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• W2113145881 abstract "The role of the extracellular domain of the voltage-dependent Ca2+ channel α2δ subunit in assembly with the α1Csubunit was investigated. Transiently transfected tsA201 cells processed the α2δ subunit properly as disulfide linkages and cleavage sites between the α2 and δ subunits were shown to be similar to native channel protein. Coimmunoprecipitation experiments demonstrated that in the absence of δ subunits, α2 subunits do not assemble with α1 subunits. Furthermore, the transmembrane and cytoplasmic sequences in δ can be exchanged with those of an unrelated protein without any effect on the association between the α2δ and α1 proteins. Extracellular domains of the α2δ subunit are also shown to be responsible for increasing the binding affinity of [3H]PN200-110 (isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-([3H]methoxycarbonyl)-pyridine-3-carboxylate) for the α1C subunit. Investigation of the corresponding interaction site on the α1 subunit revealed that although tryptic peptides containing repeat III of native α1S subunit remain in association with the α2δ subunit during wheat germ agglutinin chromatography, repeat III by itself is not sufficient for assembly with the α2δ subunit. Our results suggest that the α2δ subunit likely interacts with more than one extracellular loop of the α1 subunit. The role of the extracellular domain of the voltage-dependent Ca2+ channel α2δ subunit in assembly with the α1Csubunit was investigated. Transiently transfected tsA201 cells processed the α2δ subunit properly as disulfide linkages and cleavage sites between the α2 and δ subunits were shown to be similar to native channel protein. Coimmunoprecipitation experiments demonstrated that in the absence of δ subunits, α2 subunits do not assemble with α1 subunits. Furthermore, the transmembrane and cytoplasmic sequences in δ can be exchanged with those of an unrelated protein without any effect on the association between the α2δ and α1 proteins. Extracellular domains of the α2δ subunit are also shown to be responsible for increasing the binding affinity of [3H]PN200-110 (isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-([3H]methoxycarbonyl)-pyridine-3-carboxylate) for the α1C subunit. Investigation of the corresponding interaction site on the α1 subunit revealed that although tryptic peptides containing repeat III of native α1S subunit remain in association with the α2δ subunit during wheat germ agglutinin chromatography, repeat III by itself is not sufficient for assembly with the α2δ subunit. Our results suggest that the α2δ subunit likely interacts with more than one extracellular loop of the α1 subunit. The α2δ subunit has been identified in every voltage-dependent Ca2+ channel purified to date from various mammalian tissues, including skeletal muscle (1Takahashi M. Seagar M.J. Jones J.F. Reber B.F.X. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5478-5482Crossref PubMed Scopus (323) Google Scholar, 2Leung A.T. Imagawa T. Campbell K.P. J. Biol. Chem. 1987; 262: 7943-7946Abstract Full Text PDF PubMed Google Scholar), brain (3Witcher 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 (181) Google Scholar, 4Liu H. De Waard M. Scott V.E.S. Gurnett C.A. Lennon V.A. Campbell K.P. J. Biol. Chem. 1996; 271: 13804-13810Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and heart (5Cooper C.L. Vandaele S. Barhanin J. Fosset M. Lazdunski M. Hosey M.M. J. Biol. Chem. 1987; 262: 509-512Abstract Full Text PDF PubMed Google Scholar, 6Tokumaru H. Shojaku S. Takehara H. Hirashima N. Abe T. Saisu H. Kirino Y. J. Neurochem. 1995; 65: 831-836Crossref PubMed Scopus (9) Google Scholar). Structurally, the α2δ subunit is a heavily glycosylated 175-kDa protein that is encoded by a single gene that is post-translationally cleaved to yield the disulfide-linked α2 and δ proteins (7De Jongh K.S. Warner C. Catterall W.A. J. Biol. Chem. 1990; 265: 14738-14741Abstract Full Text PDF PubMed Google Scholar, 8Jay S.D. Sharp A.H. Kahl S.D. Vedvick T.S. Harpold M.M. Campbell K.P. J. Biol. Chem. 1991; 266: 3287-3293Abstract Full Text PDF PubMed Google Scholar). Experimental evidence supports a single transmembrane topology of the α2δ subunit in which all but the transmembrane sequence and 5 carboxyl-terminal amino acids are extracellular (9Brickley K. Campbell V. Berrow N. Leach R. Norman R.I. Wray D. Dolphin A.C. Baldwin S.A. FEBS Lett. 1995; 364: 129-133Crossref PubMed Scopus (47) Google Scholar, 10Gurnett C.A. De Waard M. Campbell K.P. Neuron. 