issue: 38 Linguagem: Inglês

10.1074/jbc.272.38.24024

1083-351X

DaNong Chen, James W. Patrick,

Cholinesterase and Neurodegenerative Diseases

When expressed in Xenopus oocytes, the rat α7 subunit forms homo-oligomeric nicotinic acetylcholine receptors, which are blocked by α-bungarotoxin. Since the pharmacological and physiological properties of the α7 receptor expressed in oocytes are similar to those of the α-bungarotoxin-sensitive nicotinic currents recorded from neuronal preparations and the distribution patterns of α7 mRNA and α-bungarotoxin-binding sites in the rat brain are very similar, α7 is thought to be the main component of the α-bungarotoxin-binding nicotinic receptor in the mammalian brain. However, while α7 is found in purified α-bungarotoxin-binding complexes from rat brain or PC12 cells, other proteins copurify with it. Therefore, the question whether α7 forms a homo-oligomeric α-bungarotoxin-binding nicotinic receptor in the mammalian brain remains. We have developed and characterized affinity-purified polyclonal antibodies and used these antibodies in Western blot analyses of α-bungarotoxin-binding proteins purified from rat brains. We report here that our experimental data support the current working hypothesis that the α-bungarotoxin-binding nicotinic receptor is a homo-oligomer of α7 subunits in the rat brain. When expressed in Xenopus oocytes, the rat α7 subunit forms homo-oligomeric nicotinic acetylcholine receptors, which are blocked by α-bungarotoxin. Since the pharmacological and physiological properties of the α7 receptor expressed in oocytes are similar to those of the α-bungarotoxin-sensitive nicotinic currents recorded from neuronal preparations and the distribution patterns of α7 mRNA and α-bungarotoxin-binding sites in the rat brain are very similar, α7 is thought to be the main component of the α-bungarotoxin-binding nicotinic receptor in the mammalian brain. However, while α7 is found in purified α-bungarotoxin-binding complexes from rat brain or PC12 cells, other proteins copurify with it. Therefore, the question whether α7 forms a homo-oligomeric α-bungarotoxin-binding nicotinic receptor in the mammalian brain remains. We have developed and characterized affinity-purified polyclonal antibodies and used these antibodies in Western blot analyses of α-bungarotoxin-binding proteins purified from rat brains. We report here that our experimental data support the current working hypothesis that the α-bungarotoxin-binding nicotinic receptor is a homo-oligomer of α7 subunits in the rat brain. α-Bungarotoxin (α-BTX), 1The abbreviations used are: α-BTX, α-bungarotoxin; nAChR, nicotinic acetylcholine receptor; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: α-BTX, α-bungarotoxin; nAChR, nicotinic acetylcholine receptor; PAGE, polyacrylamide gel electrophoresis. a snake toxin that binds to the muscle-type nicotinic acetylcholine receptor (nAChR), also binds to the surface of sympathetic ganglion cells, cultured PC12 cells (rat pheochromocytoma cells), and brain membranes. The α-BTX-binding sites in these preparations are distinct subpopulations of the neuronal nAChRs. This conclusion is based on the observations that 1) the α-BTX-sensitive nAChRs have a relatively low affinity for nicotine in comparison with the α-BTX-insensitive receptors (1Wonnacott S. J. Neurochem. 1986; 47: 1706-1712Crossref PubMed Scopus (65) Google Scholar); 2) the anatomical distribution of α-BTX-binding sites and nicotine-binding sites in the brain is highly distinct (2Clarke P.B. Schwartz R.D. Paul S.M. Pert C.B. Pert A. J. Neurosci. 1985; 5: 1307-1315Crossref PubMed Google Scholar); 3) α-BTX binds to the surface of PC12 cells, but fails to block nicotinic agonist-mediated responses in these cells (3Patrick J. Stallcup B. J. Biol. Chem. 1977; 252: 8629-8633Abstract Full Text PDF PubMed Google Scholar, 4Stallcup W.B. J. Physiol. ( Lond .). 1979; 286: 525-540Crossref PubMed Scopus (109) Google Scholar, 5Lukas R.J. J. Pharmacol. Exp. Ther. 1990; 253: 51-57PubMed Google Scholar); and 4) α-BTX-sensitive and -insensitive nicotinic receptors in PC12 cells and in the brain can be distinguished immunologically (6Patrick J. Stallcup W.B. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4689-4692Crossref PubMed Scopus (122) Google Scholar, 7Whiting P.J. Schoepfer R. Swanson L.W. Simmons D.M. Lindstrom J.M. Nature. 1987; 327: 515-518Crossref PubMed Scopus (62) Google Scholar).The molecular basis for these differences have been revealed by cloning the genes encoding subunits of the neuronal nAChRs (for reviews, see Ref. 8McGehee D.S. Role L.W. Annu. Rev. Physiol. 