Expression of a cDNA for a neuronal calcium channel alpha 1 subunit enhances secretion from adrenal chromaffin cells.

A synthetic oligonucleotide was used to isolate mouse brain cDNA clones coding for a brain isoform of the alpha 1 subunit of the voltage-sensitive Ca2+ channel. Twenty-six independent cDNA clones were isolated and sequenced. All the cDNA clones reported here showed high homology to the rat brain class C cDNA sequence (Snutch, T. P., Tomlinson, W. J., Leonard, J. P., and Gilbert, M. M. (1991) Neuron 7, 45-57). Comparison of the individual mouse brain class C (mbC) cDNA sequences indicated the presence of four regions within the alpha 1 subunit coding sequence where alternative splicing can take place in mouse brain and raise the possibility that combinatorial arrangement of these splice variants could give rise to a heterogenous class of mbC transcripts. Northern blot analysis demonstrated that mbC mRNA sequences could be detected in highest abundance in mouse heart, at lower levels in mouse brain and spinal cord, and not at all in liver or skeletal muscle. An expression vector for one isoform of the mbC alpha 1 subunit cDNA was constructed using the human cytomegalovirus promoter to direct expression, and this expression vector was used in a novel transfection assay of primary cultures of bovine adrenal chromaffin cells. Transfection of the mbC alpha 1 subunit expression vector increased the secretion of human growth hormone derived from a cotransfected human growth hormone expression vector after stimulation with elevated K+ or the dihydropyridine agonist, Bay K8644. These experiments suggest that this isoform of the mbC alpha 1 subunit is functional in the transfected chromaffin cells and that the number of Ca2+ channels is a limiting component in the secretion from chromaffin cells in culture.

A synthetic oligonucleotide was used to isolate mouse brain cDNA clones coding for a brain isoform of the a1 subunit of the voltage-sensitive Ca2+ channel. Twentysix independent cDNA clones were isolated and sequenced. All the cDNA clones reported here showed high homology to the rat brain class C cDNA sequence (Snutch, T. P., Tomlinson, W. J., Leonard, J. P., and Gilbert, M. M. (1991) Neuron 7 , 45-57). Comparison of the individual mouse brain class C (mbC) cDNA sequences indicated the presence of four regions within the a1 subunit coding sequence where alternative splicing can take place in mouse brain and raise the possibility that combinatorial arrangement of these splice variants could give rise to a heterogenous class of mbC transcripts. Northern blot analysis demonstrated that mbC mRNA sequences could be detected in highest abundance in mouse heart, at lower levels in mouse brain and spinal cord, and not at all in liver or skeletal muscle. An expression vector for one isoform of the mbC a1 subunit cDNA was constructed using the human cytomegalovirus promoter to direct expression, and this expression vector was used in a novel transfection assay of primary cultures of bovine adrenal chromaffin cells. Transfection of the mbC a1 subunit expression vector increased the secretion of human growth hormone derived from a cotransfected human growth hormone expression vector after stimulation with elevated K+ or the dihydropyridine agonist, Bay K8644. These experiments suggest that this isoform of the mbC a1 subunit is functional in the transfected chromaffin cells and that the number of Ca2+ channels is a limiting component in the secretion from chromaffin cells in culture.
Voltage-sensitive Ca2+ channels are found in the plasma membrane of many cell types where they strictly regulate the entry of extracellular calcium into the cell (Tsien et al., 1991;Miller, 1992). Influx of Ca2+ through these channels can regulate such diverse cellular functions as muscle contraction (Catterall, 1991a), neurotransmitter release (Miller, 1990), and gene expression (Day and Maurer, 1990;Murphy et al., * This work was supported by National Institutes of Health Research Grant DK27959 (to R. W. H.) and a grant from the Lucille P. Markey Charitable Trust (to M. D. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession numbeds) LO1 776.  (Tsien et al., 1991). Skeletal muscle contains the highest concentration of voltage-sensitive Caz+ channels, and these channels are of the L type (Catterall, 1991a). Biochemical characterization of the skeletal muscle Ca2+ channel has shown it to consist of five individual subunits designated a l , 012, p, y, and 6. The a1 subunit of the skeletal muscle Ca2+ is the largest of these five subunits and is an integral membrane protein. The a1 subunit also forms the ion-selective pore through which Ca2+ flows into the cell (Catterall, 1991b). The skeletal muscle a1 subunit is phosphorylated by CAMP-dependent protein kinase, and this phosphorylation alters the properties of the channel (Curtis and Catterall, 1985). Binding sites for dihydropyridine-related agonists such as Bay K8644 and