1996; 16: 431-440Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 11Wiser O. Trus M. Tobi D. Halevi S. Giladi E. Atlas D. FEBS Lett. 1996; 379: 15-20Crossref PubMed Scopus (48) Google Scholar). Coexpression of mRNA encoding the Ca2+ channel α2δ subunit has been shown to modify many properties of the α1 subunit, including increasing the macroscopic current amplitude (12Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takeshima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (765) Google Scholar, 13Mori Y. Friedrich T. Kim M. Mikami A. Nakai J. Ruth P. Bosse E. Hofmann F. Flockerzi V. Furuichi T. Tikoshiba K. Imoto K. Tanabe T. Numa S. Nature. 1991; 350: 398-402Crossref PubMed Scopus (705) Google Scholar), accelerating the activation (14Bangalore R. Mehrke G. Gingrich K. Hofmann F. Kass R.S. Am. J. Physiol. 1996; 270: H1521-H1528PubMed Google Scholar) and inactivation kinetics, and shifting the voltage dependence of activation to more hyperpolarizing potentials. 1R. Felix and K. P. Campbell, unpublished observations. 1R. Felix and K. P. Campbell, unpublished observations. However, the physical structures and molecular interactions that mediate these effects are entirely unknown. Interaction sites on the pore-forming α subunit have been identified for several voltage-dependent ion channel auxiliary subunits, including the Ca2+ and K+ channel β subunits. The binding site of the Ca2+ channel β subunit has been localized to a region of approximately 18 amino acids in the α1 subunit I-II cytoplasmic linker (15Pragnell M.P. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Crossref PubMed Scopus (545) Google Scholar), and the corresponding interaction site on the β subunit has also been described (16De Waard M. Pragnell M. Campbell K.P. Neuron. 1994; 13: 495-503Abstract Full Text PDF PubMed Scopus (222) Google Scholar). Interaction sites between K+ channel α and β subunits have been mapped to the amino-terminal A and B box (17Yu W. Xu J. Li M. Neuron. 1996; 16: 441-453Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 18Sewing S. Roeper J. Pongs O. Neuron. 1996; 16: 455-463Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) near the cytoplasmic region that is also responsible for the subfamily-specific assembly of α subunit multimers (19Shen V. Pfaffinger P. Neuron. 1995; 14: 625-633Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 20Li M. Jan Y.N. Jan L.Y. Science. 1992; 257: 1225-1240Crossref PubMed Scopus (395) Google Scholar). Unlike the previously described cytoplasmic interactions, assessment of interactions between two transmembrane proteins has generally been more challenging. Transmembrane proteins such as the Ca2+channel α2δ subunit are often extensively glycosylated, which may preclude the use of bacterial, insect, or in vitroexpression systems because glycosylation is frequently species-dependent. Likewise, the expression and correct formation of disulfide linkages is also difficult to reproduce in anin vitro expression system. Also, although there are reports of successful uses of the two-hybrid yeast expression system to map interaction sites of two transmembrane proteins (21Colonna T.E. Huynh L. Fambrough D.M. J. Biol. Chem. 1997; 272: 12366-12372Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), these have often been performed on a more limited basis after initial investigations localized interaction domains using mammalian expression systems. Using transiently transfected human tsA201 cells, we have implicated the extracellular domain of the α2δ subunit in the assembly with the α1 subunit and have also shown that this region is responsible for modulation of dihydropyridine binding affinity to the α1 subunit. tsA201 cells (SV40 large T antigen transformed HEK 293 cells) (Cell Genesis, Foster City, CA) were maintained at 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mml-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. Transfections were