1995; 57: 521-546Crossref PubMed Scopus (896) Google Scholar). In the rat, 10 genes encoding neuronal nAChR subunits have been identified to date. Seven encode α subunits (α2–α7 and α9), and three encode β subunits (β2–β4). When expressed inXenopus oocytes, only α7 and α9 form homo-oligomeric nAChRs, and activation of these receptors can be blocked by α-BTX (9Seguela P. Wadiche J. Dineley-Miller K. Dani J.A. Patrick J.W. J. Neurosci. 1993; 13: 596-604Crossref PubMed Google Scholar,10Elgoyhen A.B. Johnson D.S. Boulter J. Vetter D.E. Heinemann S. Cell. 1994; 79: 705-715Abstract Full Text PDF PubMed Scopus (741) Google Scholar). Since in situ hybridization has shown the α9 subunit to be present in hair cells but not in the brain and the α9 receptor expressed in oocytes has unique pharmacological properties that are similar to those of the cholinergic receptor observed in vertebrate cochlear hair cells, the α9 receptor is thought to be involved primarily in the cholinergic efferent innervation of the cochlear hair cells (10Elgoyhen A.B. Johnson D.S. Boulter J. Vetter D.E. Heinemann S. Cell. 1994; 79: 705-715Abstract Full Text PDF PubMed Scopus (741) Google Scholar). On the other hand, α7 mRNA is widely expressed in the rat brain and is thought to be the main component of the neuronal α-BTX-binding nAChR (9Seguela P. Wadiche J. Dineley-Miller K. Dani J.A. Patrick J.W. J. Neurosci. 1993; 13: 596-604Crossref PubMed Google Scholar).It is unclear, however, whether the rat neuronal α-BTX-binding nAChR consists of only the α7 subunit. Data supporting this notion are as follows. 1) α7 forms homo-oligomeric ligand-gated ion channels when expressed in oocytes, and the function of these receptors can be blocked by nanomolar concentrations of α-BTX (9Seguela P. Wadiche J. Dineley-Miller K. Dani J.A. Patrick J.W. J. Neurosci. 1993; 13: 596-604Crossref PubMed Google Scholar, 11Gerzanich V. Anand R. Lindstrom J. Mol. Pharmacol. 1994; 45: 212-220PubMed Google Scholar, 12Couturier S. Bertrand D. Matter J.M. Hernandez M.C. Bertrand S. Millar N. Valera S. Barkas T. Ballivet M. Neuron. 1990; 5: 847-856Abstract Full Text PDF PubMed Scopus (815) Google Scholar); 2) the distribution of α7 mRNA in the adult rat brain is similar to that of the α-125I-BTX-binding sites previously documented (2Clarke P.B. Schwartz R.D. Paul S.M. Pert C.B. Pert A. J. Neurosci. 1985; 5: 1307-1315Crossref PubMed Google Scholar); 3) both α7 mRNA and the α-BTX-binding sites are transiently expressed in primary sensory cortical and thalamic regions of developing rat and mouse brains (13Fuchs J.L. Brain Res. 1989; 501: 223-234Crossref PubMed Scopus (62) Google Scholar, 14Broide R.S. O'Connor L.T. Smith M.A. Smith J.A. Leslie F.M. Neuroscience. 1995; 67: 83-94Crossref PubMed Scopus (120) Google Scholar); 4) currents elicited from the α7 receptor expressed in oocytes have fast kinetic profiles (12Couturier S. Bertrand D. Matter J.M. Hernandez M.C. Bertrand S. Millar N. Valera S. Barkas T. Ballivet M. Neuron. 1990; 5: 847-856Abstract Full Text PDF PubMed Scopus (815) Google Scholar), similar to those of α-BTX-blockable nicotinic currents recorded from cultured rat hippocampal neurons that have α7 expressed on the surface (15Radcliffe K.A. Rajan A. Zarei M.M. Yakehiro M. Hsu J.Y. Dani J.A. Soc. Neurosci. Abstr. 1996; 22: 1264Google Scholar); and 5) the α7 receptor expressed in oocytes has high Ca2+ permeability (9Seguela P. Wadiche J. Dineley-Miller K. Dani J.A. Patrick J.W. J. Neurosci. 1993; 13: 596-604Crossref PubMed Google Scholar), which is also observed with the α-BTX-sensitive nicotinic receptor found in cultured hippocampal neurons (16Albuquerque E.X. Pereira E.F. Castro N.G. Alkondon M. Reinhardt S. Schroder H. Maelicke A. Ann. N. Y. Acad. Sci. 1995; 757: 48-72Crossref PubMed Scopus (145) Google Scholar).However, α-BTX-binding proteins purified from neuronal preparations show more than one band on SDS gels stained with Coomassie Blue, possibly because the neuronal α-BTX-binding nAChR is a hetero-oligomer (17Kemp G. Bentley L. McNamee M.G. Morley B.J. Brain Res. 1985; 347: 274-283Crossref PubMed Scopus (32) Google Scholar, 18Gotti C. Ogando A.E. Clementi F. Neuroscience. 1989; 32: 759-767Crossref PubMed Scopus (13) Google Scholar, 19Whiting P. Lindstrom J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 595-599Crossref PubMed Scopus (245) Google Scholar, 20Conti-Tronconi B.M. Dunn S.M. Barnard E.A. Dolly J.O. Lai F.A. Ray N. Raftery M.