Neuron,

Vol.

7, 45-57,

July,

1991,

Copyright

0

1991

by Cell

Press

Distinct Calcium Channels Are Generated by Alternative Splicing and Are Differentially Expressed in the Mammalian CNS Terry P. Snutch,* W . Jeffrey Tomlinson,* John P. Leonard,+ and Mary M. Gilbert* *Biotechnology Laboratory and Departments of Neuroscience and Zoology University of British Columbia Vancouver, British Columbia Canada V6T 1 W 5 +Department of Biological Sciences University of Illinois at Chicago Chicago, Illinois 60680

Summary A number of pharmacologically and electrophysiologically distinct voltage-dependent Ca2+ channels have been identified in mammalian neurons. Two rat brain Ca*+ channel al subunits (rbC-I and rbC-II) have been isolated by molecular cloning and shown to be highly related (95%) to the cardiac dihydropyridine-sensitive Ca*+ channel. The rbC-II protein is distinct from rbC-l in that it contains a 3 amino acid insert in the putative cytoplasmic loop between domains II and III and a 28 amino acid substitution corresponding to the third transmembrane segment 63) of the fourth domain. W e show that rbC-l and rbC-II transcriptsaregenerated by alternative splicing and that they are differentially expressed in the rat CNS. Introduction The rapid entry of Caz+ into excitable cells is mediated by voltage-sensitive Ca *+ channels. In the nervous system Ca*+ currents contribute to the electrical properties of neurons, including repetitive firing patterns, pacemaker activity, and the regulation of Ca2+-dependent ion channels, both directly as currents and indirectly following an increase in intracellular Ca2+ concentration (for reviews see Miller, 1987; Tsien et al., 1988). In addition,an increase in intracellularCa2+concentration at the presynaptic nerve terminal triggers the releaseof neurotransmitter intothe synaptic cleft. Ca*+ entry also affects cellular events by modulating Ca*+-dependent enzymes, and in developing neurons, Ca*+ plays a role in neurite outgrowth and growth cone migration (Kater et al., 1988). Given both the variety of physiological functions that Ca2+ entry regulates and the wide array of cell types that possess Ca*+ channels, it is perhaps not surprising that diverse types of Ca*+ channels have been described (for example, Nowycky et al., 1985; Fox et al., 1987; Llinds et al., 1989). The various Caz+ channels can be distinguished according to their voltage characteristics, single-channel properties, ionic selectivity, and pharmacological properties (designated T-, L-, N-, and P-type Ca2+ channels). While electrophysiological studies demonstrate that individual