performed using the calcium phosphate method on 50–70% confluent cells. Generally 30 μg of each channel subunit DNA (for 150-mm dish) was added to 1.25 ml of 250 mm sterile filtered CaCl2. An equal volume of 2 × sterile HEBS (274 mm NaCl, 40 mmHEPES, 12 mm dextrose, 10 mm KCl, 1.4 mm Na2HPO4, adjusted to final pH 7.05) was added drop by drop to the Ca2+/DNA mixture with constant agitation. The precipitate was allowed to form for 30 min and added dropwise to the plated cells. The medium was changed the next day. The cDNA encoding the rat brain α2δ subunit and truncated forms were all transferred to pcDNA3 (Invitrogen) and have been described previously (10Gurnett C.A. De Waard M. Campbell K.P. Neuron. 1996; 16: 431-440Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). The α1S repeat III was created by polymerase chain reaction utilizing a forward primer beginning at nucleotide 2544 and a reverse primer beginning at nucleotide 3489. A Kozak initiation start consisting of CCACCATGG (where the methionine start site is underlined) was created in the forward primer along with aKpnI site for insertion into polylinker of pcDNA3. The reverse primer contained an in frame termination site and aXbaI site for ligation. tsA201 cells were harvested 48 h after transfection by washing two times with 10 ml of phosphate-buffered saline and collected by centrifugation at 3,000 rpm for 5 min. Cell membranes were prepared immediately by resuspending cell pellet from one 150-mm plate in 20 ml of ice-cold hypotonic lysis buffer (10 mm Tris, pH 7.4 with 0.64 mmbenzamidine, and 0.23 mmPMSF). 2The abbreviations used are: PMSF, phenylmethanesulfonyl fluoride; [3H]PN200-110, isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-([3H]methoxycarbonyl)-pyridine-3-carboxylate; WGA, wheat germ agglutinin. After a 15-min incubation on ice, swollen cells were disrupted by five strokes with a Dounce homogenizer. Lysed cells were centrifuged at 3,000 rpm for 10 min at 4 °C. The supernatant was then centrifuged at 35,000 rpm for 37 min to collect the membranes. The membrane pellet was resuspended in 1 ml of Buffer I (0.3 m sucrose, 20 mm Tris, pH 7.4, 1.0 mm PMSF, and 0.75 mm benzamidine) and passed through a 28 gauge needle. Binding assays were performed in 50 mm Tris, pH 7.4, 0.23 mm PMSF, 0.64 mm benzamidine, and 1.0 mg/ml bovine serum albumin (binding buffer) in a final assay volume of 500 μl. For saturation analysis, 0.05–2 nm (+)-[3H]PN200-110 (Amersham Corp.) were incubated in the dark with 80 μg of membrane protein for 60 min at 37 °C. Nonspecifically bound ligand was determined by the addition of 50 μm nitrendipine. Specific binding sites were determined by subtracting nonspecific binding from total binding. Radiolabeled membranes were washed three times with 5 ml of ice-cold binding buffer on a GF/B glass fiber filter (Whatman) using a Brandel cell harvester (Brandel, Gaithersburg, MD). Data were fitted by a single-site binding model applying nonlinear regression analysis using GraFit software (Trithacus Software, Staines, UK). Cell membranes (500 μg) were solubilized in 1 ml of total volume of 1% (w/v) digitonin and 1m NaCl (final concentrations) for 1 h at 4 °C on a rolling platform. Protease inhibitors were added at a concentration of 0.23 mm PMSF and 0.64 mm benzamidine. Solubilized protein was isolated by centrifugation at 50,000 rpm for 15 min at 4 °C and subsequently diluted 2-fold with ice-cold double distilled H20. Solubilized protein was added to 30 μl of protein G-Sepharose that had been preincubated overnight with sheep 41 antiserum (α1C II-III loop) (4Liu H. De Waard M. Scott V.E.S. Gurnett C.A. Lennon V.A. Campbell K.P. J. Biol. Chem. 1996; 271: 13804-13810Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) or IIF7 ascites (2Leung A.T. Imagawa T. Campbell K.P. J. Biol. Chem. 1987; 262: 7943-7946Abstract Full Text PDF PubMed Google Scholar). Rabbit skeletal muscle triads (22Sharp A.H. Imagawa T. Leung A.T. Campbell K.P. J. Biol. Chem. 1987; 262: 12309-12315Abstract Full Text PDF PubMed Google Scholar) were resuspended to a concentration of 4 mg/ml in Buffer I. Trypsin concentrations were varied from 1:1000 to 1:10 (trypsin:protein), and the incubation time was variable between 0 and 160 min at 37 °C. The