γ Subunit of Voltage-activated Calcium Channels (2003)

Voltage-activated Ca2+ channels play a major role in many fundamental physiological processes including neurotransmission, muscle contraction, intracellular signaling, hormone secretion, and development. Therefore, an understanding of the structure and regulation of Ca2+ channels is critical for the comprehension of these physiological phenomena. Five types of high voltage-activated Ca2+ channels (named L-, N-, P-, Q-, and R-type) and one type of low voltage-activated Ca2+ channel (known as T-type) have been identified based on the pharmacological and biophysical characteristics of native currents. Voltage-activated Ca2+ channels (called Ca2+ channel or Cav hereafter) consist of four subunits: α1, β, α2δ, and γ. The α1 subunit is the main subunit responsible for ion conduction, voltage sensing, and binding of Ca2+ channel-specific drugs and toxins. Ten α1 subunits have been cloned and classified as α11.1 (α1S), α11.2 (α1C), α11.3 (α1D), α11.4 (α1F), α12.1 (α1A), α12.2 (α1B), α12.3 (α1E), α13.1 (α1G), α13.2 (α1H), and α13.3 (α1I). The β and α2δ subunits are auxiliary proteins that modulate the properties of the Ca2+ current (1Catterall W.A. Annu. Rev. Biochem. 1995; 64: 493-531Crossref PubMed Scopus (777) Google Scholar, 2De Waard M. Gurnett C.A. Campbell K.P. Narahashi T. Structural and Functional Diversity of Voltage-activated Calcium Channels. 4. Plenum Press, New York1996: 41-87Google Scholar, 3Jones S.W. J. Bioenerg. Biomembr. 1998; 30: 299-312Crossref PubMed Scopus (115) Google Scholar). Four isoforms of the β subunit (β1, β2, β3, and β4) and four isoforms of the α2δ subunit (α2δ-1, α2δ-2, α2δ-3, and α2δ-4) have been identified. An additional auxiliary subunit, γ, initially detected in skeletal muscle Ca2+ channels, has been recently associated with neuronal Ca2+ channels (4Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett II, F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Crossref PubMed Scopus (488) Google Scholar, 5Kang M.G. Chen C.C. Felix R. Letts V.A. Frankel W.N. Mori Y. Campbell K.P. J. Biol. Chem. 2001; 276: 32917-32924Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 6Sharp A.H. Black III, J.L. Dubel S.J. Sundarraj S. Shen J.P. Yunker A.M. Copeland T.D. McEnery M.W. Neuroscience. 2001; 105: 599-617Crossref PubMed Scopus (65) Google Scholar). The study of Ca2+ channel regulation by auxiliary subunits has been focused on β and α2δ subunits and demonstrated positive regulation of functional activities of Ca2+ channels by these proteins. The β and α2δ subunits can increase current amplitude and cause various changes in current kinetics and voltage dependence by increasing the number of channels at the plasma membrane and/or causing allosteric modulations of the α1 subunits (7Walker D. De Waard M. Trends Neurosci. 1998; 21: 148-154Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). The skeletal γ subunit (γ1) was first purified and characterized through biochemical studies with the other subunits of skeletal muscle Ca2+ channels (8Campbell K.P. Leung A.T. Sharp A.H. Trends Neurosci. 1988; 11: 425-430Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 9Sharp A.H. Campbell K.P. J. Biol. Chem. 1989; 264: 2816-2825Abstract Full Text PDF PubMed Google Scholar) followed by cloning of its cDNA (10Jay S.D. Ellis S.B. McCue A.F. Williams M.E. Vedvick T.S. Harpold M.M. Campbell K.P. Science. 1990; 248: 490-492Crossref PubMed Scopus (191) Google Scholar, 11Powers P.A. Liu S. Hogan K. Gregg R.G. J. Biol. Chem. 1993; 268: 9275-9279Abstract Full Text PDF PubMed Google Scholar). The γ subunit has not been as extensively studied as the β and α2δ subunits. The muscle-preferential expression of γ1 might be a reason that γ1 has not been studied in as great detail as the other auxiliary subunits. However, recent genetic studies revealed the existence of a γ subunit isoform in the brain whose lack of expression is responsible for the epileptic and ataxic phenotype of the stargazer mouse (4Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett II, F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Crossref PubMed Scopus (488) Google Scholar). The discovery of a novel neuronal γ (γ2) and its