Neuronal voltage-dependent Ca2+ channels have been isolated previously and shown to contain a primary α1 pore-forming subunit as well as auxiliary α2δ and β subunits, in addition to an uncharacterized 95 kDa protein. In the present study, using multiple approaches, we have extensively characterized the molecular structure of the 95 kDa protein. Separation of the P/Q- and N-type neuronal Ca2+ channels showed that the 95 kDa protein is associated exclusively with the ω-Conotoxin MVIIC receptor of the P/Q-type channels. Analysis of purified synaptic plasma membranes and the isolated P/Q-type channels, using α1A-specific antibodies, suggested a structural relationship between the α1A subunit and the 95 kDa protein. This finding was supported by protein–protein interaction data, which revealed that the β subunit can associate with the 95 kDa protein in addition to the α1A subunit. Changes in electrophoretic mobility after enzymatic treatment with Endo F indicated that the 95 kDa protein is glycosylated. Furthermore, microsequencing of the 95 kDa protein yielded 13 peptide sequences, all of which are present in the first half of the α1A subunit up to amino acid 829 of the cytoplasmic linker between repeats II and III. Taken together, our results strongly suggest that the 95 kDa glycoprotein associated with the P/Q-type Ca2+ channels is a short form of the α1A subunit.
- P/Q-type Ca2+ channels
- 95 kDa subunit
- α1A subunit
- N-type Ca2+channel
- β subunit
- ω-Conotoxin MVIIC
- ω-Conotoxin GVIA
Voltage-dependent Ca2+ channels are essential for controlling cell–cell communication in the CNS (for review, see Wheeler et al., 1994a; Dunlap et al., 1995). In particular, N- and P/Q-type Ca2+ channels have been shown to play a central role in regulating neurotransmitter release (Luebke et al., 1993; Takahashi and Momiyama, 1993; Turner et al., 1993; Wheeler et al., 1994b) via direct interactions with proteins of the synaptic vesicle docking/fusion complex at the nerve terminal (Sheng et al., 1994, 1996;Mochida et al., 1996; Rettig et al., 1996). These channels can be distinguished electrophysiologically and pharmacologically by using two naturally occurring neurotoxins, ω-Conotoxin GVIA (ω-Ctx GVIA;Boland et al., 1994; Turner and Dunlap, 1995) and ω-Conotoxin MVIIC (ω-Ctx MVIIC; Hillyard et al., 1992; Turner and Dunlap, 1995;McDonough et al., 1996).
Extensive biochemical studies have shown that neuronal N- and P/Q-type Ca2+ channels are heteromultimeric complexes of α1, α2δ, and β subunits (Ahlijanian et al., 1990; McEnery et al., 1991; Witcher et al., 1993a,b; Leveque et al., 1994; Martin-Moutot et al., 1995; Liu et al., 1996). Analysis of the subunit composition also has shown the presence of a fourth polypeptide (95–110 kDa) of unknown properties (McEnery et al., 1991; Witcher et al., 1993a,b; Leveque et al., 1994).
Functional differences between N- and P/Q-type Ca2+channels are attributed to several factors, including the expression of distinct α1 subunit proteins and the selective association of auxiliary subunits. Molecular cloning has revealed different neuronal Ca2+ channel α1genes (Snutch and Reiner, 1992). cDNA of these α1subunits has been isolated, and its functional expression has revealed properties that fall into the P/Q and N categories (α1Aand α1B, respectively; Mori et al., 1991; Fujita et al., 1993). Likewise, the molecular properties of the N-type channels have been closely examined recently by using a monoclonal antibody raised specifically against the cytoplasmic II–III loop of the α1B subunit (Scott et al., 1996). The results of this investigation have revealed extensive β subunit heterogeneity of the N-type channels. Parallel biochemical studies on P/Q-type channels, using 125I-ω-Ctx MVIIC and a polyclonal antibody to the α1A subunit, disclosed similar diversity of β subunits associated with these channels (Liu et al., 1996).