reaction was terminated by the addition of 1 mm PMSF. Trypsin digested triads were then solubilized in 1% digitonin and 0.5 mNaCl for 1 h at 4 °C followed by centrifugation at 70,000 rpm for 30 min. Solubilized protein was added to WGA-Sepharose (Sigma) and incubated for 3 h at 4 °C. Bound protein was eluted in 300 mm N-acetylglucosamine in Buffer I. For sucrose gradient fractionation, samples were concentrated to 600 μl in a YM100 (Amicon) concentration unit. Samples were loaded onto 16-ml linear gradients of 5–20% (w/w) sucrose in 100 mm NaCl, 50 mm Tris, pH 7.4, and 0.1 mm PMSF. Gradients were centrifuged in a Beckman SW 28 rotor with SW 28.1 buckets for 2 h at 150,000 rpm at 4 °C. Gradients were then fractionated (1.2 ml) from the top with an Isco gradient fractionator, and 100 μl of each was analyzed on a 5–16% SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue or immunoblotted with monoclonal antibody IIF7 or rabbit 136 polyclonal against the Ca2+ channel α2 subunit (23Gurnett C.A. Kahl S.D. Anderson R.D. Campbell K.P. J. Biol. Chem. 1995; 270: 9035-9038Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Monoclonal antibodies IIF7 and IIC12 (2Leung A.T. Imagawa T. Campbell K.P. J. Biol. Chem. 1987; 262: 7943-7946Abstract Full Text PDF PubMed Google Scholar) were used to screen 2 × 104 clones of α1S subunit epitope library in Y1090 Escherichia coli. Inserts were amplified from pure phage positives by polymerase chain reaction using primers directed to λgt11 phage arms. These were directly inserted into a T-vector (made from Bluescript Sk− plasmid) for sequencing. All inserts were sequenced using either the dideoxy chain termination method Sequenase II (U. S. Biochemical Corp.) or automated sequencer (Applied Biosystems, Inc.). Because of the extensive post-translational processing events involved in the formation of the α2δ subunit (N-linked glycosylation, disulfide linkages, and subunit cleavage), we chose to utilize the mammalian tsA201 cell line for expression. The endogenous proteolytic cleavage between the α2 and δ subunits was investigated by Western blot analysis of membranes from cells transfected with the full-length α2δ subunit. In nonreducing conditions, an antibody directed against the α2 subunit recognized a protein of 175 kDa in both skeletal muscle triads and in membranes prepared from tsA201 cells transfected with the full-length α2δ subunit (Fig. 1). One apparent difference between native and transfected α2δ protein is that the transfected α2δ protein ran as a broader band. This may reflect larger amounts of incompletely processed forms including untrimmed glycosylation and immature noncleaved protein that often result from transient expression. Cells expressing only the α2subunit produced a protein migrating at 150 kDa in both the presence and the absence of reducing agents, which is consistent with the addition of more than 40 kDa of N-linked oligosaccharide. When identical cell membranes were electrophoresed in reducing conditions, native skeletal muscle α2δ subunit shifted to an apparent molecular mass of 150 kDa. Likewise, there was a noticeable shift in molecular mass of the transfected α2δ protein, suggesting that the cleavage site and disulfide linkages are similar to native protein. To test the involvement of the extracellular domain of the α2δ subunit in the interaction with the α1 subunit, coimmunoprecipitation experiments were performed using cells transfected with both α1C subunit and either the full-length α2δ subunit or any one of the truncated α2δ subunit constructs (Fig.2 A). Cell membranes were solubilized in 1% digitonin and 1 m NaCl prior to immunoprecipitation. The full-length α2δ subunit assembles with the α1C subunit as demonstrated by its coimmunoprecipitation with an anti-α1C antibody and detection by Western blot analysis with an anti-α2 antibody (Fig. 2 B). No α2δ protein was immunoprecipitated from control untransfected cells. In addition, no α2δ protein was precipitated from cells in the absence of the α1 subunit (data not shown). Truncation of 450 extracellular amino-terminal amino acids of the α2δ subunit abolished the ability of this protein to assemble with the α1C subunit, despite its abundance in the starting material. Likewise, the