Extracellular Interaction of the Voltage-dependent Ca2+ Channel alpha 2delta and alpha 1 Subunits*

(Received for publication, March 17, 1997, and in revised form, May 14, 1997)

Christina A. Gurnett Dagger , Ricardo Felix § and Kevin P. Campbell par

From the Department of Physiology and Biophysics and the  Department of Neurology, Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The role of the extracellular domain of the voltage-dependent Ca2+ channel alpha 2delta subunit in assembly with the alpha 1C subunit was investigated. Transiently transfected tsA201 cells processed the alpha 2delta subunit properly as disulfide linkages and cleavage sites between the alpha 2 and delta  subunits were shown to be similar to native channel protein. Coimmunoprecipitation experiments demonstrated that in the absence of delta  subunits, alpha 2 subunits do not assemble with alpha 1 subunits. Furthermore, the transmembrane and cytoplasmic sequences in delta  can be exchanged with those of an unrelated protein without any effect on the association between the alpha 2delta and alpha 1 proteins. Extracellular domains of the alpha 2delta 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 alpha 1C subunit. Investigation of the corresponding interaction site on the alpha 1 subunit revealed that although tryptic peptides containing repeat III of native alpha 1S subunit remain in association with the alpha 2delta subunit during wheat germ agglutinin chromatography, repeat III by itself is not sufficient for assembly with the alpha 2delta subunit. Our results suggest that the alpha 2delta subunit likely interacts with more than one extracellular loop of the alpha 1 subunit.


INTRODUCTION

The alpha 2delta subunit has been identified in every voltage-dependent Ca2+ channel purified to date from various mammalian tissues, including skeletal muscle (1, 2), brain (3, 4), and heart (5, 6). Structurally, the alpha 2delta 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 alpha 2 and delta  proteins (7, 8). Experimental evidence supports a single transmembrane topology of the alpha 2delta subunit in which all but the transmembrane sequence and 5 carboxyl-terminal amino acids are extracellular (9-11).

Coexpression of mRNA encoding the Ca2+ channel alpha 2delta subunit has been shown to modify many properties of the alpha 1 subunit, including increasing the macroscopic current amplitude (12, 13), accelerating the activation (14) and inactivation kinetics, and shifting the voltage dependence of activation to more hyperpolarizing potentials.1 However, the physical structures and molecular interactions that mediate these effects are entirely unknown.

Interaction sites on the pore-forming alpha  subunit have been identified for several voltage-dependent ion channel auxiliary subunits, including the Ca2+ and K+ channel beta  subunits. The binding site of the Ca2+ channel beta  subunit has been localized to a region of approximately 18 amino acids in the alpha 1 subunit I-II cytoplasmic linker (15), and the corresponding interaction site on the beta  subunit has also been described (16). Interaction sites between K+ channel alpha  and beta  subunits have been mapped to the amino-terminal A and B box (17, 18) near the cytoplasmic region that is also responsible for the subfamily-specific assembly of alpha  subunit multimers (19, 20).

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 alpha 2delta subunit are often extensively glycosylated, which may preclude the use of bacterial, insect, or in vitro expression systems because glycosylation is frequently species-dependent. Likewise, the expression and correct formation of disulfide linkages is also difficult to reproduce in an in 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 (21), 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 alpha 2delta subunit in the assembly with the alpha 1 subunit and have also shown that this region is responsible for modulation of dihydropyridine binding affinity to the alpha 1 subunit.


EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

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 mM L-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 mM HEPES, 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.

Construction of Plasmids for Mammalian Cell Transfection

The cDNA encoding the rat brain alpha 2delta subunit and truncated forms were all transferred to pcDNA3 (Invitrogen) and have been described previously (10). The alpha 1S 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 a KpnI site for insertion into polylinker of pcDNA3. The reverse primer contained an in frame termination site and a XbaI site for ligation.

Cell Membrane Preparation

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 mM benzamidine, and 0.23 mM PMSF).2 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

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).