relevance to epilepsy and ataxia has drawn much attention to the study of Ca2+ channel γ subunits. Subsequently, six additional γ subunit isoforms have been identified through DNA data base searches based on sequence homology to γ1 and γ2 (12Black III, J.L. Lennon V.A. Mayo Clin. Proc. 1999; 74: 357-361Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 13Burgess D.L. Davis C.F. Gefrides L.A. Noebels J.L. Genome Res. 1999; 9: 1204-1213Crossref PubMed Scopus (55) Google Scholar, 14Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar, 15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar, 16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). Furthermore, mutations in Ca2+ channel subunits, including γ mutations, have recently been implicated in a number of human and/or animal neurological disorders, drawing even more attention to the study of Ca2+ channel modulation by its auxiliary subunits. All eight γ subunit isoforms (γ1–8) are characterized by four predicted transmembrane domains, intracellular N and C termini, and the first extracellular loop that includes a signature motif (GLWXXC), a conserved N-glycosylation site, and a pair of conserved cysteine residues that may form a disulfide linkage in the first extracellular loop (Fig. 1). Phylogenetic analyses have suggested that all γ subunits are derived from a single ancestral gene through gene duplication (15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar, 16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). Based on the phylogenetic analyses, sequence homologies, and tissue distribution, the γ subunits have been categorized into two groups: skeletal γ (γ1 and γ6) and neuronal γ (γ2–5 and γ7–8) (15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar, 16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). The γ1 and γ6 subunits are classified as skeletal muscle γ subunits based on their predominant expression in skeletal muscle and relatively short C terminus as well as the high sequence homology (62.5% similarity) between them. In addition, a short form of γ6 constituted by two instead of four transmembrane domains was reported (15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar, 16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). The γ2–5 and γ7–8 subunits are classified as neuronal γ subunits based on their predominant expression in neuronal tissues, relatively long C terminus, and the sequence homology (29.5–90.7% similarity) among them. The neuronal γ subunits can be subdivided into two groups. The γ2–4 and γ8 share higher homology than γ5 and γ7. PDZ binding motifs at the end of C termini of the neuronal γ subunits also differentiate the two groups of neuronal γ subunits. The γ2–4 and γ8 have the same PDZ binding motif (R(R/K)TTPV) at the end of C termini, which differs from the potential PDZ binding motif (S(T/S)SPC) of the γ5 and γ7 (16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). The genes of γ5 and γ7 subunits share a distinct intron/exon configuration, a five-exon structure rather than the four-exon structure of the other γ genes (16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar, 17Moss F.J. Viard P. Davies A. Bertaso F. Page K.M. Graham A. Canti C. Plumpton M. Plumpton C. Clare J.J. Dolphin A.C. EMBO J. 2002; 21: 1514-1523Crossref PubMed Scopus (69) Google Scholar). In addition, γ8 differs from the other neuronal γ subunits because of its unusual translation initiation codon (–3 AXXCTGG +4) and as it contains the longest C terminus (16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). The N- and C-terminal regions of γ8 also have extensive C/G nucleotide-rich regions (15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar). There has been confusion about the sequences of γ5 and γ7. The amino acid sequence of the mouse γ5 reported by Klugbauer et al. (14Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar) was quite different from that of human γ5 reported by Burgess et al. (13Burgess D.L. Davis C.F. Gefrides L.A. Noebels J.L. Genome Res. 1999; 9: 1204-1213Crossref PubMed Scopus (55) Google Scholar, 15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar). Chu et al. (16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar) performed