neurons express multiple subtypes of Ca2+ channels, the exact number and physiological roles of Ca*+ channel subtypes remain to be determined. Biochemical analysis of the skeletal muscleT-tubule dihydropyridine (DHP)-sensitive Ca2+channel (L-type) shows that under reducing conditions it is composed of five distinct subunits (a,, a2, s, y, and 6; Campbell et al., 1988; Catterall et al., 1988). Expression of cDNAs encoding the al subunit in myotubes from dysgenic mice (Tanabe et al., 1988) and in mouse L cells (PerezReyes et al., 1989) results in functional DHP-sensitive Ca*+ channels. Recently, cDNA cloning of Ca2+ channel a1 subunits from other tissues has revealed that distinct al subunits are expressed. The primary structures of smooth muscle (Biel et al., 1990) and heart (Mikami et al., 1989) al subunits are more highly related to each other than to the skeletal muscle L-type Ca2+ channel (Tanabe et al., 1987). Microinjection of synthetic RNA derived from the heart and smooth muscle al subunit cDNAs into Xenopus oocytes results in the synthesis of functional DHP-sensitive L-type Ca2+ channels (Mikami et al., 1989; Biel et al., 1990). In both instances, coinjection of RNA coding for the a2 subunit resulted in increased whole-cell currents. Several lines of evidence suggest that the brain expresses L-type Ca*+ channels similar in composition to the skeletal muscle Ca2+ channel complex. RNA studies with cloned probes for the a2 and [\ subunits reveal that transcripts homologous to the skeletal muscle complex are expressed in brain (Ellis et al., 1988; Ruth et al., 1989). In addition, a monoclonal antibody against the skeletal muscle a2 subunit immunoprecipitates a DHP receptor complex from brain homogenates that contains al-, a2-, b-, and &like subunits (Ahlijanian et al., 1990; Westenbroek et al., 1990). In contrast to the L-type Ca2+ channel, the subunit composition of T-, N-, and P-type Ca*+ chamnels has not been described. The functional differences between Ca*+ channel subtypesmaybeduetoanumberoffactors,including the expression of distinct a1 subunit proteins, the selective expression of structural subunits or modulatory proteins, the existence of cell-specific posttranslational processing pathways, and differences in the local lipid membrane environment. W e have taken a molecular genetic approach to examine the structural and functional basis of Ca2+ channel diversity in the mammalian CNS. W e have previously described the isolation of a number of rat brain cDNAs that encode a heterogeneous family of proteins related to the al subunit of DHP-sensitive Ca*+ channels (Snutch et al., 1990). Southern blot and Northern blot analyses showed that the rat brain Ca2+ channels can be grouped into four distinct subfamilies (desi,gnated rat brain A, B, C, and D classes). In the present study, we report the isolation and characterization of 2 cDNAs

Differential 47

Figure

Calcium

1. cDNA

Channel

Cloning

Expression

and Sequence

of rbC-I and rbC-II

(A) Schematic map of overlapping cDNAs isolated from a rat brain cDNA library. rbC-30 and rbC- 61 encode the rbC-l-type Ca2+ channel; rbC-2040 and rbC-2019 encode the rbC-II-type Ca2+ channel. The open box represents the protein-coding region of the rbC transcripts. The asterisk above rbC-61 represents a premature stop in this cDNA. The scale is in kilobases. (6) The nucleotide sequence of the rbC-I transcript and the deduced amino acid sequence of the rbC-I and rbC-II proteins. The deduced amino acid sequences of the two rat brain proteins are compared with the rabbit cardiac L-type Ca*+ channel sequence (Mnkami et al., 1989). Amino acids that are identical to rbC-I are shown by dashes. Gaps are indicated by spaces. The numbers at the right of each line are the nucleotide numbers for rbC-I DNA sequence (top) and the rbC-I protein sequence (below). The putative transmembrane segments (Sl-S6) of the four homology domains (I-IV) are indicated by lines above the DNA sequence. The premature stop in the rbC-I sequence is noted by an asterisk at amino acid position 1758. The 5 serine residues that are putative recognition sites for protein kinase A are noted by asterisks above the DNA sequence (Ser-1545, Ser-1597, Ser-1670, Ser-1818, and Ser-1898).