α2subunit expressed in the absence of the δ subunit was also unable to coimmunoprecipitate with the α1C subunit. We also investigated the role of the transmembrane domain of the δ subunit in assembly with the α1C subunit (Fig.2 B). Substitution of the transmembrane domain from adhalin, an unrelated type I transmembrane protein (recently renamed α-sarcoglycan), did not appear to alter the ability of the protein to assemble with the α1C subunit. In this chimera, the 5 cytoplasmic amino acids of the α2δ protein were also substituted with adhalin sequence. Therefore, we conclude that neither intracellular or transmembrane sequences of the α2δ subunit are required for interaction with the α1subunit. The region of the α2δ subunit responsible for modulation of dihydropyridine binding to the α1C subunit was also investigated. Although [3H]PN200-110 binding to whole cell tsA201 cell membranes was often low and nonsaturable when α1C was transfected in the absence of any auxiliary subunit, several experiments resulted in significant and saturable binding that allowed us to determine the binding affinity (K d) and binding capacity (B max) using saturation analysis (TableI). Cells expressing α1C alone had an average B max of 94.6 ± 51.7 fmol/mg (n = 4).Table IComparison of K d and saturable [3 H]PN200–110 binding to total microsomes from tsA201 cells transfected with α 1C and α 2 δ constructsMembranesK d ± S.E.Saturable/totalnmα1C1.34 ± 0.794 /7α1Cα2δ1-aPaired t test, p < 0.05 versus α1C.0.16 ± 0.085 /5α1Cα20.65 ± 0.263 /5α1CNΔ28–4731.31 ± 0.743 /5α1Cα2δAd1-aPaired t test, p < 0.05 versus α1C.0.21 ± 0.164 /4tsA201 cells were transfected with the cDNAs encoding Ca2+channel subunits in the combinations indicated. K d values were calculated from saturation binding data using GraFit. Data are presented as the means ± S.E. Also shown are the number of experiments (separate transfections) in which saturable binding was measured and the total number of experiments that were performed.1-a Paired t test, p < 0.05 versus α1C. Open table in a new tab tsA201 cells were transfected with the cDNAs encoding Ca2+channel subunits in the combinations indicated. K d values were calculated from saturation binding data using GraFit. Data are presented as the means ± S.E. Also shown are the number of experiments (separate transfections) in which saturable binding was measured and the total number of experiments that were performed. Coexpression of the full-length α2δ subunit with the α1C subunit resulted in a significant increase in binding, most of which could be accounted for by a significant mean increase in the binding affinity (Table I). Binding was saturable in all experiments. There appeared to be little effect of the α2δ subunit on B max(B max = 133 ± 73.5 fmol/mg), although there was significant error between experiments in theB max depending on the transfection efficiency. Likewise, Western blot analysis on whole cell membranes from transfected cells showed no effect of coexpression of the α2δ subunit on the protein expression of the α1 subunit (data not shown). The binding affinity, however, was not affected by the differences in transfection efficiency. As expected, when the α2 subunit was coexpressed with the α1C subunit, there was no effect on [3H]PN200-110 binding. This is consistent with coimmunoprecipitation experiments that demonstrated the inability of the α2 subunit to associate with α1 in the absence of the δ subunit. However, coexpression of the α2δAd chimera, in which the transmembrane domain of the α2δ subunit was replaced with that of adhalin, increased [3H]PN200-110 binding affinity to approximately the same extent as full-length α2δ protein. Because the α1 subunit is very large and difficult to express, we chose an alternative approach to identify regions interacting with the α2δ subunit. Our approach was to trypsinize skeletal muscle microsomes containing native dihydropyridine receptors and follow the α1S subunit fragments remaining in association with the α2δ subunit during WGA affinity chromatography. By taking advantage of the selective ability of the glycosylated α2δ subunit to bind WGA, any α1S fragment identified is presumed to bind WGA only through its interaction with the α2δ subunit. With