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5208-5212Crossref PubMed Scopus (106) Google Scholar). In the chick brain, some α-BTX-binding proteins are hetero-oligomers composed of both α7 and α8 subunits (21Gotti C. Hanke W. Maury K. Moretti M. Ballivet M. Clementi F. Bertrand D. Eur. J. Neurosci. 1994; 6: 1281-1291Crossref PubMed Scopus (83) Google Scholar). Although a rat homologue of chick α8 has not been identified, it is possible that α7 associates with other neuronal nicotinic receptor subunits to form hetero-oligomeric α-BTX-binding receptors in the rat brain. There are areas in the rat brain where the expression of other receptor subunit genes overlaps with the expression of the α7 gene (22Dineley-Miller K. Patrick J. Brain Res. Mol. Brain Res. 1992; 16: 339-344Crossref PubMed Scopus (172) Google Scholar). Furthermore, different subunits can express in a single cell. PC12 cells, for example, have α-BTX-binding sites on their surface and express mRNAs encoding α3, α5, β2, and β4 in addition to α7 (23Rogers S.W. Mandelzys A. Deneris E.S. Cooper E. Heinemann S. J. Neurosci. 1992; 12: 4611-4623Crossref PubMed Google Scholar).To determine whether the α7 subunit in the α-BTX-binding nAChR from rat brain is associated with other known neuronal nicotinic receptor subunits, we developed affinity-purified polyclonal antibodies and used them in Western blot analyses of affinity-purified α-BTX-binding sites. We found no evidence that α7 is associated with other subunits in the α-BTX-binding nAChR. The data from our experiments support the current working hypothesis that, in the mammalian brain, the α-BTX-binding nAChR is a homo-oligomer of α7 subunits.DISCUSSIONWe conducted experiments to determine the subunit composition of the α-BTX-binding nAChR in the rat brain. Using Western blot analysis, we showed that only the α7 subunit was detected in neuronal α-BTX-binding receptors purified from all four brain fractions. In addition, we calculated the ratio of α-BTX-binding sites to the α7 subunit in the receptors from these fractions and showed that it was constant (0.20). Therefore, the most straightforward interpretation for these observations is that the rat neuronal α-BTX-binding nAChR is a homo-oligomer of α7 subunits with one detectable α-BTX-binding site.Several groups have reported that other proteins copurify with α7 from brains using α-BTX affinity columns (17Kemp G. Bentley L. McNamee M.G. Morley B.J. Brain Res. 1985; 347: 274-283Crossref PubMed Scopus (32) Google Scholar, 35Gotti C. Briscini L. Verderio C. Oortgiesen M. Balestra B. Clementi F. Eur. J. Neurosci. 1995; 7: 2083-2092Crossref PubMed Scopus (40) Google Scholar, 36Gotti C. Ogando A.E. Hanke W. Schlue R. Moretti M. Clementi F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3258-3262Crossref PubMed Scopus (45) Google Scholar). Therefore, it is possible that in vivo, the neuronal α-BTX-binding nAChR is a hetero-oligomer of α7 plus other subunits. We performed Western blot analyses and showed that α3, α5, α6, β2, β3, and β4 were not detected in the α-BTX-binding nAChR purified from rat brains. Although the antibodies used in our experiments did not recognize α2 and α4, both subunits are unlikely to be present in the neuronal α-BTX-binding nAChR due to the observations that α2 mRNA is expressed in very restricted brain areas and α4 mRNA is hardly detected in the hippocampus, where α-BTX-binding sites are abundant (2Clarke P.B. Schwartz R.D. Paul S.M. Pert C.B. Pert A. J. Neurosci. 1985; 5: 1307-1315Crossref PubMed Google Scholar, 37Wada E. Wada K. Boulter J. Deneris E. Heinemann S. Patrick J. Swanson L.W. J. Comp. Neurol. 1989; 284: 314-335Crossref PubMed Scopus (924) Google Scholar), and that neither α2 nor α4 mRNAs are found in PC12 cells, which also express α-BTX-binding sites (23Rogers S.W. Mandelzys A. Deneris E.S. Cooper E. Heinemann S. J. Neurosci. 1992; 12: 4611-4623Crossref PubMed Google Scholar).A rat homologue of the chicken α8 subunit, which also binds α-BTX, has not been found. Nevertheless, it is unlikely that an α8-like subunit is present in the rat neuronal α-BTX-binding nAChR. As shown in Fig. 1, anti-α7-rp, a polyclonal antibody against the N terminus of α7, also recognizes the N termini of α3, α6, β2, and β4, subunits that share only 40% amino acid identity with α7 in the N-terminal region. On the other hand, the N termini of the chicken α7 and α8 subunits share 78% amino acid identity. Therefore, if an α8-like subunit were in the rat neuronal α-BTX-binding nAChR, it is likely that the antibody anti-α7-rp would recognize it on Western blots. Chicken and rat α7 subunits