Immunoprecipitation

Cell membranes (500 µg) were solubilized in 1 ml of total volume of 1% (w/v) digitonin and 1 M 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 (alpha 1C II-III loop) (4) or IIF7 ascites (2).

Trypsin Digestion of Triads and Sucrose Gradient Fractionation

Rabbit skeletal muscle triads (22) 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 M NaCl 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 alpha 2 subunit (23).

Determination of Monoclonal Antibody Recognition Epitopes

Monoclonal antibodies IIF7 and IIC12 (2) were used to screen 2 × 104 clones of alpha 1S subunit epitope library in Y1090 Escherichia coli. Inserts were amplified from pure phage positives by polymerase chain reaction using primers directed to lambda gt11 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.).


RESULTS

Because of the extensive post-translational processing events involved in the formation of the alpha 2delta 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 alpha 2 and delta  subunits was investigated by Western blot analysis of membranes from cells transfected with the full-length alpha 2delta subunit. In nonreducing conditions, an antibody directed against the alpha 2 subunit recognized a protein of 175 kDa in both skeletal muscle triads and in membranes prepared from tsA201 cells transfected with the full-length alpha 2delta subunit (Fig. 1). One apparent difference between native and transfected alpha 2delta protein is that the transfected alpha 2delta 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 alpha 2 subunit 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 alpha 2delta subunit shifted to an apparent molecular mass of 150 kDa. Likewise, there was a noticeable shift in molecular mass of the transfected alpha 2delta protein, suggesting that the cleavage site and disulfide linkages are similar to native protein.


Fig. 1. Comparison of native skeletal muscle alpha 2delta subunit and alpha 2delta expressed transiently in tsA201 cells. Skeletal muscle triads (100 µg) (Triads) or total membranes of tsA201 cells transfected with either the full-length alpha 2delta subunit (alpha 2delta ) or alpha 2 protein (alpha 2) (200 µg each) were subjected to SDS-polyacrylamide gel electrophoresis on a 5-16% gradient gel under nonreducing and reducing conditions. Transfer was stained with polyclonal antibody against the alpha 2delta subunit (Rabbit 136) and developed using enhanced chemiluminescence (Amersham Corp.). Molecular mass markers appear on the left.
[View Larger Version of this Image (64K GIF file)]

To test the involvement of the extracellular domain of the alpha 2delta subunit in the interaction with the alpha 1 subunit, coimmunoprecipitation experiments were performed using cells transfected with both alpha 1C subunit and either the full-length alpha 2delta subunit or any one of the truncated alpha 2delta subunit constructs (Fig. 2A). Cell membranes were solubilized in 1% digitonin and 1 M NaCl prior to immunoprecipitation. The full-length alpha 2delta subunit assembles with the alpha 1C subunit as demonstrated by its coimmunoprecipitation with an anti-alpha 1C antibody and detection by Western blot analysis with an anti-alpha 2 antibody (Fig. 2B). No alpha 2delta protein was immunoprecipitated from control untransfected cells. In addition, no alpha 2delta protein was precipitated from cells in the absence of the alpha 1 subunit (data not shown). Truncation of 450 extracellular amino-terminal amino acids of the alpha 2delta subunit abolished the ability of this protein to assemble with the alpha 1C subunit, despite its abundance in the starting material. Likewise, the alpha 2 subunit expressed in the absence of the delta  subunit was also unable to coimmunoprecipitate with the alpha 1C subunit.