Although extensive studies on the α1, α2δ, and β subunits of voltage-dependent Ca2+ channels have been performed (for review, seeCatterall, 1995; De Waard et al., 1996), little is known about the structure of the 95 kDa protein associated with neuronal Ca2+ channels. To investigate the molecular properties of this protein, we separated the N- and P/Q-type channels, using site-directed monoclonal antibodies. This not only has allowed us to examine the subunit composition of both of these channel types but also has permitted us to investigate the structure of the 95 kDa protein by a number of biochemical techniques. The identity of this protein ultimately was established by protein microsequencing. Hence, using multiple approaches, we show conclusively in this report that the 95 kDa glycoprotein associates with the P/Q-type Ca2+ channels and is a short form of the α1A subunit.
MATERIALS AND METHODS
Materials. 125I-ω-Ctx GVIA, [35S]methionine, and the enhanced chemiluminescence (ECL) kit were obtained from Amersham Life Science (Arlington Heights, IL). 125I-ω-Ctx MVIIC was from DuPont NEN (Boston, MA). Digitonin, purchased from ICN Biomedicals (Costa Mesa, CA), was purified as previously described (Leung et al., 1987). Other biochemicals used were protein G-Sepharose (Pharmacia Biotech, Piscataway, NJ), horseradish peroxidase-conjugated secondary antibodies (Boehringer Mannheim, Indianapolis, IN), Hydrazide Avidgel (Unisyn Technologies, Hopkinton, MA), and peptide N-glycosidase F (Endo F; Oxford GlycoSystems, Bedford, MA). All other chemicals were of reagent grade.
Separation of the N- and P/Q-type Ca2+channels. Rabbit brain membranes (∼1 gm of protein) were prepared as detailed elsewhere (Witcher et al., 1993b), and the channels were extracted by solubilization in 10 mmHEPES-NaOH, pH 7.4, 0.5 m NaCl, and five protease inhibitors (0.23 mm phenylmethylsulphonyl fluoride, 0.64 mm benzamidine, 1 mm leupeptin, 0.7 mm pepstatin A, and 76.8 nm aprotinin) containing 0.6% digitonin for 1 hr at 4°C. After centrifugation at 35,000 rpm for 37 min in a 45 Ti rotor, the detergent extract was diluted twofold with ice-cold deionized water and applied to a heparin agarose column (50 ml) preequilibrated with 5 column volumes of buffer A (10 mm HEPES-NaOH, pH 7.4, 0.1 m NaCl, and protease inhibitors) containing 0.1% digitonin at a flow rate of 5 ml/min. The column was washed extensively with buffer A and eluted in the same buffer containing 0.7 m NaCl, collecting 75 ml (heparin pool). Then the enriched channels were incubated overnight with the monoclonal antibody (mAb) CC 18 (α1B-specific antibody; Scott et al., 1996) coupled to Hydrazide Avidgel (∼2 ml of settled resin) prepared according to the manufacturer’s instructions. The resin was washed extensively with buffer A and eluted with 50 mm glycine-HCl, pH 2.5, containing 0.6 m NaCl and 0.1% (w/v) digitonin (7.5 ml). The channels were neutralized immediately with 2 m Tris-HCl, pH 8.0 (1 ml). Then the void of the mAb CC 18 column (which is devoid of N-type channels) was incubated for 3 hr at 4°C with the mAb VD21 (β subunit-specific antibody; Witcher et al., 1993a) in a Hydrazide Avidgel column (∼2 ml of settled resin), washed, and eluted as mentioned above. The isolated N- and P/Q-type channels were concentrated in a Centricon 30 (Amicon, Beverly, MA) by centrifugation at 5000 rpm for 2 hr at 4°C. The subunit composition was analyzed by SDS-PAGE and immunoblotting.