a comprehensive analysis of the difference and suggested a new version of the γ5 sequence (16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). Based on the new study it seems that the mouse γ5 reported by Klugbauer et al. (14Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar) does not actually belong to the γ family and that the C-terminal sequence of human γ5 by Burgess et al. (13Burgess D.L. Davis C.F. Gefrides L.A. Noebels J.L. Genome Res. 1999; 9: 1204-1213Crossref PubMed Scopus (55) Google Scholar, 15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar) should be modified (16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). The C-terminal sequence of γ7 was also modified as γ5 by two recent studies (16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar, 17Moss F.J. Viard P. Davies A. Bertaso F. Page K.M. Graham A. Canti C. Plumpton M. Plumpton C. Clare J.J. Dolphin A.C. EMBO J. 2002; 21: 1514-1523Crossref PubMed Scopus (69) Google Scholar). Therefore, the new sequences of γ5 and γ7 genes are different from those of the original reports. The new sequences of γ5 and γ7 have much longer C termini than those of the originals, resulting in C-terminal length similar to that of the other neuronal γ subunits. In addition, it has been suggested that γ may belong to the claudin family, a group of cell adhesion proteins in tight junctions (15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar, 18Tomita S. Nicoll R.A. Bredt D.S. J. Cell Biol. 2001; 153: F19-F24Crossref PubMed Google Scholar). Claudins are similar to neuronal γ subunits in that they have four predicted transmembrane domains, a C-terminal PDZ binding domain, and the first extracellular loop that includes a signature motif (GLWXXC) and a pair of conserved cysteine residues. On the other hand, the phylogenetic relationship of the γ subunits to claudin families is much lower compared with that among γ subunits (15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar). However, the possibility exists that indeed the γ family may be a subfamily of the claudin superfamily. Tissue distribution of γ subunits has been studied mainly at the mRNA level through RT-PCR 1The abbreviations used are: RT, reverse transcriptase; EM, electron microscopy; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate. or Northern blot analyses. In the case of some neuronal γ subunits, there are additional data from in situ hybridization, immunohistochemical, and Western blot analyses. In general, the expression pattern of each γ subunit varies considerably among tissues. The mRNA of γ1 is primarily expressed in skeletal muscle (10Jay S.D. Ellis S.B. McCue A.F. Williams M.E. Vedvick T.S. Harpold M.M. Campbell K.P. Science. 1990; 248: 490-492Crossref PubMed Scopus (191) Google Scholar, 11Powers P.A. Liu S. Hogan K. Gregg R.G. J. Biol. Chem. 1993; 268: 9275-9279Abstract Full Text PDF PubMed Google Scholar, 15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar, 16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar) although mild mRNA expression of γ1 in other tissues has also been reported (10Jay S.D. Ellis S.B. McCue A.F. Williams M.E. Vedvick T.S. Harpold M.M. Campbell K.P. Science. 1990; 248: 490-492Crossref PubMed Scopus (191) Google Scholar, 15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar, 16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). At the protein level, γ1 was detected in skeletal muscle but not in the brain or the heart (9Sharp A.H. Campbell K.P. J. Biol. Chem. 1989; 264: 2816-2825Abstract Full Text PDF PubMed Google Scholar, 19Wissenbach U. Bosse-Doenecke E. Freise D. Ludwig A. Murakami M. Hofmann F. Flockerzi V. J. Biol. Chem. 1998; 379: 45-50Crossref Scopus (19) Google Scholar). Like γ1, γ6 also showed robust expression in skeletal muscle and mild expression in the other tissues (15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar, 16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). In addition, RT-PCR results showed that the short form γ6 has a different tissue distribution from that of the long form γ6 (16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). The analyses of neuronal γ subunits (γ2–5 and γ7–8) showed predominant expression of their mRNA in the brain. However, in contrast to the brain-restricted expression of γ3 and γ5, the other neuronal γ subunits are expressed in various tissues (15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar, 16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). γ2 and γ8 are expressed in testis whereas the expression of γ4 is in lung and prostate tissue (15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar, 20Green P.J. Warre R. Hayes P.D. McNaughton N.C. Medhurst A.D. Pangalos M. Duckworth D.M. Randall A.D. J. Physiol. (Lond.). 