have the same molecular mass (38Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (406) Google Scholar). If an α8-like protein existed in the rat brain and were the same size as chicken α8, it might also be expected to migrate as a 60-kDa protein on SDS gels (38Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (406) Google Scholar). The blots probed with anti-α7-rp were carefully examined at different exposure times, and no immunoreactive protein band of 60 kDa was observed. The α9 subunit, expressed only in epithelial cells, is unlikely to be present in the α-BTX-binding nAChR in the brain (10Elgoyhen A.B. Johnson D.S. Boulter J. Vetter D.E. Heinemann S. Cell. 1994; 79: 705-715Abstract Full Text PDF PubMed Scopus (741) Google Scholar).Therefore, the results from our Western blot analyses indicate that subunits other than α7 are unlikely to be present in the α-BTX-binding nAChR, consistent with the current working hypothesis that the α-BTX-binding nAChR is a homo-oligomer of α7 subunits in the rat brain. A plausible explanation for the proteins copurifying with α7 is that they may be associated proteins such as cytoskeletal proteins and/or degradation products of α7.The number of α-BTX-binding sites per rat neuronal α-BTX-binding nAChR is not known. However, given the assumption that the receptor is a homo-oligomer of five α7 subunits, several predictions can be made. 1) If each α7 subunit binds α-BTX in the mature receptor, the predicted number would be five; 2) if only a correctly folded α7 dimer forms an α-BTX-binding site and two such dimers plus a monomer assemble to form a mature α7 pentamer, then the predicted number would be two; 3) if only one α-BTX-binding site is formed on a mature α7 receptor or if a receptor has five possible α-BTX-binding sites, but binding of the first α-BTX sterically prevents further α-BTX binding, the predicted number would be one.We calculated the molar ratio of α-BTX-binding sites to the α7 subunit in α-BTX-binding nAChRs from all brain fractions (Fig. 7) and found that it is 0.2. This suggests that the number of detectable α-BTX-binding sites per pentameric α7 receptor is one, consistent with the third prediction above. Since, under certain experimental conditions, only one of the two α-BTX-binding sites of the muscle-type nAChR can be detected (39Conti-Tronconi B.M. Tang F. Walgrave S. Gallagher W. Biochemistry. 1990; 29: 1046-1054Crossref PubMed Scopus (37) Google Scholar, 40Sine S.M. Taylor P. J. Biol. Chem. 1981; 256: 6692-6699Abstract Full Text PDF PubMed Google Scholar), it is possible that the α7 receptor consists of two or more α-BTX-binding sites, but only one is detected under our experimental conditions. In either case, the estimated molar ratio supports the current working hypothesis that the α-BTX-binding nAChR is a homo-oligomer of α7 subunits in the rat brain. α-Bungarotoxin (α-BTX), 1The abbreviations used are: α-BTX, α-bungarotoxin; nAChR, nicotinic acetylcholine receptor; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: α-BTX, α-bungarotoxin; nAChR, nicotinic acetylcholine receptor; PAGE, polyacrylamide gel electrophoresis. a snake toxin that binds to the muscle-type nicotinic acetylcholine receptor (nAChR), also binds to the surface of sympathetic ganglion cells, cultured PC12 cells (rat pheochromocytoma cells), and brain membranes. The α-BTX-binding sites in these preparations are distinct subpopulations of the neuronal nAChRs. This conclusion is based on the observations that 1) the α-BTX-sensitive nAChRs have a relatively low affinity for nicotine in comparison with the α-BTX-insensitive receptors (1Wonnacott S. J. Neurochem. 1986; 47: 1706-1712Crossref PubMed Scopus (65) Google Scholar); 2) the anatomical distribution of α-BTX-binding sites and nicotine-binding sites in the brain is highly distinct (2Clarke P.B. Schwartz R.D. Paul S.M. Pert C.B. Pert A. J. Neurosci. 1985; 5: 1307-1315Crossref PubMed Google Scholar); 3) α-BTX binds to the surface of PC12 cells, but fails to block nicotinic agonist-mediated responses in these cells (3Patrick J. Stallcup B. J. Biol. Chem. 1977; 252: 8629-8633Abstract Full Text PDF PubMed Google Scholar, 4Stallcup W.B. J. Physiol. ( Lond .). 1979; 286: 525-540Crossref PubMed Scopus (109) Google Scholar, 5Lukas R.J. J. Pharmacol. Exp. Ther. 1990; 253: 51-57PubMed Google Scholar); and 4) α-BTX-sensitive and -insensitive nicotinic receptors in PC12 cells and in the brain can be distinguished immunologically (6Patrick J. Stallcup W.B. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4689-4692Crossref PubMed Scopus (122) Google Scholar, 7Whiting P.J. Schoepfer R. Swanson L.W. Simmons D.M. Lindstrom J.M. Nature. 1987; 327: 515-518Crossref PubMed Scopus (62) Google Scholar). The molecular basis for these differences have been revealed by cloning the genes encoding subunits of the neuronal nAChRs (for reviews, see Ref. 8McGehee D.S. Role L.W. Annu. Rev. Physiol. 1995; 57: 521-546Crossref PubMed Scopus (896) Google Scholar). In the rat, 10 genes encoding neuronal nAChR subunits have been identified to date. Seven encode α subunits (α2–α7 and α9), and three encode β subunits (β2–β4). When expressed inXenopus oocytes, only α7 and α9 form homo-oligomeric nAChRs, and activation of these receptors can be blocked by α-BTX (9Seguela P. Wadiche J. Dineley-Miller K. Dani J.A. Patrick J.W. J. Neurosci. 1993; 13: 596-604Crossref PubMed Google Scholar,10Elgoyhen A.B. Johnson D.S. Boulter J. Vetter D.E. Heinemann S. Cell. 1994; 79: 705-715Abstract Full Text PDF PubMed Scopus (741) Google Scholar). Since in situ hybridization has shown the α9 subunit to be present in hair cells but not in the brain and the α9 receptor expressed in oocytes has unique pharmacological properties that are similar to those of the cholinergic receptor observed in vertebrate cochlear hair cells, the α9 receptor is thought to be involved primarily in the cholinergic efferent innervation of the cochlear hair cells (10Elgoyhen A.B. Johnson D.S. Boulter J. Vetter D.E. Heinemann S. Cell. 1994; 79: 705-715Abstract Full Text PDF PubMed Scopus (741) Google Scholar). On the other hand, α7 mRNA is widely expressed in the rat brain and is thought to be the main component of the neuronal α-BTX-binding nAChR (9Seguela P. Wadiche J. Dineley-Miller K. Dani J.A. Patrick J.W. J. Neurosci. 1993; 13: 596-604Crossref PubMed Google Scholar). It is unclear, however, whether the rat neuronal α-BTX-binding nAChR consists of only the α7 subunit. Data supporting this notion are as follows. 1) α7 forms homo-oligomeric ligand-gated ion channels when expressed in oocytes, and the function of these receptors can be blocked by nanomolar concentrations of α-BTX (9Seguela P. Wadiche J. Dineley-Miller K. Dani J.A. Patrick J.W. J. Neurosci. 1993; 13: 596-604Crossref PubMed Google Scholar, 11Gerzanich V. Anand R. Lindstrom J. Mol. Pharmacol. 1994; 45: 212-220PubMed Google Scholar, 12Couturier S. Bertrand D. Matter J.M. Hernandez M.C. Bertrand S. Millar N. Valera S. Barkas T. Ballivet M. Neuron. 1990; 5: 847-856Abstract Full Text PDF PubMed Scopus (815) Google Scholar); 2) the distribution of α7 mRNA in the adult rat brain is similar to that of the α-125I-BTX-binding sites previously documented (2Clarke P.B. Schwartz R.D. Paul S.M. Pert C.B. Pert A. J. Neurosci. 1985; 5: 1307-1315Crossref PubMed Google Scholar); 3) both α7 mRNA and the α-BTX-binding sites are transiently expressed in primary sensory cortical and thalamic regions of developing rat and mouse brains (13Fuchs J.L. Brain Res. 1989; 501: 223-234Crossref PubMed Scopus (62) Google Scholar, 14Broide R.S. O'Connor L.T. Smith M.A. Smith J.A. Leslie F.M. Neuroscience. 1995; 67: 83-94Crossref PubMed Scopus (120) Google Scholar); 4) currents elicited from the α7 receptor expressed in oocytes have fast kinetic profiles (12Couturier S. Bertrand D. Matter J.M. Hernandez M.C. Bertrand S. Millar N. Valera S. Barkas T. Ballivet M. Neuron. 1990; 5: 847-856Abstract Full Text PDF PubMed Scopus (815) Google Scholar), similar to those of α-BTX-blockable nicotinic currents recorded from cultured rat hippocampal neurons that have α7 expressed on the surface (15Radcliffe K.A. Rajan A. Zarei M.M. Yakehiro M. Hsu J.Y. Dani J.A. Soc. Neurosci. Abstr. 1996; 22: 1264Google Scholar); and 5) the α7 receptor expressed in oocytes has high Ca2+ permeability (9Seguela P. Wadiche J. Dineley-Miller K. Dani J.A. Patrick J.W. J. Neurosci. 1993; 13: 596-604Crossref PubMed Google Scholar), which is also observed with the α-BTX-sensitive nicotinic receptor found in cultured hippocampal neurons (16Albuquerque E.X. Pereira E.F. Castro N.G. Alkondon M. Reinhardt S. Schroder H. Maelicke A. Ann. N. Y. Acad. Sci. 1995; 757: 48-72Crossref PubMed Scopus (145) Google Scholar). However, α-BTX-binding proteins purified from neuronal preparations show more than one band on SDS gels stained with Coomassie Blue, possibly because the neuronal α-BTX-binding nAChR is a hetero-oligomer (17Kemp G. Bentley L. McNamee M.G. Morley B.J. Brain Res. 1985; 347: 274-283Crossref PubMed Scopus (32) Google Scholar, 18Gotti C. Ogando A.E. Clementi F. Neuroscience. 