ω-Ctx binding to neuronal Ca2+ channels.N- and P/Q-type channel binding activity was assayed with125I-ω-Ctx GVIA and 125I-ω-Ctx MVIIC, respectively, at different stages throughout the separation, as detailed elsewhere (Liu et al., 1996). Briefly, aliquots (50 μl) of solubilized membrane extracts were resuspended in a total volume of 300 μl of binding buffer (10 mm HEPES, pH 7.4, 0.2 mg/ml bovine serum albumin, 100 mm NaCl, and protease inhibitors) and incubated with a saturating concentration (1 nm) of125I-ω-Ctx GVIA for 1 hr at room temperature. The receptor–ligand complexes were collected and washed rapidly with ice-cold binding buffer on Whatman GF/B filters with a cell harvester (Brandel, Gaithersburg, MD). Nonspecific binding was determined by the addition of 100-fold excess nonradioactive ω-Ctx GVIA 10–15 min before the addition of the radiolabeled toxin. Specific binding was calculated by subtracting nonspecific binding from total binding. ω-Ctx MVIIC binding was assayed similarly; however, in this case the binding buffer also contained 0.1 mm EDTA and 0.1 mm EGTA.
SDS-PAGE and immunoblot analysis. Samples were analyzed by SDS-PAGE on 3–12% gradient gels, using the Laemmli buffer system (Laemmli, 1970). After electrophoresis the SDS gels were transferred to nitrocellulose membranes and immunoblotted as described previously (Witcher et al., 1993a; Scott et al., 1996). In short, the membranes were incubated overnight with immunoaffinity-purified polyclonal antibodies to fusion proteins containing specific regions of the α1 subunit (amino acids 779–969, Sheep 37) or the 95 kDa protein (Sheep 46; Witcher et al., 1993a). The specific protein bands were detected with either the horseradish peroxidase or ECL detection method (according to the manufacturers’ instructions). Synaptic plasma membranes were prepared from rabbit brain by using the method of Jones and Matus (1974).
Overlay assay of the [35S]-β3subunit on the isolated P/Q-type Ca2+ channel.The Ca2+ channel β3 subunit was translated in vitro and labeled with [35S]methionine, using a TNT-coupled reticulocyte lysate system (Promega, Madison, WI) as described elsewhere (Pragnell et al., 1994). P/Q-type channels from a single purification were concentrated, and the subunits were resolved on a 3–12% SDS gel. After electrophoresis the proteins were transferred onto nitrocellulose, and the membrane was blocked by incubation for 1 hr in blocking buffer (5% bovine serum albumin and 0.5% fat-free milk prepared in PBS, pH 7.5). Then the membrane was incubated with [35S]-β3 subunit (1 × 106 cpm) overnight at 4°C in the blocking buffer. To remove unbound [35S]-β3 subunit, we washed the membrane extensively with blocking buffer for 1 hr at room temperature before completely drying and exposing it to x-ray film.
Deglycosylation of the P/Q-type Ca2+ channels.P/Q-type Ca2+ channels from one isolation procedure were concentrated to 100 μl, using a centrifugal concentrator (Centricon 30; Amicon) and boiled in the presence of 1% SDS for 3 min to fully unfold and denature the protein. The sample was diluted fivefold to a final concentration of 0.2% SDS with water. Triton X-100 (final concentration 1%) and protease inhibitors were added before deglycosylation with 3 U of Endo F and incubation at 37°C overnight. The reaction was halted by the addition of Laemmli sample buffer, followed by SDS-PAGE analysis and Western blotting, using an anti-95 kDa protein-specific antibody (affinity-purified sheep polyclonal antibodies) directed to the II–III loop of the α1 (amino acids 779–969; Witcher et al., 1993a).