antagonists such as nifedipine are also found on the a1 subunit (Nakayama et al., 1991). The structure of neuronal Ca2+ channels is not as clearly understood as the skeletal channel due to their lower abundance and molecular heterogeneity. Molecular biological studies suggest the expression of at least four different genes for the a1 subunit in brain (Snutch et al., 1990), and alternative splicing has been demonstrated for two of these transcripts Starr et al., 1991). The four classes of a1 subunit in rat brain have been designated rbA, rbB, rbC, and rbD (Snutch et al., 1990). Here we report the cloning of cDNAs for isoforms of the mouse brain a1 subunit and that the expression one of these isoforms in bovine adrenal chromaffin cells facilitates secretion in a manner consistent with the production of functional Ca2+ channels. Biomedical Core Facility). Oligonucleotide B was extended using oligonucleotide A as template using ["'PIdATP and the Klenow fragment of DNA polymerase I. The extended and radiolabeled oligonucleotide B was denatured and used to screen a mouse brain Xgtll cDNA library as described (Olsen and Uhler, 1991). Initially, a single cDNA clone designated as MC1 was isolated, subcloned into pTZ18U (U. S. Biochemical Corp.) and sequenced using dideoxynucleotides (Sanger et al., 1977). T7 RNA polymerase (BRL) was then used to generate a "P-labeled RNA complementary to the MC1 cDNA sequence and this RNA was used to rescreen the mouse brain cDNA library. Ultimately the cDNA library was screened six times sequentially, and 26 independent cDNA clones were isolated and sequenced. Sequences were analyzed using the GCG Sequence Analysis software (Devereux et al., 1984). RNA Isolation and Northern Blot Analysis-Total RNA was isolated from adult C57B6 mouse tissue using guanidinium isothiocyanate as described (Chirgwin et al., 1979). RNA was electrophoresed 22728 FIG. 1. Schematic diagram of mouse brain type C a1 subunit cDNA clones. Twenty-six independent mouse brain clones were isolated by sequential screening of a mouse brain hgt 11 cDNA library as described under "Materials and Methods." The individual mouse brain cDNA clones (black, cross-hatched bars below) are indicated by number below the composite sequence indicated by the white bar a t the top of the figure. The four homologous, internally repeated sequences are indicated by small black bars below the composite sequence bar. Nucleotide differences between individual cDNA clones are indicated as follows: dash indicates an insert in the indicated cDNA clones; asterisk indicates cDNA clones with deletions with respect to overlapping cDNA clones; the loop below clone 103 indicates the presence of intronic sequence, and the dashed line indicates sequence within a cDNA that shows no homology to known a1 subunit sequences. through a 0.8% agarose gel in 2 M formaldehyde as described and transferred to Nytran (Olsen and Uhler, 1991). The blot was hybridized with an antisense '"P-labeled RNA probe derived from the 1.1kbp' ApaI/EcoRI fragment of the MC1 cDNA subcloned into the pGEM4 vector (Promega) using the SP6 promoter as described (Olsen and Uhler, 1991).
Transfection of Bouine Chromaffin Cells and Measurement of Exocytosis-Primary dissociated chromaffin cells from bovine adrenal medulla were prepared by collagenase digestion, purified by differential plating (Waymire et al., 1983), and maintained as monolayer cultures in collagen-coated dishes (10,000,000 cells/60-mm diameter dish) (Terbush and Holz, 1990). The cells are highly purified, being greater than 90% chromaffin cells with virtually no detectable fibroblasts. Chromaffin cells were transfected with the appropriate plasmid DNAs using calcium phosphate (BRL) and shocked 4 h after addition of the DNA precipitate with 10% glycerol for 3 min. The pXGH5 plasmid construct contains the coding region for human growth hormone and has been described previously (Selden et al., 1986). Four days after transfection, the cells were washed with physiological salt solution ( P s s ; 142 mM NaCI, 5.6 mM KCI, 3.6 mM NaHCO:I, 2.2 mM CaC12, 5.6 mM glucose, 0.5 mM sodium ascorbate, and 15 mM HEPES buffer (pH 7.4)) and then incubated for 15 min in Pss or in high K+ PSS (PSS containing 20 mM KC1 and 130 mM NaC1). Secretion of endogenous catecholamines was determined by a fluorescence assay as described (Holz et a/., 1982) and secretion of human growth hormone was determined by radioimmunoassay (Nichols Institute). Secretion was calculated as the percentage of the total cellular catecholamine or growth hormone that was released into the medium and expressed as mean f standard error.
Transfection with a plasmid coding for @-galactosidase and subsequent staining for @-galactosidase activity (Sanes et al., 1986) revealed that 0.1-1% of the cells were transfected by this method. The cells which stained as @-galactosidase-positive had normal chromaffin cell morphology as has been previously reported (Ross et al., 1990).