1989; 32: 759-767Crossref PubMed Scopus (13) Google Scholar, 19Whiting P. Lindstrom J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 595-599Crossref PubMed Scopus (245) Google Scholar, 20Conti-Tronconi B.M. Dunn S.M. Barnard E.A. Dolly J.O. Lai F.A. Ray N. Raftery M.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5208-5212Crossref PubMed Scopus (106) Google Scholar). In the chick brain, some α-BTX-binding proteins are hetero-oligomers composed of both α7 and α8 subunits (21Gotti C. Hanke W. Maury K. Moretti M. Ballivet M. Clementi F. Bertrand D. Eur. J. Neurosci. 1994; 6: 1281-1291Crossref PubMed Scopus (83) Google Scholar). Although a rat homologue of chick α8 has not been identified, it is possible that α7 associates with other neuronal nicotinic receptor subunits to form hetero-oligomeric α-BTX-binding receptors in the rat brain. There are areas in the rat brain where the expression of other receptor subunit genes overlaps with the expression of the α7 gene (22Dineley-Miller K. Patrick J. Brain Res. Mol. Brain Res. 1992; 16: 339-344Crossref PubMed Scopus (172) Google Scholar). Furthermore, different subunits can express in a single cell. PC12 cells, for example, have α-BTX-binding sites on their surface and express mRNAs encoding α3, α5, β2, and β4 in addition to α7 (23Rogers S.W. Mandelzys A. Deneris E.S. Cooper E. Heinemann S. J. Neurosci. 1992; 12: 4611-4623Crossref PubMed Google Scholar). To determine whether the α7 subunit in the α-BTX-binding nAChR from rat brain is associated with other known neuronal nicotinic receptor subunits, we developed affinity-purified polyclonal antibodies and used them in Western blot analyses of affinity-purified α-BTX-binding sites. We found no evidence that α7 is associated with other subunits in the α-BTX-binding nAChR. The data from our experiments support the current working hypothesis that, in the mammalian brain, the α-BTX-binding nAChR is a homo-oligomer of α7 subunits. DISCUSSIONWe conducted experiments to determine the subunit composition of the α-BTX-binding nAChR in the rat brain. Using Western blot analysis, we showed that only the α7 subunit was detected in neuronal α-BTX-binding receptors purified from all four brain fractions. In addition, we calculated the ratio of α-BTX-binding sites to the α7 subunit in the receptors from these fractions and showed that it was constant (0.20). Therefore, the most straightforward interpretation for these observations is that the rat neuronal α-BTX-binding nAChR is a homo-oligomer of α7 subunits with one detectable α-BTX-binding site.Several groups have reported that other proteins copurify with α7 from brains using α-BTX affinity columns (17Kemp G. Bentley L. McNamee M.G. Morley B.J. Brain Res. 1985; 347: 274-283Crossref PubMed Scopus (32) Google Scholar, 35Gotti C. Briscini L. Verderio C. Oortgiesen M. Balestra B. Clementi F. Eur. J. Neurosci. 1995; 7: 2083-2092Crossref PubMed Scopus (40) Google Scholar, 36Gotti C. Ogando A.E. Hanke W. Schlue R. Moretti M. Clementi F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3258-3262Crossref PubMed Scopus (45) Google Scholar). Therefore, it is possible that in vivo, the neuronal α-BTX-binding nAChR is a hetero-oligomer of α7 plus other subunits. We performed Western blot analyses and showed that α3, α5, α6, β2, β3, and β4 were not detected in the α-BTX-binding nAChR purified from rat brains. Although the antibodies used in our experiments did not recognize α2 and α4, both subunits are unlikely to be present in the neuronal α-BTX-binding nAChR due to the observations that α2 mRNA is expressed in very restricted brain areas and α4 mRNA is hardly detected in the hippocampus, where α-BTX-binding sites are abundant (2Clarke P.B. Schwartz R.D. Paul S.M. Pert C.B. Pert A. J. Neurosci. 1985; 5: 1307-1315Crossref PubMed Google Scholar, 37Wada E. Wada K. Boulter J. Deneris E. Heinemann S. Patrick J. Swanson L.W. J. Comp. Neurol. 1989; 284: 314-335Crossref PubMed Scopus (924) Google Scholar), and that neither α2 nor α4 mRNAs are found in PC12 cells, which also express α-BTX-binding sites (23Rogers S.W. Mandelzys A. Deneris E.S. Cooper E. Heinemann S. J. Neurosci. 