Microsequencing of the 95 kDa protein. Amino acid sequence information was obtained from the 95 kDa protein, using two different methods. The first involved sequencing the 95 kDa protein from a polyvinylidene difluoride transfer membrane (Immobilon-P; Millipore, Bedford, MA). Briefly, the molecular composition of the P/Q-type channels was resolved by SDS-PAGE and the gel electrotransferred to the Immobilon-P membrane. The protein band corresponding to the 95 kDa protein was excised. Then the immobilized protein was digested with trypsin. Peptides that were released were separated by reverse-phase HPLC on a 2.1 × 100 mm RP-300 column (Perkin-Elmer, Foster City, CA), using a mobile phase of 0.1% trifluoroacetic acid. Elution was accomplished with a 100 min gradient of 0–70% acetonitrile. Peptides were subjected to automated Edman degradation, using an Applied Biosystems model 477A amino acid sequencer (Foster City, CA) with standard manufacturer’s programming and chemicals. The second method for acquisition of peptide sequence information involved the generation of peptides by digesting the 95 kDa protein band directly excised from the gel, using endoproteinase lys-C (from Acromobacter lyticus; Wako Chemicals, Richmond, VA). The resulting peptides were separated on a Hewlett Packard model 1090 HPLC (Wilmington, DE). Then peptide fractions were analyzed on an Applied Biosystems model 477A amino acid sequencer as described above. In both cases the resultant sequences were screened against the GenBank CDS database, using a BLAST search to determine the homology with any other known proteins.
Separation of the neuronal N- and P/Q-type Ca2+ channels
N- and P/Q-type Ca2+ channels were separated by immunoaffinity chromatography by using the α1B-specific monoclonal antibody (mAb CC18) in combination with the β subunit-specific monoclonal antibody (mAb VD21), and the subunit composition of these two neuronal Ca2+channels was examined. The proteins were extracted from rabbit brain membranes and applied to a heparin agarose column. After elution from the heparin column with high salt, the preparation of channels was incubated with the α1B antibody resin overnight; then the void was collected. Conditions in which 125I-ω-Ctx MVIIC binds to the P/Q-type Ca2+ channels and not the N-type channels have been determined previously (Liu et al., 1996), and these exact conditions have been applied throughout the present study. We assayed binding of 125I-ω-Ctx GVIA and125I-ω-Ctx MVIIC to the N- and P/Q-type channels, respectively, in both the heparin eluate and the void from the α1B column (Fig. 1). Because the α1 subunit that is present in the N-type calcium channels is α1B, virtually all of the125I-ω-Ctx GVIA specific binding observed originally in the heparin pool (Fig. 1 A, left bar) bound to the α1B Hydrazide Avidgel column. In consequence, only a minimal fraction of the 125I-ω-Ctx GVIA binding was found in the void of that column (Fig. 1 A, middle bar). Analogously, when the isolated N-type channels were applied to a β subunit column antibody, the void of that column showed no125I-ω-Ctx GVIA specific binding, indicating that all of the channels in the preparation bound the column (Fig. 1 A, right bar). In contrast, equal amounts of the P/Q-type channels, as determined by high-affinity 125I-ω-Ctx MVIIC binding, were present in both the heparin pool (Fig. 1 B, left bar) and the α1B column void (Fig. 1 B, middle bar). These results clearly indicate that the P/Q-type channels (α1A subunit-containing channels) did not bind to the α1B Hydrazide Avidgel column. However, when the isolated P/Q-type channels were applied to the β subunit-specific (VD21) antibody column, nearly all of the ω-Ctx MVIIC binding activity bound (Fig. 1 B, right bar), and only a small fraction of the binding activity was found in the void as expected. In summary, all of the ω-Ctx GVIA binding, which represents N-type Ca2+ channels, binds to the α1B column, whereas the ω-Ctx MVIIC binding, which represents the P/Q channels, is present in the void of the α1B column. The VD21 column, on the other hand, binds the remainder of the ω-Ctx MVIIC binding.
After elution from each respective antibody column with glycine containing buffer solution, the subunit composition of the isolated N- and P/Q-type channels was examined by SDS-PAGE, followed by Western blotting. Proteins in both isolated channel preparations were examined by using polyclonal antibodies to the II–III loop of the α1 subunit (Sheep 37; Witcher et al., 1993a). In the case of the N-type channel, these antibodies recognized primarily a protein with a molecular size of 210 kDa (α1B; Fig.2 A, left). In contrast, the isolated P/Q-type channels showed a more complex pattern of immunoblotting. In this case, the same antibodies reacted with distinct proteins ranging in size from 95 to 220 kDa (Fig. 2 A, right), suggesting the presence of different isoforms of the α1A subunit. Earlier immunoblotting studies with partially purified channels identified an α2δ subunit and four different β subunits (β1b, β2, β3, and β4; Liu et al., 1996). Herein, a 95 kDa protein was shown to be associated with the P/Q-type and not the N-type channels, as was previously reported (McEnery et al., 1991; Witcher et al., 1993a,b; Leveque et al., 1994).