encoding rat brain class C (rbC) Ca’+ channels. We find that these two rbC variants are encoded by the same gene and offer direct evidence that alternative splicing events provide a general mechanism for generating Caz+ channel diversity in the CNS. Results Primary Structure of Rat Brain Class C CaZ’ Channels We previously isolated and determined the partial DNA sequence of a rat brain cDNA (rbC-61) found to be highly related to the rabbit cardiac DHP-sensitive Ca2’ channel (Snutch et al., 1990). To isolate the remaining portion of the rbC Cal’ channel, a 2.4 kb EcoRl fragment from rbC-61 was used to probe a rat brain cDNA library enriched for cDNAs greater than 4 kb in size. An additional 18 clones were isolated and, by Northern blot analysis and partial DNA sequence, were shown to encode rbC-type Ca” channels (data not shown). Initially,2overlapping cDNAs, rbC-30and rbC-61, were selected for further study (Figure IA). Sequential deletions were prepared using the exonuclease III/S1 nuclease method (Henikoff, 1984), and the DNA sequence of one strand of each clone was determined by the dideoxy method using doublestranded templates (Biggin et al., 1983). The DNA sequences were confirmed by sequencing the second strand of each clone using synthetic oligonucleotide primers. The rbC-30 and rbC-61 cDNAs are identical in the 1353 bp region of overlap between the 2 clones, and the full-length sequencederived iscalled rbC-I (Figure IB). The complete nucleotide sequence of rbC-I consists of 7848 bp. The first in-frame ATG of rbC-I is preceded by a 5’ nontranslated region of 753 bp. This ATG is within a sequence (S-TGGCCAATCC-3’) that partially matches the consensus sequence for initiation (Kozak, 1984). The first ATG is followed by two open reading frames of 5271 bp (1757 amino acids) and 1146 bp (382 amino acids). The termination codon (TAA) separating the two reading frames is the codon for tyrosine (TAC) in the cardiac (Mikami et al., 1989) and lung (Biel et al., 1990) L-type Cal’ channels. We interpret the premature stop in rbC-I to be an artifact of the cDNA cloning. Indeed, the initial characterization of a third rbC-I cDNA (rbC-612) shows that it contains a tyrosine at this position (data not shown). The termination codon (TAG) at the 3’ end of the second reading frame aligns with the 3’ ends of the cardiac and lung L-type Cal+ channels and is followed by a 3’ nontranslated region of 675 bp. A poly(A) addition site or a poly(A) tail was not observed within the 3’ noncoding region of rbC-I. The predicted primary sequence of the rbC-I peptide shows that it is similar in overall structure to voltage-gated Na+and Ca2’channeIs(Nodaet al., 1986; Auld et al., 1988; Tanabe et al., 1988; Mikami et al., 1989). It is composed of four homologous domains (I-IV) of 229-282 residues, each of which contains between six and eight putative transmembrane seg-

ments. In the fourth transmembrane segment ot eat h domain (S4) every third or fourth residue is positively charged (arganine or lysine) dnd is thought to represent the voltage sensor of the molecule (Stuhmer et al., 1989). The putative cytoplasmic carboxyl segment of rbC-i contains a total of five potential cAMPdependent protein kinase sites (Figure IB). All of these sites are conserved among rbC-I and the rabbit cardiac and lung L-type Cal+ channels. While the overall primary structure of rbC-I is 95% identical to that ot the cardiac- L-type Ca” channel, there are a number of notable regions of difference: rbC-I i\ truncated by 30 amino dcids at the amino terminus, and the first 16 residues are different from the cardiac sequence: there are tour clusters of amino acid substitutions (of between 3 and 7 residues) in the tour domains, including one that alters the S2 segment of domain III; and there are a number of substitutions clustered in the tarboxyl terminus (Figure IB). In an attempt to identify other rbC-I cDNAs that did not possess the premature stop after residue 1757, we found that the majority of rbC sequences were in tact different from rbC-I. Two overlapping cDNAs (rbC 2040and rbC2019; Figure IA) were sequenced in their entirety dnd found to be 99% identical to rbC-I (de noted rb( -II: Figure 1B). The differences between rbC-I dnd rhC-II include 5 single amino acid substitutions, a .1 amino acid insertion (Pro-Ala-Arg) in the loop region separating domains II and III, and a stretch ot 13 substitutions in a 28 amino stretch representing the S3 segment of domain IV. At the DNA level, the rb(‘-I and rbC-II S3 regions are 43% different (36 differences in 84 bp). The same 13 amino acid substitutions found in the rb(‘-II domain IV S3 segment are also found in the rabbit lung L-type Cal+ channel (Biel et al., 1990) and the rat aorta (Koch et dl.. 1990). Attempts to express the rbC-II product in Xenopus oocytes injected with synthetic RNA have proven unsuccessful (datd not shown). Hybrid Depletion of Ca” Channels Expressed in Xenopus Oocytes The reldtlonship between the rbC transc-ripts and L-type Cd-” channels expressed in Xenopus oocytrs after injection of cellular RNA was investigated using a hybrid depletion technique. Figure 2A shows that rat heart RNA injected into oocytes induces the synthesis ot a slowly inactivating Ca” channel that 15 blocked completely by application of 50 PM nifedlpineat all voltages. The peak current-voltage relationship revealed a maximum at +I0 mV. To examine the relationship between the rbC transcripts and the car disc L-type Ca” channel, we hybridized sense or ant{sense oligonucleotides (18-mers) for the rbC gene to rat heart RNA and then coinjected the mixtures into oocytes. The antisense oligonucleotide chosen was conserved between rbC-I and rbC-II and does not cross-react with any other rat brain Cal+ channels (Snutch et al., 1990). After an incubation of 3 days, the oocytes were tested for expression of voltage-gated