Fig. 2. Coimmunoprecipitation of full-length and truncated alpha 2delta subunits with the alpha 1C subunit expressed in tsA201 cells. A, shown are the full-length rat brain alpha 2delta subunit, amino-terminal truncation NDelta 28-473, carboxyl-terminal truncation consisting of the alpha 2 protein, and chimera containing transmembrane of adhalin (alpha 2delta Ad). The plasma membrane is shown as a vertical rectangle. The vertical dashed line indicates the cleavage site between the alpha 2 and delta  subunits. Also shown is the antibody recognition site of Rabbit 136 at amino acids 839-856. B, cell membranes (500 µg) from tsA201 cells transfected with cDNAs encoding either the alpha 1C subunit alone or in combination with full-length alpha 2delta subunit, NDelta 29-473, alpha 2, or alpha 2delta Ad were solubilized in 1% digitonin and 1 M NaCl and incubated with protein G-Sepharose preincubated with a polyclonal antibody against the alpha 1C subunit (sheep 41). Sepharose beads were washed extensively, and immunoprecipitating protein was resolved using 5-16% SDS-polyacrylamide gel electrophoresis under reducing conditions. Shown are Western blots of the starting solubilized material (200 µg) (Start) and the alpha 1C immunoprecipitates (alpha 1C I.P.). Both were incubated with a polyclonal antibody against the alpha 2delta subunit (Rabbit 136) and developed using enhanced chemiluminescence.
[View Larger Version of this Image (28K GIF file)]

We also investigated the role of the transmembrane domain of the delta  subunit in assembly with the alpha 1C subunit (Fig. 2B). Substitution of the transmembrane domain from adhalin, an unrelated type I transmembrane protein (recently renamed alpha -sarcoglycan), did not appear to alter the ability of the protein to assemble with the alpha 1C subunit. In this chimera, the 5 cytoplasmic amino acids of the alpha 2delta protein were also substituted with adhalin sequence. Therefore, we conclude that neither intracellular or transmembrane sequences of the alpha 2delta subunit are required for interaction with the alpha 1 subunit.

The region of the alpha 2delta subunit responsible for modulation of dihydropyridine binding to the alpha 1C subunit was also investigated. Although [3H]PN200-110 binding to whole cell tsA201 cell membranes was often low and nonsaturable when alpha 1C 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 (Kd) and binding capacity (Bmax) using saturation analysis (Table I). Cells expressing alpha 1C alone had an average Bmax of 94.6 ± 51.7 fmol/mg (n = 4).

Table I. Comparison of Kd and saturable [3H]PN200-110 binding to total microsomes from tsA201 cells transfected with alpha 1C and alpha 2delta constructs

tsA201 cells were transfected with the cDNAs encoding Ca2+ channel subunits in the combinations indicated. Kd 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.

Membranes Kd ± S.E. Saturable/total

nM
 alpha 1C 1.34  ± 0.79 4 /7
 alpha 1Calpha 2delta a 0.16  ± 0.08 5 /5
 alpha 1Calpha 2 0.65  ± 0.26 3 /5
 alpha 1CNDelta 28-473 1.31  ± 0.74 3 /5
 alpha 1Calpha 2delta Ada 0.21  ± 0.16 4 /4

a Paired t test, p < 0.05 versus alpha 1C.

Coexpression of the full-length alpha 2delta subunit with the alpha 1C 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 alpha 2delta subunit on Bmax (Bmax = 133 ± 73.5 fmol/mg), although there was significant error between experiments in the Bmax depending on the transfection efficiency. Likewise, Western blot analysis on whole cell membranes from transfected cells showed no effect of coexpression of the alpha 2delta subunit on the protein expression of the alpha 1 subunit (data not shown). The binding affinity, however, was not affected by the differences in transfection efficiency.

As expected, when the alpha 2 subunit was coexpressed with the alpha 1C subunit, there was no effect on [3H]PN200-110 binding. This is consistent with coimmunoprecipitation experiments that demonstrated the inability of the alpha 2 subunit to associate with alpha 1 in the absence of the delta  subunit. However, coexpression of the alpha 2delta Ad chimera, in which the transmembrane domain of the alpha 2delta subunit was replaced with that of adhalin, increased [3H]PN200-110 binding affinity to approximately the same extent as full-length alpha 2delta protein.