A potential alternative explanation for the presence of these antigenically similar proteins in the purified P/Q-type channel preparations could be proteolysis that occurred during the isolation procedure; however, several lines of evidence contest this possibility. The significant sequence homology between the α1A and the α1B subunits would suggest that proteolysis probably would occur similarly on both α1 subunits. Moreover, freshly purified synaptic plasma membranes when probed with the same antibody contained similarly reactive proteins (Fig.2 B). Furthermore, the 95 kDa protein is a predominant species in both the membrane and purified preparations. More importantly, inclusion or omission of protease inhibitors (data not shown) did not affect the relative quantities of these antigenic variants. These antibody data suggest that the 95 kDa protein associated with P/Q-type neuronal channels is structurally related to the α1A subunit.
Ca2+ channel β subunit interacts with the 95 kDa protein
To investigate the relationship between the α1subunit and the 95 kDa protein further, we performed a β subunit interaction assay. This assay was developed in a previous study (Pragnell et al., 1994), which identified a region in the I–II loop in all known α1 subunits that can bind Ca2+ channel β subunits. The β3subunit in a pcDNA3 expression vector containing a T7 RNA polymerase site was translated in vitro in the presence of [35S]methionine, and the translation product was analyzed by SDS-PAGE and autoradiography. The left panel in Figure3 A shows the migration of the polypeptide identified as the translated product, which exhibited a molecular mass similar to that expected from its amino acid sequence (∼58 kDa). Then the [35S]-β3 probe was incubated with the P/Q-type channels, after separation of the subunits by electrophoresis on a 3–12% gradient gel and transfer onto nitrocellulose membrane (Fig. 3 A, right). The resulting overlay identified a number of proteins of similar molecular size to those identified in immunoblotting studies above (Fig. 2 A, right), suggesting that each of these proteins contained the site of interaction between the α1 and β subunits of voltage-dependent Ca2+ channels. The overlay assay also revealed a protein of slightly smaller molecular weight (∼75 kDa) that appeared not to be recognized by the α1antibodies (Fig. 2) and might represent the C-terminal part of α1A subunit. Accordingly, it has been shown that a second β subunit interaction site exists within the C terminus of the neuronal α1E subunit distinct from the previously known interaction domain located between repeat I and II of α1subunits (Tareilus et al., 1997). More interestingly, the presence of a second β interaction site also has been observed recently in the C terminus of the α1A subunit (Walker et al., 1997).
95 kDa polypeptide is a glycoprotein
Another biochemical property of the 95 kDa protein was investigated by deglycosylation studies with the isolated P/Q-type channels and Western blotting of the resultant sample. When a 95 kDa-specific antibody was used, the blot showed a distinct shift in the mobility of the 95 kDa protein to 90 kDa on a 3–12% gradient gel after Endo F treatment (Fig. 3 B). Interestingly, there are two potential glycosylation sites on the α1A subunit, on Asn 283 and Asn 1665. The first of these sites is present within the sequence of the 95 kDa subunit, which explains the shift in molecular size observed in the blot. These data provided the first indication that an α1 subunit of voltage-dependent Ca2+ channels is glycosylated.