Differential 49

Calcium

Channel

Expression

HEART

-23.1

u

Kb

-

9.3 Kb

-

6.6 Kb

-

4.4 Kb

50 NA FiRure 3. Southern 40 MS

B

HEART

Blot Analysis

of Rat Genomic

DIVA

Autoradiograph of hybridization of rbC-l- and rbC-II-specific probes to rat genomic DNA. Rat liver DNA (IO pg) was digested to completion with each of the following enzymes: EcoRI, Pstl, Hindlll, and EcoRV. After electrophoresis, the DNAwas blotted to Nytran membrane and probed with 32P-labeled oligonucleotides specific for the rbC-I and rbC-II S3 segments. The results show that the rbC-I and rbC-II S3 segment probes hybridize to an identical pattern of rat genomic fragments. Size markers (in kilobases) are Hindlll-digested phage A. DNA.

ANTISENSE

SENSE 40 NA 40 ns Figure 2. Hybrid Oocytes

Depletion

of L-type Ca2+ Channels

in Xenopus

(A) Total rat heart RNA was microinjected into Xenopus oocytes, and after 3 days, the currents were measured using a twomicroelectrode voltage clamp. Peak Ba2+ currents were measured by stepping from a holding potential of -80 mV to an optimal test potential of +I0 mV. The lower trace (-DHP) represents the inward Ba*+current of untreated cells. The upper trace (+DHP) shows that the Ba?+current is sensitive to block by50 uM nifedipine treatment. (6) Total rat heart RNA was hybridized with sense or antisense oligonucleotides (18mers; see Experimental Procedures), and the mixture was then coinjected into Xenopus oocytes. The traces show that preincubation with the antisense rbC oligonucleotide inhibits the expression of the DHP-sensitive Ca2+ channel. The traces in (A) and (B) were leak subtracted.

Ca2+ and K+ conductances. Figure 28 shows that the sense rbC oligonucleotide had no appreciable effect on expression of the heart L-type Caz+ channel. However, the rbC antisense oligonucleotide blocked approximately 90% of the expression of the heart L-type CaZ+ current measured at the peak of the currentvoltage curve. The Ba2+ current amplitudes for 5 cells in the presence of sense oligonucleotide were 33 f 8, 48 f 12, and 37 f 12 nA at 0, +lO, and +20 mV, respectively. The Baz+current amplitudes for 5 cells in the presense of antisense oligonucleotides were 3 + 2,4 f 2, and 2 + 1 nA at 0, +lO, and +20 mV, respectively. None of the oligonucleotides used affected the