2001; 533: 467-478Crossref Scopus (34) Google Scholar). Finally, the expression of γ7 has been detected in various tissues (16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar) although a recent study suggested a brain-specific expression of γ7 (17Moss F.J. Viard P. Davies A. Bertaso F. Page K.M. Graham A. Canti C. Plumpton M. Plumpton C. Clare J.J. Dolphin A.C. EMBO J. 2002; 21: 1514-1523Crossref PubMed Scopus (69) Google Scholar). Similarly, Western blot data from mouse brain showed specific expression of γ2, γ3, and γ4 proteins only in neuronal tissues (6Sharp A.H. Black III, J.L. Dubel S.J. Sundarraj S. Shen J.P. Yunker A.M. Copeland T.D. McEnery M.W. Neuroscience. 2001; 105: 599-617Crossref PubMed Scopus (65) Google Scholar). Regional differences of neuronal γ expression within the brain have been studied at the mRNA and protein levels through RT-PCR, in situ, immunohistochemical, and/or Western blot analyses. The RT-PCR and in situ hybridization analyses of mouse brain showed highest expression of γ2 in cerebellum (4Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett II, F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Crossref PubMed Scopus (488) Google Scholar, 14Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar, 20Green P.J. Warre R. Hayes P.D. McNaughton N.C. Medhurst A.D. Pangalos M. Duckworth D.M. Randall A.D. J. Physiol. (Lond.). 2001; 533: 467-478Crossref Scopus (34) Google Scholar). The γ3 expression was strong in the hippocampus, cerebral cortex, amygdala, and nucleus accumbens (14Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar, 20Green P.J. Warre R. Hayes P.D. McNaughton N.C. Medhurst A.D. Pangalos M. Duckworth D.M. Randall A.D. J. Physiol. (Lond.). 2001; 533: 467-478Crossref Scopus (34) Google Scholar). In addition, γ3 expression in cerebellum was reported by an in situ hybridization study (21Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). The γ4 expression was high in the caudate putamen, olfactory bulb, and habenulae (14Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar). The γ2 and γ4 expression in the cerebellum originates predominantly from the Purkinje cell layer and not from the granular cell layer (14Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar). Likewise, the regional expression of the neuronal γ proteins has been analyzed through immunohistochemistry of mouse brain, and the results are consistent, to a certain degree, with the mRNA expression patterns (6Sharp A.H. Black III, J.L. Dubel S.J. Sundarraj S. Shen J.P. Yunker A.M. Copeland T.D. McEnery M.W. Neuroscience. 2001; 105: 599-617Crossref PubMed Scopus (65) Google Scholar). The γ2 proteins express highest in the cerebellum and cortex. The γ3 protein expression levels are highest in the cortex. The γ4 proteins express highest in the cortex, midbrain, hippocampus, and striatum (6Sharp A.H. Black III, J.L. Dubel S.J. Sundarraj S. Shen J.P. Yunker A.M. Copeland T.D. McEnery M.W. Neuroscience. 2001; 105: 599-617Crossref PubMed Scopus (65) Google Scholar). Finally, partial purification of the Ca2+ channel complex from rabbit cerebellum showed that the protein expression of γ3 in cerebellum is as strong as that of the whole brain (5Kang M.G. Chen C.C. Felix R. Letts V.A. Frankel W.N. Mori Y. Campbell K.P. J. Biol. Chem. 2001; 276: 32917-32924Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Subcellular expression of the γ subunits has been studied through immunofluorescence and electron microscopy (EM) studies. Immunofluorescence studies showed that γ2, γ3, and γ4 can go to the plasma membrane by themselves (20Green P.J. Warre R. Hayes P.D. McNaughton N.C. Medhurst A.D. Pangalos M. Duckworth D.M. Randall A.D. J. Physiol. (Lond.). 