Molecular identity of the 95 kDa protein
To establish the identity of the 95 kDa polypeptide precisely, we performed protein microsequencing on this subunit after large-scale isolation of the P/Q-type channels. After SDS-PAGE analysis to resolve the subunits, two alternative methods were used to obtain protein sequence information from the 95 kDa protein. The first method involved transferring the 95 kDa protein from a SDS gel onto an Immobilon-P membrane and excising the 95 kDa band previously stained with Coomassie blue. Peptides were released with trypsin and resolved by reverse-phase HPLC. Sequence analysis was performed with an automated amino acid sequencer. This yielded a number of signals (peptides 1, 4, 6, 8, 10–12; Fig. 4), which, when screened in a BLAST search, showed strong identity with the amino acid sequence of the α1A subunit cloned from rabbit brain (Table1).
To investigate this further, we implemented a second method to obtain additional peptide sequence information. Similarly, the sample was subjected to SDS-PAGE, the gel piece containing the 95 kDa protein was excised, and peptides were generated by in-gel digestion, using endoproteinase lys-C. The resulting fragments were resolved by reverse-phase HPLC before protein microsequencing, which yielded six different peptide sequences (peptides 2, 3, 5, 7, 9, 13; Fig. 4).
Consistent with the results obtained in Western blotting, analysis of these signals showed that 10 of the 13 peptides presented 100% amino acid identity with the sequence of the P/Q-type calcium channel α1A subunit (Table 1). Only in three cases were there discrepancies with the protein sequence of the α1Asubunit, and in all cases the divergence can be attributed to ambiguous amino acid identification during the microsequencing process. Notably, all 13 sequences were confined to the first half of the α1A subunit up to the middle of the cytoplasmic II–III loop (Fig. 5). These data provide convincing evidence that the 95 kDa protein associated with the P/Q-type Ca2+ channels is a member of the α1A subunit family. Interestingly, the determined molecular size of this short form of the α1A subunit from the start of the N-terminal sequence (peptide 1) to the end of peptide 13 was 93,784 Da, which is close to that estimated for this protein from SDS-PAGE analysis.
Neuronal Ca2+ channels were purified first from rat (McEnery et al., 1991; Leveque et al., 1994) and rabbit (Witcher et al., 1993a) brain and shown to contain α1B, α2δ, and β subunits, together with a specific protein of 110 or 95 kDa, respectively. Although these purification protocols have greatly increased our understanding of neuronal Ca2+ channels, in view of the recently realized structural similarities between the N- and P/Q-type Ca2+ channels (Liu et al., 1996; Rettig et al., 1996; Scott et al., 1996; Sheng et al., 1996), it is entirely possible that these purified materials may contain a mixture of both channel types. Unfortunately, because of the lack of specific radiolabeled high-affinity ligands that bind to the P/Q-type channels, this was not realized previously (McEnery et al., 1991; Witcher et al., 1993a;Leveque et al., 1994). Recently, however, the conditions for measuring the binding of 125I-ω-Ctx MVIIC specifically to P/Q-type channels were established (Liu et al., 1996), thereby allowing the detection of these channels throughout the present study (see Fig. 1). Moreover, a monoclonal antibody that exclusively reacts with the α1B subunit was developed (Scott et al., 1996). This also has proven very useful for specifically separating the N-type from the P/Q-type Ca2+ channels, consequently permitting the subunit composition of each of these channel types to be assessed. Surprisingly, the 95 kDa protein that previously was proposed to be associated with the N-type channel was shown in this study to associate exclusively with the P/Q-type channels after immunoaffinity enrichment (see Fig. 2).
The properties of this protein were examined further, using a number of different biochemical approaches from immunoblotting to protein–protein interaction assays and, finally, to protein microsequencing (Figs. 3, 4). The data that arose from these studies established a close structural relationship between the 95 kDa protein and the α1A subunit of P/Q-type channels. Interestingly, when the α1A subunit was identified originally, Northern blot analysis revealed the presence of two transcripts, one with a size of ∼9 kb and a second with a size of 4.4 kb (Snutch et al., 1990). It would appear from these data that the 95 kDa protein described in the present study could, in fact, be a splice variant of the α1A subunit because it contains approximately the first half of the full-length α1A subunit. However, it cannot be excluded that this 95 kDa subunit could have arisen because of post-translational proteolytic processing. This is supported by a recent study that revealed that the NMDA receptor-activated proteolytic processing of the brain α1C subunit yielded neuronal L-type Ca2+ channels that have modified kinetic properties (Hell et al., 1996). Given these data, it is tempting to speculate that the association of two 95 kDa proteins could form a functional Ca2+ channel equivalent to that generated by the full-length α1A subunit. This possibility is particularly interesting because both of the 95 kDa proteins can bind a β subunit (Fig. 3 A), potentially yielding a Ca2+ channel with novel kinetic properties.