expression of K+currents induced by heart RNA. The K+ currents were measured to control for oocyte to oocyte variability in expression levels, and Ba2+ currents were then normalized to K+ current amplitudes from the same cell for quantitative comparison of sense versus antisense expression levels. The ratio of Ba*+current to K+current was 0.61 + 0.11 for the sense oligonucleotide and 0.08 f 0.04 for the antisense oligonucleotide (N = 5 oocytes each; Student’s t-test, P < 0.005). A Single Gene Encodes rbC-I and rbC-II CaZi Channels To examine the molecular relationship between rbC-I and rbC-II more closely, we performed a Southern blot analysis of rat genomic DNA. Figure 3 shows that oligonucleotide probes specific for the domain IV S3 segmentsof either rbC-I or rbC-II hybridizetoan identical pattern of rat genomic DNA fragments. This result is consistent with two formal possibilities: that rbC-I and rbC-II transcripts are derived from two highly similar but distinct genes, or that the rbC-I and rbC-II transcripts are derived from the samegene and are generated by an alternative splicing mechanism. To distinguish between these two possibilities, weanalyzed the rbC genomic sequences using the polymerase chain reaction (PCR). We postulated that if the distinct S3 segments were exons in a single rbC gene and the distance between the two exons was less than 10 kb, then a genomic DNA fragment could be amplified bythe PCR. Four unique oligonucleotide primers were synthesized against the 3’ends of the rbC-I and rbC-II S3 segments and then used in pairwise combinations to amplify rat genomic DNA (Figure 4A). PCR amplification using the rbC-II primer 48-B and the rbC-I primer61-BAdid not result in an amplified prod-

DOMAIN I" S3 REGION

A

-0.60 -0.31 Figure 4. Polymerase

Chain

Reaction

Analysis

of Rat Gnomlc

DNA

(A) Schematic diagram of the PCR strategy to determine the genomic organization OI the rbC:Ll and rt,C-Ii domain IV \ i segments. IWU sense oligonucleotides (61-B and 48-B) and two antisense oligonucleotides (61-BA and 48.BA) within the rbC-I and rbC-Ii S3 segment\ were synthesized using an automatic DNA synthesizer. The oligonucleotides (‘I-mers) were used in combination as primers to amplify rat genomic DNA (250 ng) using the PCR (32 cycles). The pairwise combinations were 6’lLB together with 4%BA and 48-B together with 61-BA. (B) Negative imageofan ethidium bromide-stained gel ot theamplification produc I\ ot the reactIon\ described above. The combindtlon of primers 48-B and 6%BA was not able to amplify rat genomic DNA. In contrast, the primer pair of 61-B and 48-BA amplified all approximately 740 bp fragment in the rat genome. The data illustrate that the rh(‘-I and rbC-II S3 segments arc colinear in the rat genome.

uct (Figure4B). However, amplification using the rbC-I primer 61-B and the rbC-II primer 48-BA produced an approximately 740 bp product. This result is consistent with the two S3 segments being adjacent exons approximately 630 bp apart in the rat genome. The result of this experiment also determined the relative orientation of the two exons in the rat genome, since only oligonucleotides 61-B and 48-BA produced overlapping strands (see Figure 4). To confirm the PCR result, a rat genomic DNA library in the vector h Dash (Stratagene) was screened with radiolabeled probes for both the rbC-I and the rbC-II domain IV S3 segments. Eight positive genomic clones were isolated and shown to hybridize to both

rbC-l- and rb(:-l I-specltlc probes (data not shown). By restrictron map analysis, the 8 h clones were found to represent a 26 kb stretch of overlapping rat genomlc DNA. A 3.0 kb Xbal fragment was common to all genomic clones and hybridized to both rbC-I and rbC-II S3 segment probes. This fragment was subcloned into the plasmid vector Bluescript (Stratagene) for further analysis. Figure 5 shows the DNA sequence of a portion of the genomic Xbal fragment. The results show that the rbC-I and rbC-II S3 segments are separated by an intron of 632 bp in the rat genome. The boundaries of the two S3 segments are highly similar to the consefisus sequences for intron-exon splice junctions (McKeown, 1990). Thus, the rat genome encodes a single

ctgqcttttctqccccatgc=-~~ -T‘XA-TACAXTTfSWGC-~~~ BYFCDIWNTFDALIVVCSIVDIAITLVB Figure 5. Characterization