Because the alpha 1 subunit is very large and difficult to express, we chose an alternative approach to identify regions interacting with the alpha 2delta subunit. Our approach was to trypsinize skeletal muscle microsomes containing native dihydropyridine receptors and follow the alpha 1S subunit fragments remaining in association with the alpha 2delta subunit during WGA affinity chromatography. By taking advantage of the selective ability of the glycosylated alpha 2delta subunit to bind WGA, any alpha 1S fragment identified is presumed to bind WGA only through its interaction with the alpha 2delta subunit. With increasing concentrations of trypsin, an alpha 1S subunit-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 alpha 1S subunit fragments eluted from a WGA-Sepharose column (Fig. 3). Sucrose gradient fractionation was subsequently used to demonstrate cosedimentation of the alpha 1S subunit fragments with the intact full-length alpha 2delta subunit (Fig. 4). Multiple tryptic fragments of alpha 1 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).


Fig. 3. Identification of 28-kDa alpha 1S trypsin fragment from WGA-Sepharose eluate. Skeletal muscle triads were resuspended to a concentration of 4 mg/ml, and trypsin concentrations and incubation times (0 to 160 min) were varied to achieve gradually increasing digestion. Trypsinized triads were then solubilized in 1% digitonin and loaded onto a wheat germ agglutinin-Sepharose column. Bound protein was eluted with 300 mM N-acetylglucosamine. Aliquots were separated onto 5-16% SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to nitrocellulose. Transfer was stained with a monoclonal antibody IIF7 against the alpha 1S IIIS5-S6 region. The arrows indicate full-length alpha 1S protein and alpha 1S fragment (frag) of 28 kDa.
[View Larger Version of this Image (67K GIF file)]


Fig. 4. Sucrose 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 alpha 1S subunit (top) or polyclonal antibody against the alpha 2delta subunit (Rabbit 136) (bottom). Fraction number is listed on the bottom, and molecular mass is indicated at the left. Arrows indicate alpha 1S fragment (frag) and full-length alpha 2delta subunit.
[View Larger Version of this Image (68K GIF file)]

To test the ability of alpha 1S repeat III to associate with the alpha 2delta subunit, we cotransfected tsA201 cells with constructs containing only repeat III and the full-length alpha 2delta subunit. Although the alpha 2delta 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 alpha 1S subunit repeat III by itself is not sufficient to form stable interactions with the alpha 2delta subunit.


DISCUSSION

Our data support a model whereby the interaction sites between the alpha 2delta and alpha 1 subunits are entirely extracellular, because transmembrane modifications of the alpha 2delta subunit did not appear to alter coassembly with the alpha 1 subunit. Morever, our data suggest a requirement for nontransmembrane domains of the delta  subunit in determining a stable association between the alpha 2delta and alpha 1 proteins, because the alpha 2 protein by itself could not support interaction. delta  may contain the interaction site, or the tertiary structure it confers on alpha 2 through its disulfide linkages may enable alpha 2 to directly interact with the alpha 1 subunit. Low expression of delta expressed alone resulted in our inability to distinguish between these possibilities. However, delta  was shown to be able to compete with full-length alpha 2delta protein in Xenopus oocytes and inhibit its stimulatory effects on current amplitude (10), and expression studies in tsA201 cells demonstrated that coexpression of delta  can significantly modulate the biophysical properties of the alpha 1C subunit.1

Interestingly, our data are consistent with reports regarding a functionally related pair of proteins, the Na+,K+-ATPase alpha  and auxiliary beta  subunit, in which the interaction sites have also been localized to extracellular domains (24, 25). In this case, the yeast two-hybrid assay was successful in further localizing the site of interaction on the beta  subunit to the 61 amino acids most proximal to the membrane (21). Although the interaction sites between the voltage-dependent Na+ channel alpha  and beta 1 or beta 2 subunits have not been mapped, it is interesting to note that deletion of the beta 1 intracellular domain does not alter functional effects of beta 1 subunit coexpression (26), suggesting that this interaction may also be in the extracellular domain.