On the other hand, although the expression of the 95 kDa protein may not be sufficient to support channel function, the possibility exists that it may regulate the functional expression of the full-length α1A subunit. Because the 95 kDa protein binds the β subunit, in this scenario the truncated 95 kDa protein would compete with the full-length α1A subunit for this auxiliary subunit. Because the β subunit has been implicated in determining the membrane localization in addition to modulating properties of channel conduction of the α1 subunit (for review, see Catterall, 1995), this alternative hypothesis predicts that the coexpression of the 95 kDa protein would affect the functional expression of the full-length α1A subunit. Likewise, the 95 kDa protein also might contribute to determining Ca2+ current diversity in neurons by interacting with the channel-forming subunits and modifying their biophysical properties and/or altering the α1 subunit modulation by second messengers.
Genetic studies very recently have linked the α1A subunit of the P/Q-type Ca2+ channels to several animal seizure models and human neurological disorders (Fletcher et al., 1996;Ophoff et al., 1996; Doyle et al., 1997; Zhuchenko et al., 1997). Interestingly, mutations within the sequence of the α1Asubunit have been found in patients suffering from familial hemiplegic migraine and episodic ataxia type 2 (Ophoff et al., 1996). In line with this finding, it is interesting to note that one of the mutations linked to episodic ataxia type 2 resulted in a frame shift and a premature stop at exon 22, in the IIIS1 region, generating a truncated α1A subunit approximate in size to the 95 kDa protein. This is the first report on the occurrence of a short form of a Ca2+ channel α1 subunit that is linked to a human disorder. Future studies on the functional properties of the 95 kDa protein associated with P/Q-type channels could provide insights into possible mechanisms by which this protein may be linked to migraine.
In conclusion, this study has extensively characterized the molecular structure of the 95 kDa protein of native P/Q-type Ca2+ channels by several approaches. Collectively, all of the information obtained in this study on the structure of the 95 kDa protein allows a model to be proposed containing the α1 interaction domain (AID), the glycosylation site on Asn 283, part of the II–III loop (amino acids 779–969), and the 13 peptide sequences (Fig. 5). These data will be essential for the discovery and development of therapeutic agents for the pharmacological treatment of disorders linked to the voltage-dependent Ca2+ channel α1A subunit of the neuronal P/Q-type Ca2+ channels.
R.F. is supported by a Human Frontier Science Program postdoctoral fellowship. K.P.C. is an Investigator of the Howard Hughes Medical Institute (HHMI). The protein sequencing was performed by Dr. C. A. Slaughter and C. Moomaw at the HHMI Biopolymers Facility (University of Texas Southwestern Medical Center, Dallas, TX) and by R. F. Cook and N. Thorngren at the Biopolymers Laboratory, Center for Cancer Research (MIT, Cambridge, MA). Monoclonal antibody CC 18 was raised in collaboration with Dr. V. A. Lennon (Mayo Clinic, Rochester, MN). We thank J. C. Miller for his expert technical assistance. We are also grateful to C. Leveille, D. Venzke, and Drs. G. Biddlecome, K. Bielefeldt, and C. A. Gurnett for critically reading this manuscript.
Correspondence should be addressed to Dr. Kevin P. Campbell, Howard Hughes Medical Institute, University of Iowa College of Medicine, 400 Eckstein Medical Research Building, Iowa City, IA 52242.
Dr. Scott’s present address: Department of Neurological and Urological Disease Research (D47C), Abbott Laboratories, Abbott Park, IL 60064.