of Domain

IV S3 Segment

TAT--AC&C

Exons

(A) Schematic map of a portion of the genomic clone h D2 containing the rbC-I and rbC-II S3 variants. The map shows the 3.0 kb Xbal restriction fragment in clone I Dash D2. The 3.0 kb Xbal fragment hybridized to both rbC-I and rbC-II probes and was subcloned into the plasmid vector Bluescript. The DNA sequence was determined. The scale is in basepairs. (B) Nucleotide sequence of the genomic clone in the region containing the rbC-I and rbC-II S3 variants (between the arrows shown in [A]). The two S3 segments are highlighted, and the deduced amino acid sequences are shown below. The two S3 exons are separated by a 632 bp intron. The remaining 5’ and 3’ flanking regions of the D2 7, clone do not contain any additional coding regions.

Differential Calcium Channel Expression 51

DOtlAIN IV 53 REGION

A rbC-I

c

12Kb

C

8 Kb

rbC-II

48-B -

. . -.

. . . . T

Figure 6. Northern Blot Analysis of rbC Expression in the CNS Autoradiograph of hybridization of an rbC cDNA probe to rat brain RNAs. RNA was prepared from the CNS of young adult male rats using the atlas of Paxinos and Watson (1986) as a guide. Total brain RNA (30 pg per lane) was denatured, electrophoresed through an agarose gel containing 1.1 M formaldehyde, and then blotted to Hybond-N membrane. The probe was a 2.0 kb EcoRl fragment of rbC-61 and would cross-hybridize to both rbC-I and rbC-II variants. Sizesare in kilobases and weredetermined using RNA standards (Bethesda Research Laboratories). Autoradiography was for 5 days with an intensifying screen.

class C-type Ca*+ channel gene that is alternatively spliced to generate rat brain rbC-I and rbC-II Ca*+ channels.

Differential

Expression of rbC-I and rbC-II

The regional distribution of rbC Ca*+ channel expression was examined by Northern blot analysis. Hybridization of a probe that cross-hybridizes with both rbC-I and rbC-II cDNAs shows that two transcripts of approximately 8 and 12 kb are expressed throughout the rat CNS (Figure 6). In most brain regions the 8 kb transcript is the most prevalent of the two transcripts, although the relative level of expression of the 8 and 12 kb transcripts does vary somewhat in the different regions. The two rbC transcripts are most prevalent in the olfactory bulb and cerebellum and are lowest in the pans/medulla and spinal cord. We note that the regional distribution of rbC transcripts does not exactly correlate with autoradiographic mapping of DHP-binding sites in the rat brain (Cortes et al., 1984). Examination of thedevelopmental expression pattern of the rbC gene shows that both transcripts are present in rat brain from the earliest stage tested (embryonic day 16) through to the adult (data not shown). PCR analysis was utilized to distinguish between the rbC-I and rbC-ll transcripts in the rat CNS. To avoid potential problems with selective amplification of transcripts, we used a primer pair that was identical in both rbC-I and rbC-II for amplification and then probed the PCR products with radiolabeled rbC-I and rbC-II domain IV SS-specific oligonucleotides. Initially, first strand cDNA was synthesized from brain RNA samples using primer #8 as the downstream primer, and the resulting cDNA was then used as a substrate for amplification using primers #8 and #I4 (Figure 7A). The PCR products were separated on an agarose gel and then transferred to a nylon membrane. One-half of each blot was then probed with radiolabeled oligos specific for the rbC-I and rbC-I I S3