Repeat III of the Ca2+ channel alpha 1S subunit appears to interact strongly with the alpha 2delta 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 alpha 2delta subunit after extensive trypsinization, we were unable to reconstitute the interaction between this small region and alpha 2delta in an expression system. This suggests that multiple regions of the alpha 1 subunit may be involved in assembly with the alpha 2delta subunit. In analogous studies on the voltage-dependent Na+ channel, multiple domains within the carboxyl-terminal half of the skeletal muscle Na+ channel alpha  subunit were shown to be required for functional response of the coexpressed Na+ channel beta 1 subunit on inactivation kinetics (27).

Association of the alpha 2delta subunit with the carboxyl-terminal half of the alpha 1 subunit is consistent with the significant effects that we and others have measured of the alpha 2delta subunit on dihydropyridine binding affinity (28). The membrane spanning segments IIIS6 and IVS6 of the alpha 1S subunit have recently been shown to contain amino acids critical for dihydropyridine binding (29), although the S5-S6 extracellular linkers of the III and IV repeats also confer dihydropyridine sensitivity (30). The extracellular domain of the alpha 2delta 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 alpha 1 subunit, we can speculate on the exact sites of interaction. Most extracellular loops of the alpha 1 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 alpha 2delta subunit, are the S5-S6 linkers, which also contain the pore. Experimental evidence regarding the folding pattern of the alpha 1 subunit suggests that the S5-S6 regions of all four repeats closely interact to form the central pore (31). 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 alpha 2delta 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 alpha 2delta subunit on WGA chromatography.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Supported by an American Heart Association predoctoral fellowship (Iowa affiliate).
§   Supported by a postdoctoral fellowship from the Human Frontier Science Program.
par    Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Inst., University of Iowa College of Medicine, 400 Eckstein Medical Research Bldg., Iowa City, IA 52242. Tel.: 319-335-7867; Fax: 319-335-6957; E-mail: kevin-campbell{at}uiowa.edu; www address: www-camlab.physlog.uiowa.edu.
1   R. Felix and K. P. Campbell, unpublished observations.
2   The 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.

ACKNOWLEDGEMENTS

We thank the late Dr. Xiangyang Wei (Medical College of Georgia) for providing the alpha 1C cDNA clone and Dr. Terry Snutch (University of British Columbia) for the alpha 2delta 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.