2001; 533: 467-478Crossref Scopus (34) Google Scholar, 22Rousset M. Cens T. Restituito S. Barrere C. Black III, J.L. McEnery M.W. Charnet P. J. Physiol. (Lond.). 2001; 532: 583-593Crossref Scopus (60) Google Scholar). However, subcellular expression of γ7 in the COS-7 cell was not associated with the plasma membrane (17Moss F.J. Viard P. Davies A. Bertaso F. Page K.M. Graham A. Canti C. Plumpton M. Plumpton C. Clare J.J. Dolphin A.C. EMBO J. 2002; 21: 1514-1523Crossref PubMed Scopus (69) Google Scholar). The EM study suggested predominant postsynaptic localization of γ2, γ3, and γ4 and weak expression of these γ subunits at the presynapse and the soma of neurons (6Sharp A.H. Black III, J.L. Dubel S.J. Sundarraj S. Shen J.P. Yunker A.M. Copeland T.D. McEnery M.W. Neuroscience. 2001; 105: 599-617Crossref PubMed Scopus (65) Google Scholar, 21Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). A recent subcellular fractionation study also supports the EM results: dominant expression of the γ2 and γ3 in postsynaptic membrane and far less expression in the presynaptic membrane (23Kang M.G. Campbell K.P. Biophys. J. 2002; 82 (abstr.): 26Google Scholar). Relatively less information is available about the γ subunits at the protein level compared with the other Ca2+ channel subunits. The small size and high hydrophobicity of the γ protein limit the choice of peptides for the development of antibodies against a γ subunit that do not cross-react with the other γ subunits. The γ1 subunit was found to be a 32-kDa protein (in reduced SDS-PAGE) co-purified with Ca2+ channels (Cav1.1) from rabbit skeletal muscle (8Campbell K.P. Leung A.T. Sharp A.H. Trends Neurosci. 1988; 11: 425-430Abstract Full Text PDF PubMed Scopus (227) Google Scholar). Immunoprecipitation with antibodies specific for α11.1 and β subunit supports the association of γ1 with Cav1.1 (9Sharp A.H. Campbell K.P. J. Biol. Chem. 1989; 264: 2816-2825Abstract Full Text PDF PubMed Google Scholar). Since γ1 was co-purified with Ca2+ channels of skeletal muscle, there has been little doubt about the association of γ1 with Cav1.1 in native tissue. However, because the neuronal γ subunit was not initially co-purified with Cav2.2 (24Witcher D.R. De Waard M. Sakamoto J. Franzini-Armstrong C. Pragnell M. Kahl S.D. Campbell K.P. Science. 1993; 261: 486-489Crossref PubMed Scopus (183) Google Scholar), the association of neuronal γ with Ca2+ channels has been controversial for several years since the finding of the γ2 (stargazin) through the study of stargazer mice (4Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett II, F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Crossref PubMed Scopus (488) Google Scholar). However, two recent biochemical studies have shown the association of neuronal γ subunits with neuronal Ca2+ channels. The association of γ2 and γ3 with Cav2.1 and Cav2.2 was demonstrated by co-sedimentation of γ2 and γ3 with neuronal Ca2+ channel subunits and by immunoprecipitation of γ2 and γ3 by antibodies specific for either α12.1/2 or β of Ca2+ channels (5Kang M.G. Chen C.C. Felix R. Letts V.A. Frankel W.N. Mori Y. Campbell K.P. J. Biol. Chem. 2001; 276: 32917-32924Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In this study, the molecular sizes of the γ2 and γ3 proteins in native tissue are reported as 38 and 34 kDa (in reduced SDS-PAGE), respectively (5Kang M.G. Chen C.C. Felix R. Letts V.A. Frankel W.N. Mori Y. Campbell K.P. J. Biol. Chem. 2001; 276: 32917-32924Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The association of γ2, γ3, and γ4 with α12.2 was also suggested by immunoprecipitation of α12.2 by antibodies recognizing γ2, γ3, and γ4 (6Sharp A.H. Black III, J.L. Dubel S.J. Sundarraj S. Shen J.P. Yunker A.M. Copeland T.D. McEnery M.W. Neuroscience. 2001; 105: 599-617Crossref PubMed Scopus (65) Google Scholar). In addition, the co-sedimentation of γ2 and γ3 with neuronal Ca2+ channel subunits (5Kang M.G. Chen C.C. Felix R. Letts V.A. Frankel W.N. Mori Y. Campbell K.P. J. Biol. Chem. 2001; 276: 32917-32924Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) was demonstrated through partial purification of neuronal Ca2+ channels performed utilizing wheat germ agglutinin chromatography instead of heparin chromatography, which was used for initial purification of neuronal Ca2+ channels (24Witcher D.R. De Waard M. Sakamoto J. Franzini-Armstrong C. Pragnell M. Kahl S.D. Campbell K.P. Science. 