increasing concentrations of trypsin, an α1Ssubunit-specific monoclonal antibody that recognizes an epitope within the first extracellular loop of the IIIS5-IIIS6 linker (amino acid 955–1005) (IIF7) detected 28- and 18-kDa α1S subunit fragments eluted from a WGA-Sepharose column (Fig. 3). Sucrose gradient fractionation was subsequently used to demonstrate cosedimentation of the α1S subunit fragments with the intact full-length α2δ subunit (Fig. 4). Multiple tryptic fragments of α1 did not bind WGA and were identified in the starting material, including carboxyl-terminal fragments (identified by monoclonal antibody IIC12) and fragments of repeat I and II and the II-III loop (identified by polyclonal antibody sheep DHPR) (data not shown).Figure 4Sucrose gradient fractionation of trypsinized skeletal muscle WGA eluate. Trypsinized skeletal muscle triads were loaded onto WGA-Sepharose and eluted with 300 mm N-acetylglucosamine. Samples were loaded onto 5–20% linear sucrose gradients, centrifuged, and fractionated from the top. Alternate fractions were analyzed on a 5–16% SDS-polyacrylamide gradient gel and immunoblotted with either monoclonal antibody IIF7 against the α1S subunit (top) or polyclonal antibody against the α2δ subunit (Rabbit 136) (bottom). Fraction number is listed on thebottom, and molecular mass is indicated at theleft. Arrows indicate α1S fragment (frag) and full-length α2δ subunit.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test the ability of α1S repeat III to associate with the α2δ subunit, we cotransfected tsA201 cells with constructs containing only repeat III and the full-length α2δ subunit. Although the α2δ subunit and repeat III were well expressed, we were unable to detect stable interactions between these two proteins using coimmunoprecipitation assays after solubilization in 1% digitionin and 1 m NaCl (data not shown). This suggests that expression of the α1S subunit repeat III by itself is not sufficient to form stable interactions with the α2δ subunit. Our data support a model whereby the interaction sites between the α2δ and α1 subunits are entirely extracellular, because transmembrane modifications of the α2δ subunit did not appear to alter coassembly with the α1 subunit. Morever, our data suggest a requirement for nontransmembrane domains of the δ subunit in determining a stable association between the α2δ and α1proteins, because the α2 protein by itself could not support interaction. δ may contain the interaction site, or the tertiary structure it confers on α2 through its disulfide linkages may enable α2 to directly interact with the α1 subunit. Low expression of δ expressed alone resulted in our inability to distinguish between these possibilities. However, δ was shown to be able to compete with full-length α2δ protein in Xenopus oocytes and inhibit its stimulatory effects on current amplitude (10Gurnett C.A. De Waard M. Campbell K.P. Neuron. 1996; 16: 431-440Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), and expression studies in tsA201 cells demonstrated that coexpression of δ can significantly modulate the biophysical properties of the α1C subunit.1 Interestingly, our data are consistent with reports regarding a functionally related pair of proteins, the Na+,K+-ATPase α and auxiliary β subunit, in which the interaction sites have also been localized to extracellular domains (24Hamrick M. Renaud K.J. Fambrough D.M. J. Biol. Chem. 1993; 268: 24367-24373Abstract Full Text PDF PubMed Google Scholar, 25Lemas M.V. Hamrick M. Takeyasu K. Fambrough D.M. J. Biol. Chem. 1994; 269: 8255-8259Abstract Full Text PDF PubMed Google Scholar). In this case, the yeast two-hybrid assay was successful in further localizing the site of interaction on the β subunit to the 61 amino acids most proximal to the membrane (21Colonna T.E. Huynh L. Fambrough D.M. J. Biol. Chem. 1997; 272: 12366-12372Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Although the interaction sites between the voltage-dependent Na+ channel α and β1 or β2 subunits have not been mapped, it is interesting to note that deletion of the β1intracellular domain does not alter functional effects of β1 subunit coexpression (26Chen C. Cannon S. Pfluegers Arch. 1995; 431: 186-195Crossref PubMed