1992; 12: 4611-4623Crossref PubMed Google Scholar).A rat homologue of the chicken α8 subunit, which also binds α-BTX, has not been found. Nevertheless, it is unlikely that an α8-like subunit is present in the rat neuronal α-BTX-binding nAChR. As shown in Fig. 1, anti-α7-rp, a polyclonal antibody against the N terminus of α7, also recognizes the N termini of α3, α6, β2, and β4, subunits that share only 40% amino acid identity with α7 in the N-terminal region. On the other hand, the N termini of the chicken α7 and α8 subunits share 78% amino acid identity. Therefore, if an α8-like subunit were in the rat neuronal α-BTX-binding nAChR, it is likely that the antibody anti-α7-rp would recognize it on Western blots. Chicken and rat α7 subunits have the same molecular mass (38Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (406) Google Scholar). If an α8-like protein existed in the rat brain and were the same size as chicken α8, it might also be expected to migrate as a 60-kDa protein on SDS gels (38Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (406) Google Scholar). The blots probed with anti-α7-rp were carefully examined at different exposure times, and no immunoreactive protein band of 60 kDa was observed. The α9 subunit, expressed only in epithelial cells, is unlikely to be present in the α-BTX-binding nAChR in the brain (10Elgoyhen A.B. Johnson D.S. Boulter J. Vetter D.E. Heinemann S. Cell. 1994; 79: 705-715Abstract Full Text PDF PubMed Scopus (741) Google Scholar).Therefore, the results from our Western blot analyses indicate that subunits other than α7 are unlikely to be present in the α-BTX-binding nAChR, consistent with the current working hypothesis that the α-BTX-binding nAChR is a homo-oligomer of α7 subunits in the rat brain. A plausible explanation for the proteins copurifying with α7 is that they may be associated proteins such as cytoskeletal proteins and/or degradation products of α7.The number of α-BTX-binding sites per rat neuronal α-BTX-binding nAChR is not known. However, given the assumption that the receptor is a homo-oligomer of five α7 subunits, several predictions can be made. 1) If each α7 subunit binds α-BTX in the mature receptor, the predicted number would be five; 2) if only a correctly folded α7 dimer forms an α-BTX-binding site and two such dimers plus a monomer assemble to form a mature α7 pentamer, then the predicted number would be two; 3) if only one α-BTX-binding site is formed on a mature α7 receptor or if a receptor has five possible α-BTX-binding sites, but binding of the first α-BTX sterically prevents further α-BTX binding, the predicted number would be one.We calculated the molar ratio of α-BTX-binding sites to the α7 subunit in α-BTX-binding nAChRs from all brain fractions (Fig. 7) and found that it is 0.2. This suggests that the number of detectable α-BTX-binding sites per pentameric α7 receptor is one, consistent with the third prediction above. Since, under certain experimental conditions, only one of the two α-BTX-binding sites of the muscle-type nAChR can be detected (39Conti-Tronconi B.M. Tang F. Walgrave S. Gallagher W. Biochemistry. 1990; 29: 1046-1054Crossref PubMed Scopus (37) Google Scholar, 40Sine S.M. Taylor P. J. Biol. Chem. 1981; 256: 6692-6699Abstract Full Text PDF PubMed Google Scholar), it is possible that the α7 receptor consists of two or more α-BTX-binding sites, but only one is detected under our experimental conditions. In either case, the estimated molar ratio supports the current working hypothesis that the α-BTX-binding nAChR is a homo-oligomer of α7 subunits in the rat brain. We conducted experiments to determine the subunit composition of the α-BTX-binding nAChR in the rat brain. Using Western blot analysis, we showed that only the α7 subunit was detected in neuronal α-BTX-binding receptors purified from all four brain fractions. In addition, we calculated the ratio of α-BTX-binding sites to the α7 subunit in the receptors from these fractions and showed that it was constant (0.20). Therefore, the most straightforward interpretation for these observations is that the rat neuronal α-BTX-binding nAChR is a homo-oligomer of α7 subunits with one detectable α-BTX-binding site. Several groups have reported that other proteins copurify with α7 from brains using α-BTX affinity columns (17Kemp G. Bentley L. McNamee M.G. Morley B.J. Brain Res. 1985; 347: 274-283Crossref PubMed Scopus (32) Google Scholar, 35Gotti C. Briscini L. Verderio C. Oortgiesen M. Balestra B. Clementi F. Eur. J. Neurosci. 1995; 7: 2083-2092Crossref PubMed Scopus (40) Google Scholar, 36Gotti C. Ogando A.E. Hanke W. Schlue R. Moretti M. Clementi F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3258-3262Crossref PubMed Scopus (45) Google Scholar). Therefore, it is possible that in vivo, the neuronal α-BTX-binding nAChR is a hetero-oligomer of α7 plus other subunits. We performed Western blot analyses and showed that α3, α5, α6, β2, β3, and β4 were not detected in the α-BTX-binding nAChR purified from rat brains. Although the antibodies used in our experiments did not recognize α2 and α4, both subunits are unlikely to be present in the neuronal α-BTX-binding nAChR due to the observations that α2 mRNA is expressed in very restricted brain areas and α4 mRNA is hardly detected in the hippocampus, where α-BTX-binding sites are abundant (2Clarke P.B. Schwartz R.D. Paul S.M. Pert C.B. Pert A. J. Neurosci. 