L

659 bp -

B

rbC-I

M-659

bp

rbC-II

-659

bp

Figure 7. Differential Expression of rbC-I and rbC-II Transcripts (A) Schematic diagram for the analysis of rbC-I and rbC-II transcript levels using the PCR. One oligonucleoGde (#B) was used to prime first strand cDNA synthesis of various rat brain total RNAs (2 vg per reaction). Subsequently, a second primer was added(#14)and,inconjunctionwith thefirst,wasusedtoamplify the cDNA products by the PCR (32 cycles). PCR products were detected by hybridization of radiolabeled oligonucleotide probes specific for the rbC-I and rbC-II S3 segments. The expected PCR product, including the #I4 and #8 primers, is 659 bp. (8) Autoradiograph of hybridization of S3 rbC-l- and rbC-llspecific probes. PCR products were separated on a 1.2% agarose gel, blotted to Nytran, and then probed with the rbC-I and rbC-II S3 region oligonucleotide probes. Note that both S3 variants are expressed in all regions of the rat CNS. Also, note that the relative levels of the two S3 segments vary in different regions of the CNS. Autoradiography was for 2 hr with an intensifying screen.

segments. The results show that both rbC-I and rbC-II S3 variants are expressed in all regions of the rat CNS (Figure 7B). Comparison of the relative amounts of rbC-I and rbC-II in whole rat brain, shows that the rbC-II transcript is more prevalent than the rbC-I transcript.This is in agreementwith the molecularcloning results, which show that only 16% (3 of ‘19) of the isolated brain rbC clones are of the rbC-I type. Significantly, however, the relative amounts of rbC-I and rbC-II vary between brain regions. For example, there is significantly more rbC-II transcript in the olfactory bulb, cortex, hippocampus, hypothalamus, and striaturn, whereas there is approximately equal amounts of rbC-I and rbC-II transcripts in the cerebellum and pans/medulla. In the spinal cord and in thetrigeminal nerve, the rbC-I transcript predominates. To confirm that the PCR protocol accurately reflected the abundance of rbC transcripts in brain RNAs, we performed a similar PCR amplification using synthetic sense rbC-I and rbC-ll RNAs (see Experimental Procedures). Using rbC-I and rbC-II synthetic RNAs mixed at a ratio of l:lO, the PCR amplification products were found to be proportional to the abundance of initial template

Figure 8. Tissue

Specificity

of rbC Gene

Expression

Autoradiograph of hybridization of rbC-I- and rbC-II-specrfic 53 segment probes to PCR products. RNA was isolated from various rat tissues (young adult). It was converted to first strand cDNA and subsequently amplified as described in Experimental Procedures. PCR amplification was carried out for 32 cycles. Note that except for the pituitary gland, the rbC-II S3 variant is the most abundant rbC form. Also note that the amplified produc-t from adrenal RNA is slightly smaller than that from the other RNA samples. Autoradiography was for 5 hr with an intensifying screen. With lo-fold longer exposures, rbC-I and rbC-II transcripts are also detected in spleen and testes.

and to be independent of cycle number (22-36 cycles; data not shown). Similar results have been obtained with RNA isolated using both lithium chloride-urea (Auffrayand Rougeon, 1980) and guanidinium thiocyanate-CsCI (Chirgwin et al., 1979) RNA isolation protocols. These results indicate that rbC-I and rbC-II transcripts are expressed throughout the rat CNS and that the level of expression varies among brain regions. Differential Expression of rbC Transcripts in Other Rat Tissues We took advantage of the fact that the rat genome encodes a single rbC gene to examine both the expression of rbC gene transcripts in other rat tissues and the relative levels of expression of the two domain IV S3 segments in these tissues. Using oligo #8 as the downstream primer, single-stranded cDNA was prepared from total RNA and then amplified by the PCR using oligos #I4 and #8. Hybridization with the two domain IV S3-specific probes shows that the rbC gene is transcribed in a variety of tissues (Figure 8). These include the heart, pituitary, adrenal gland, liver, and kidney. With IO-fold longer exposure times, we also detect rbC transcripts in testes and spleen. All tissues that express rbC transcripts express both alternatively spliced forms of the rbC 53 segment. As was the case for the different brain regions assayed, the rbC-II type S3 segment is the predominant rbC transcript in most tissues. However, tissue-specific differential expression is also suggested by the fact that relatively higher levels of the rbC-I type S3 transcript are expressed in the pituitary gland. We note that the amplified product from adrenal RNA is routinely shorter than that from the other tissues, suggesting that further splicing events occur to generate additional rbC Ca?+ channel variants. Of particular interest are the results concerning the expression of rbC transcripts in the heart. Figure 8 shows that the majority of rbC transcripts in the heart