RESULTS AND DISCUSSION
Isolation of Mouse Brain cDNA Clones for the a1 Subunit of the Calcium Channel-A synthetic oligonucleotide corresponding to nucleotides 3355-3401 of the rabbit skeletal muscle was used to screen a mouse brain cDNA library in Xgtll. This oligonucleotide was chosen based on the homology of ' The abbreviations used are: kbp, kilobase pair(s); kb, kilobase(s); CMV, cytomegalovirus; PSS, physiological salt solution; hGH, human growth hormone. this region of the skeletal muscle calcium channel a1 subunit to the otl subunit of the sodium channel, and it was presumed that if multiple subtypes of the calcium channel a1 subunit were found in mouse brain, this probe would have a high probability of hybridizing with these isoforms. The MC1 cDNA was isolated first and used to isolate 25 other cDNA clones as shown in Fig. 1.
Composite Sequence of the Mouse Brain a1 Subunit-The individual cDNA sequences were aligned to generate the composite cDNA sequence shown in Fig. 2. The composite mbC nucleotide sequence shows that the greatest homology to the rat brain type C-I (rbC-I) which has been shown to code for an L-type otl subunit by hybrid depletion experiments . The mouse brain cDNA sequence shows 95% nucleotide sequence identity and 99% predicted amino acid sequence identity to the corresponding rbC sequences. Because of the high homology to rbC and the likelihood that this cDNA represents the mouse homologue of rbC, we have designated this family of clones the mouse brain type C (mbC) a1 subunit.
Four regions of putative alternative RNA splicing were determined from nucleotide sequence comparisons of individual mbC cDNA clones. The first region involves the coding sequence for the sixth transmembrane domain of the first repeat (amino acids 372-404). The second region of nucleotide sequence difference represents an insertion of 75 nucleotides in the mbC sequence between the codons for amino acids 463 and 464. This insertion represents an additional 25 amino acids in the first intracellular loop between repeats I and 11. Both of these first two splicing variants have been reported previously in comparison of smooth muscle (Biel et al., 1990) and cardiac (Mikami et al., 1989), but have not been reported to occur in brain. The third region of alternative splicing corresponds to an insertion of 9 nucleotides between the codons for amino acids 780 and 781. The last region of alternative splicing corresponds to a substitution of the codons for the third membrane spanning segment of the fourth repeat (amino acids 1277-1302). The alternate spliced forms for both region 3 and 4 have been described previously and constitute the difference between isotypes rbC-I and rbC-11. In addition, region 4 splicing has been shown to be developmentally regulated in rat heart (Diebold et al., 1992). It is of interest to note that none of the cDNAs isolated corresponds to the cardiac amino terminus (Mikami et al., 1989) but only the smooth muscle variant (Biel et al., 1990). In fact, polymerase chain reaction analysis suggests that the cardiac aminoterminal sequences are not expressed in mouse brain (data not shown). Outside of the alternately spliced regions, all of  Fig. 1 is shown above with the predicted amino acid sequence below the nucleotide sequence. Transmembrane domains predicted by hydropathy plots and homology to other otl subunit sequences are indicated by dashes between individual amino acids. The four regions where alternatively spliced forms of cDNA clones were detected are indicated by the presence of a second nucleotide and amino acid sequence below (in brackets). Where differences in the predicted amino acid sequence between the mouse brain type C and the rat brain type C sequences were detected, the rat amino acid is shown beneath the mouse sequence. Two other differences in nucleotide sequence were detected in individual cDNAs; clone 124 had a T a t position -47 and clone 121 had an insert of 3 nucleotides (GAG) at position 7072. the species-specific amino acid differences between mbC and cord were used for Northern blot analysis (Fig. 3). Hybridi-rbCI occur in the carboxyl-terminal sequence. zation with antisense MC1 "'P-riboprobe showed that mbC Northern Blot Analysis of mbC Expression-Total RNA related mRNA was detected in heart, brain, and spinal cord isolated from liver, skeletal muscle, heart, brain, and spinal but was not detected in liver or skeletal muscle. In all tissues 14 kb -8.9 kb -FIC. 3 . Northern blot analysis of mbC mRNA in various tissues. Total RNA was isolated from the indicated tissues and suhjected to Northern hlot analysis as descrihed under "Materials a n d Methods." The sizes ofthe hyhridizing RNA species were calculated from RNA molecular weight standards which were electrophoresed on the same gel. which showed mbC transcripts, two distinct mRNAs were observed, an 8.9-kb and a 14-kb mRNA. Both of these RNAs were more abundant in heart than in brain or spinal cord, and in heart the 8.9-kb transcript appeared to predominate. In contrast, the larger 14-kb transcript was the predominant species in brain and spinal cord. The tissue distribution of the mbC mRNA is in agreement with the homology of the mbC nucleotide sequence with the rbC sequence and supports the notion that the mbC transcript is expressed in both cardiac and neural tissues.