1993; 261: 486-489Crossref PubMed Scopus (183) Google Scholar), suggesting a dissociation of γ2 and γ3 from the Ca2+ channel complex by heparin. Furthermore, a recent study with γ1-γ2 chimeras suggested that the first half of the γ subunit has binding domain(s) to the α1 subunit of Ca2+ channels (25Arikkath J. Chen C.C. Ahern C. Allamand V. Flanagan J.D. Coronado R. Gregg R.G. Campbell K.P. J. Biol. Chem. 2003; 278: 1212-1219Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The association of the other γ subunits with high voltage-activated Ca2+ channels has not yet been studied. No interaction between any γ subunit and low voltage-activated Ca2+ channels in native tissues has been reported. Some neuronal γ subunits (γ2, γ3, γ4, and γ8) have a PDZ binding motif (R(R/K)TTPV) at the end of the C terminus (16Chu P.J. Robertson H.M. Best P.M. Gene (Amst.). 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar). The binding of some PDZ domain-containing proteins to the PDZ binding motif of γ2 has been reported (21Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar, 26Chetkovich D.M. Chen L. Stocker T.J. Nicoll R.A. Bredt D.S. J. Neurosci. 2002; 22: 5791-5796Crossref PubMed Google Scholar, 27Choi J. Ko J. Park E. Lee J. S. E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, two of the studies suggested that the binding of to the PDZ binding motif of the γ2 subunit is by of a in the motif D.M. Chen L. Stocker T.J. Nicoll R.A. Bredt D.S. J. Neurosci. 2002; 22: 5791-5796Crossref PubMed Google Scholar, 27Choi J. Ko J. Park E. Lee J. S. E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, one of these studies also suggested an interaction between the γ2 and α-amino-3-hydroxy-5-methyl-4-isoxazole (21Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). On the other hand, the association of the and with Ca2+ channels has been demonstrated in native but γ2 was not for this association (23Kang M.G. Campbell K.P. Biophys. J. 2002; 82 (abstr.): 26Google Scholar). has been shown that the γ1 subunit is M. W.A. Proc. U. S. A. PubMed Scopus Google Scholar). The of some neuronal γ subunits has been also reported (6Sharp A.H. Black III, J.L. Dubel S.J. Sundarraj S. Shen J.P. Yunker A.M. Copeland T.D. McEnery M.W. Neuroscience. 2001; 105: 599-617Crossref PubMed Scopus (65) Google Scholar). The in the first extracellular loop of the γ subunit is conserved among all γ subunits, suggesting an functional role of the The may play an role in the association of the γ subunit with the Ca2+ channel complex as shown in the study of α2δ C.A. De Waard M. Campbell K.P. Full Text Full Text PDF PubMed Scopus Google Scholar), in the of the γ subunit, and/or in the of proteins with the γ On the other hand, a recent study suggested that the is not for the association of the γ1 subunit with the Ca2+ channel complex (25Arikkath J. Chen C.C. Ahern C. Allamand V. Flanagan J.D. Coronado R. Gregg R.G. Campbell K.P. J. Biol. Chem. 2003; 278: 1212-1219Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In addition, of the γ subunits have several potential for or in to the one in the PDZ binding motif (15Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar). in these may be in the or association of the γ subunit within the Ca2+ channel complex or with other proteins. A relatively small number of γ1 functional compared with that of β and α2δ have been which suggested mild changes in current current and/or voltage dependence of of the Ca2+ channels R. Dai S. Klugbauer N. Hofmann F. PubMed Scopus Google Scholar, Klugbauer N. F. Hofmann F. J. Physiol. PubMed Scopus Google Scholar, Y. T. A. J. P. E. Hofmann F. Flockerzi V. T. K. K. T. S. Nature. PubMed Scopus Google Scholar, D. M. Flockerzi V. Hofmann F. N. Science. PubMed Scopus Google Scholar, E. G. A.M. L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). However, it has been to a the of the γ1 subunit as the results of the functional have been Furthermore, the physiological relevance of γ1 has been