1985; 5: 1307-1315Crossref PubMed Google Scholar, 37Wada E. Wada K. Boulter J. Deneris E. Heinemann S. Patrick J. Swanson L.W. J. Comp. Neurol. 1989; 284: 314-335Crossref PubMed Scopus (924) Google Scholar), and that neither α2 nor α4 mRNAs are found in PC12 cells, which also express α-BTX-binding sites (23Rogers S.W. Mandelzys A. Deneris E.S. Cooper E. Heinemann S. J. Neurosci. 1992; 12: 4611-4623Crossref PubMed Google Scholar). A rat homologue of the chicken α8 subunit, which also binds α-BTX, has not been found. Nevertheless, it is unlikely that an α8-like subunit is present in the rat neuronal α-BTX-binding nAChR. As shown in Fig. 1, anti-α7-rp, a polyclonal antibody against the N terminus of α7, also recognizes the N termini of α3, α6, β2, and β4, subunits that share only 40% amino acid identity with α7 in the N-terminal region. On the other hand, the N termini of the chicken α7 and α8 subunits share 78% amino acid identity. Therefore, if an α8-like subunit were in the rat neuronal α-BTX-binding nAChR, it is likely that the antibody anti-α7-rp would recognize it on Western blots. Chicken and rat α7 subunits have the same molecular mass (38Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (406) Google Scholar). If an α8-like protein existed in the rat brain and were the same size as chicken α8, it might also be expected to migrate as a 60-kDa protein on SDS gels (38Schoepfer R. Conroy W.G. Whiting P. Gore M. Lindstrom J. Neuron. 1990; 5: 35-48Abstract Full Text PDF PubMed Scopus (406) Google Scholar). The blots probed with anti-α7-rp were carefully examined at different exposure times, and no immunoreactive protein band of 60 kDa was observed. The α9 subunit, expressed only in epithelial cells, is unlikely to be present in the α-BTX-binding nAChR in the brain (10Elgoyhen A.B. Johnson D.S. Boulter J. Vetter D.E. Heinemann S. Cell. 1994; 79: 705-715Abstract Full Text PDF PubMed Scopus (741) Google Scholar). Therefore, the results from our Western blot analyses indicate that subunits other than α7 are unlikely to be present in the α-BTX-binding nAChR, consistent with the current working hypothesis that the α-BTX-binding nAChR is a homo-oligomer of α7 subunits in the rat brain. A plausible explanation for the proteins copurifying with α7 is that they may be associated proteins such as cytoskeletal proteins and/or degradation products of α7. The number of α-BTX-binding sites per rat neuronal α-BTX-binding nAChR is not known. However, given the assumption that the receptor is a homo-oligomer of five α7 subunits, several predictions can be made. 1) If each α7 subunit binds α-BTX in the mature receptor, the predicted number would be five; 2) if only a correctly folded α7 dimer forms an α-BTX-binding site and two such dimers plus a monomer assemble to form a mature α7 pentamer, then the predicted number would be two; 3) if only one α-BTX-binding site is formed on a mature α7 receptor or if a receptor has five possible α-BTX-binding sites, but binding of the first α-BTX sterically prevents further α-BTX binding, the predicted number would be one. We calculated the molar ratio of α-BTX-binding sites to the α7 subunit in α-BTX-binding nAChRs from all brain fractions (Fig. 7) and found that it is 0.2. This suggests that the number of detectable α-BTX-binding sites per pentameric α7 receptor is one, consistent with the third prediction above. Since, under certain experimental conditions, only one of the two α-BTX-binding sites of the muscle-type nAChR can be detected (39Conti-Tronconi B.M. Tang F. Walgrave S. Gallagher W. Biochemistry. 1990; 29: 1046-1054Crossref PubMed Scopus (37) Google Scholar, 40Sine S.M. Taylor P. J. Biol. Chem. 1981; 256: 6692-6699Abstract Full Text PDF PubMed Google Scholar), it is possible that the α7 receptor consists of two or more α-BTX-binding sites, but only one is detected under our experimental conditions. In either case, the estimated molar ratio supports the current working hypothesis that the α-BTX-binding nAChR is a homo-oligomer of α7 subunits in the rat brain. We thank Jean-Baptiste Le Pichon, Finn Martin Göldner, and Kelly Dineley for careful reading of the manuscript.