Transient Expression of mbC1 in Bovine Adrenal Chromaffin Cells-In order to demonstrate that the mbC cDNAs code for a functional channel, an expression vector coding for the full-length mbC (Fig. 2, upper line with 25-amino acid insert between amino acids 463 and 464) was constructed using the human cytomegalovirus promoter to direct expression of the mbC cDNA. The experimental paradigm used to detect mbC expression is analogous to that which has been used previously t o detect regulation of gene transcription (Huggenvik et al.,

1991
). An expression vector for human growth hormone was cotransfected into chromaffin cells with either the parental pCMV.Neo vector or the mbC a1 subunit expression vector, pCMV.MC1. Primary bovine chromaffin cells have been used extensively in the study of exocytosis and have be used previously in transient expression experiments (Ross et al., 1990). Because transient transfection results in a high incidence of cotransfection of two distinct plasmids, those cells which express the transfected mbC c u l subunit also have a high probability of expressing human growth hormone. Furthermore, the transfected human growth hormone has been demonstrated previously to be targeted to the regulated secretory pathway (Moore and Kelly, 1985;Schweitzer and Kelly, 1985). Since calcium entry via calcium channels is a key step in exocytosis from chromaffin cells (Holz et al., 1991;Atlas, 1990;Burgoyne, 1991), we sought to determine if expression of the mbC cDNA in chromaffin cells could alter the secretion of transfected hGH.
As shown in Fig. 4 A , the secretion of catecholamines from cells cotransfected with the growth hormone expression vector (pXGH5) and either the parental pCMV.Neo expression vector or the mbC c u l subunit expression vector, pCMV.MC1, was measured after incubation with the calcium channel agonist Bay K8644 and moderately elevated K' . Measurement of catecholamine secretion measures the secretion from all cells and thus since less than 1% of cells are transfected, no effect of mbC c u l subunit transfection should be seen on catecholamine secretion. As expected, catecholamine release was small and similar in dishes with or without the cul subunit expression vector. In cells transfected with the pCMV.Neo control expression vector, hGH release, which specifically measures secretion from only the transfected cells, paralleled to catecholamine release (Fig. 4R). This is consistent with transiently expressed hGH being stored in secretory vesicles which are similar if not identical to catecholamine-containing vesicles (chromaffin granules). In contrast, hGH secretion from cells cotransfected with the plasmid for the c u l subunit showed a much greater secretory response to elevated K' and Bay K8644 than did hGH secretion from cells cotransfected with pCMV.Neo. Maximal depolarization-induced catecholamine secretion is usually attained at 56 mM K' and it typically releases 10-20% of total cellular content (Holz et al., 1982). In these experiments, suboptimal K' (20 mM) stimulated GH secretion in the presence of the cul subunit vector to the same degree that 56 mM K' normally stimulates cate-cholamine secretion in non-transfected cells. Bay K8644 in the absence of elevated K+ also induced significant secretion in cells cotransfected with the a1 subunit. In the absence of co-transfected a1 subunit, Bay K8644 does not stimulate hGH or catecholamine secretion in non-depolarized cells. The secretion induced by the combination of 20 mM KC1 and 1 FM Bay K8644 was Ca2+-dependent from the pCMV.MC1 transfected cells (Fig. 5). For example, secretion in the presence of 20 mM KC1 and 1 FM Bay K8644 was reduced from 31.9 k 0.62% to 3.1 k 0.5% of total content upon removal of Ca2+ (in the presence of 1 mM EGTA) in cells with the a1 subunit. Similarly, the Ca2+ channel antagonist D-600 inhibited secretion induced by the combination of 20 mM KC1 and 1 p~ Bay K8644 by 65-75% in transfected cells. In summary, overexpression of the mbC a1 subunit sensitizes calcium-dependent secretion to both depolarization and the effects of the dihydropyridine agonist, Bay K8644. Since a1 subunits alone do not efficiently form functional channels , it is quite likely that the tranfected a1 subunit may be recruiting other subunits of the endogenous calcium channels in the formation of the Bay K8644-sensitive channels.
These experiments are significant for several reasons. First, these experiments demonstrate that the transfected mbC cDNA produces a functional protein. Although expression of c~l subunit cDNAs in oocytes has demonstrated calcium influx (Mikami et al., 1989;Snutch et al., 1990), this report is the first to demonstrate functional expression using secretion. Secondly, these results suggest that the mbC protein is responsive to activation by Bay K8644. This is consistent with expression experiments with other isoforms of the cardiac a1 subunit in oocytes, which suggest that this isoform is L-type and responsive to dihydropyridines. Finally, these experiments suggest that Ca2+ influx through voltage-sensitive calcium channels is rate-limiting for secretion from chromaffin cells and that altering the character or the number of calcium channels can alter the rate of secretion. The mbC a1 subunit expression vector and the chromaffin cell transfection system should be useful in the detailed analysis of a1 subunit function as well as the role of this protein in regulated secretion.