REFERENCES

  1. Takahashi, M., Seagar, M. J., Jones, J. F., Reber, B. F. X., and Catterall, W. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5478-5482 [Abstract]
  2. Leung, A. T., Imagawa, T., and Campbell, K. P. (1987) J. Biol. Chem. 262, 7943-7946 [Abstract/Free Full Text]
  3. Witcher, D. R., De Waard, M., Sakamoto, J., Franzini-Armstrong, C., Pragnell, M., Kahl, S. D., and Campbell, K. P. (1993) Science 261, 486-489 [Medline] [Order article via Infotrieve]
  4. Liu, H., De Waard, M., Scott, V. E. S., Gurnett, C. A., Lennon, V. A., and Campbell, K. P. (1996) J. Biol. Chem. 271, 13804-13810 [Abstract/Free Full Text]
  5. Cooper, C. L., Vandaele, S., Barhanin, J., Fosset, M., Lazdunski, M., and Hosey, M. M. (1987) J. Biol. Chem. 262, 509-512 [Abstract/Free Full Text]
  6. Tokumaru, H., Shojaku, S., Takehara, H., Hirashima, N., Abe, T., Saisu, H., and Kirino, Y. (1995) J. Neurochem. 65, 831-836 [Medline] [Order article via Infotrieve]
  7. De Jongh, K. S., Warner, C., and Catterall, W. A. (1990) J. Biol. Chem. 265, 14738-14741 [Abstract/Free Full Text]
  8. Jay, S. D., Sharp, A. H., Kahl, S. D., Vedvick, T. S., Harpold, M. M., and Campbell, K. P. (1991) J. Biol. Chem. 266, 3287-3293 [Abstract/Free Full Text]
  9. Brickley, K., Campbell, V., Berrow, N., Leach, R., Norman, R. I., Wray, D., Dolphin, A. C., and Baldwin, S. A. (1995) FEBS Lett. 364, 129-133 [CrossRef][Medline] [Order article via Infotrieve]
  10. Gurnett, C. A., De Waard, M., and Campbell, K. P. (1996) Neuron 16, 431-440 [CrossRef][Medline] [Order article via Infotrieve]
  11. Wiser, O., Trus, M., Tobi, D., Halevi, S., Giladi, E., and Atlas, D. (1996) FEBS Lett. 379, 15-20 [CrossRef][Medline] [Order article via Infotrieve]
  12. Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S., and Numa, S. (1989) Nature 340, 230-233 [CrossRef][Medline] [Order article via Infotrieve]
  13. Mori, 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., and Numa, S. (1991) Nature 350, 398-402 [CrossRef][Medline] [Order article via Infotrieve]
  14. Bangalore, R., Mehrke, G., Gingrich, K., Hofmann, F., and Kass, R. S. (1996) Am. J. Physiol. 270, H1521-H1528 [Abstract/Free Full Text]
  15. Pragnell, M. P., De Waard, M., Mori, Y., Tanabe, T., Snutch, T. P., and Campbell, K. P. (1994) Nature 368, 67-70 [CrossRef][Medline] [Order article via Infotrieve]
  16. De Waard, M., Pragnell, M., and Campbell, K. P. (1994) Neuron 13, 495-503 [Medline] [Order article via Infotrieve]
  17. Yu, W., Xu, J., and Li, M. (1996) Neuron 16, 441-453 [Medline] [Order article via Infotrieve]
  18. Sewing, S., Roeper, J., and Pongs, O. (1996) Neuron 16, 455-463 [Medline] [Order article via Infotrieve]
  19. Shen, V., and Pfaffinger, P. (1995) Neuron 14, 625-633 [Medline] [Order article via Infotrieve]
  20. Li, M., Jan, Y. N., and Jan, L. Y. (1992) Science 257, 1225-1240 [Medline] [Order article via Infotrieve]
  21. Colonna, T. E., Huynh, L., and Fambrough, D. M. (1997) J. Biol. Chem. 272, 12366-12372 [Abstract/Free Full Text]
  22. Sharp, A. H., Imagawa, T., Leung, A. T., and Campbell, K. P. (1987) J. Biol. Chem. 262, 12309-12315 [Abstract/Free Full Text]
  23. Gurnett, C. A., Kahl, S. D., Anderson, R. D., and Campbell, K. P. (1995) J. Biol. Chem. 270, 9035-9038 [Abstract/Free Full Text]
  24. Hamrick, M., Renaud, K. J., and Fambrough, D. M. (1993) J. Biol. Chem. 268, 24367-24373 [Abstract/Free Full Text]
  25. Lemas, M. V., Hamrick, M., Takeyasu, K., and Fambrough, D. M. (1994) J. Biol. Chem. 269, 8255-8259 [Abstract/Free Full Text]
  26. Chen, C., and Cannon, S. (1995) Pfluegers Arch. 431, 186-195 [Medline] [Order article via Infotrieve]
  27. Makita, N., Bennett, P. B., and George, A. L. (1996) Circ. Res. 78, 244-252 [Abstract/Free Full Text]
  28. Wei, X., Pan, S., Lang, W., Kim, H., Schneider, T., Perez-Reyes, E., and Birnbaumer, L. (1995) J. Biol. Chem. 270, 27106-27111 [Abstract/Free Full Text]
  29. Peterson, B. Z., and Catterall, W. A. (1995) J. Biol. Chem. 270, 18201-18204 [Abstract/Free Full Text]
  30. Grabner, M., Wang, Z., Hering, S., Streissnig, J., and Glossmann, H. (1996) Neuron 16, 207-218 [Medline] [Order article via Infotrieve]
  31. MacKinnon, R. (1995) Neuron 14, 889-892 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.