Scopus (67) Google Scholar), suggesting that this interaction may also be in the extracellular domain. Repeat III of the Ca2+ channel α1S subunit appears to interact strongly with the α2δ subunit after extensive trypsinization, although we cannot exclude the involvement of other unidentified fragments (especially in repeat IV) based on our inability to recognize small tryptic fragments with specific antibodies. Interestingly, whereas repeat III remains in association with the α2δ subunit after extensive trypsinization, we were unable to reconstitute the interaction between this small region and α2δ in an expression system. This suggests that multiple regions of the α1 subunit may be involved in assembly with the α2δ subunit. In analogous studies on the voltage-dependent Na+ channel, multiple domains within the carboxyl-terminal half of the skeletal muscle Na+ channel α subunit were shown to be required for functional response of the coexpressed Na+ channel β1 subunit on inactivation kinetics (27Makita N. Bennett P.B. George A.L. Circ. Res. 1996; 78: 244-252Crossref PubMed Scopus (39) Google Scholar). Association of the α2δ subunit with the carboxyl-terminal half of the α1 subunit is consistent with the significant effects that we and others have measured of the α2δ subunit on dihydropyridine binding affinity (28Wei X. Pan S. Lang W. Kim H. Schneider T. Perez-Reyes E. Birnbaumer L. J. Biol. Chem. 1995; 270: 27106-27111Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The membrane spanning segments IIIS6 and IVS6 of the α1Ssubunit have recently been shown to contain amino acids critical for dihydropyridine binding (29Peterson B.Z. Catterall W.A. J. Biol. Chem. 1995; 270: 18201-18204Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), although the S5-S6 extracellular linkers of the III and IV repeats also confer dihydropyridine sensitivity (30Grabner M. Wang Z. Hering S. Streissnig J. Glossmann H. Neuron. 1996; 16: 207-218Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The extracellular domain of the α2δ subunit, which is capable of modulating dihydropyridine binding, may be interacting with sites at or near these dihydropyridine binding sites within the III and IV repeats. Based on several observations regarding the extracellular regions of the α1 subunit, we can speculate on the exact sites of interaction. Most extracellular loops of the α1 subunit are small in size, the smallest being only 7 amino acids. The largest extracellular loops, and thus the regions with the highest probability of interacting with the α2δ subunit, are the S5-S6 linkers, which also contain the pore. Experimental evidence regarding the folding pattern of the α1 subunit suggests that the S5-S6 regions of all four repeats closely interact to form the central pore (31MacKinnon R. Neuron. 1995; 14: 889-892Abstract Full Text PDF PubMed Scopus (222) Google Scholar). These amino acids near the pore, while neighboring each other in tertiary structure, are far apart in primary structure, and thus it may be difficult to reconstitute the structure of this region by expression of a single repeat. Based on the substantial effects of the α2δ subunit on dihydropyridine binding affinity, we predict that the smallest regions of interaction may be within the S5-S6 extracellular loops, particularly of repeat III, because this region copurifies with the α2δ subunit on WGA chromatography. We thank the late Dr. Xiangyang Wei (Medical College of Georgia) for providing the α1C cDNA clone and Dr. Terry Snutch (University of British Columbia) for the α2δ cDNA clone. We acknowledge the University of Iowa Diabetes and Endocrinology Research Center, which is funded by National Institutes of Health Grant DK25295, for cell culture reagents and the University of Iowa DNA Core Facility for DNA sequencing." @default.
• W2113145881 created "2016-06-24" @default.
• W2113145881 creator A5057403367 @default.
• W2113145881 creator A5085822063 @default.
• W2113145881 creator A5087886690 @default.
• W2113145881 date "1997-07-01" @default.
• W2113145881 modified "2023-09-30" @default.
• W2113145881 title "Extracellular Interaction of the Voltage-dependent Ca2+ Channel α2δ and α1 Subunits" @default.
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