possess the rbC-II domain IV Sj segment. This is In contrast to the published cardiac Ca?’ channel sequence that identifies the rbC-I S3 segment (Makami et al., 1989). Thus, our results suggest that a second, highly abundant rbC transcript, containing the rbC-II type domain IV S3 segment, is also expressed in the heart. This result is in agreement with recent reports describing the PCR analysis of cardiac RNA (PerezReyes et al., 1990) and the cDNA cloning of a CaL’ channel from aorta (Koch et al., 1990). Discussion Two Novel Ca” Channel at Subunits In this report, we describe two rat brain transcripts that are distinct from previously cloned Ca*+ channel a, subunits. Including the two reported here, the primary structures of six complete Cal’ channel al subunits have now been described (Tanabe et al., 1987; Mikami et al., 1989; Biel et al., 1990; Koch et al., 1990). All cloned a: subunits consist of between 1873 and 2171 amino acids and show a conserved predicted overall structure and topology. The two rbC Cal’ channels are most closely related (approximately95%i to the rabbit cardiac (Mikami et al., 1989) and lung (Biel et al., 1990) DHP-sensitive CaL- channels (L-type) and are more distantly related to the rabbit skeletal muscle L-type channel (approximately 70%). Compared with the rabbit cardiac and lung L-type CaL+ channels, the rat brain transcripts contain a number of notable differences. First, rbC-I and rbC-II contain a cluster of 3 amino acid substitutions in the segment separating S5 and S6 of the first domain. This region has been shown to be involved in toxin binding in voltage-gated Na’ channels (Noda et al., 1989). Furthermore, the analogous region in voltage-gated K’ channels (called H5) has recently been implicated in forming the pore of the channel (MacKinnon and Yellen, 1990; Hartmann et al., 1991; Yellen et al., 1991;Yool and Schwarz, 1991). Second, the rbC Cal’ channels contain two clusters of amino acid substitutions in the domain II-I I I loop. Tanabe and co-workers (1990) have elegantly demonstrated that this region of the skeletal muscle L-type CaL+ channel is involved in mediating excitation-contraction coupling. Third, rbC-I and rbC-II have 7, mostly conservative, substitutions corresponding to the S2 segment of domain II. Finally, in the putative cytoplasmic carboxyl region, rbC-I and rbC-II havea number of clustered substitutions when compared with the cardiac and lung L-type Ca” channels. Single amino acid changes have been shown to affect the functional properties of voltage-gated ion channels dramatically (Auld et al., 1990; MacKinnon and Yellen, 1990; Noda et al., 1989). Thus, it is possible that the differences between the rbC transcripts and the other Ca” channels confer distinct functional characteristics to the brain Ca” channels. The rbC-I and rbC-II primary structures differ trom each other in three distinct ways. First, there are 5 single amino acid substitutions and a premature stop

Differential 53

Calcium

Channel

Expression

in rbC-I when compared with rbC-Il. At the DNA level, 5 of the changes are the result of either G to A or A to G transitions and, given their scattered nature, are likely to represent either artifactual mutations introduced during cDNA cloning or DNA polymorphisms. The second type of difference between rbC-I and rbC-ll is the insertion of 3 amino acids in the putative cytoplasmic loop between domains II and III of rbC-Il. This insertion is found in a number of independent rbC-ll cDNAs and is unlikely to represent a cloning artifact (data not shown). The 3 amino acid insertion occurs in a region of the protein which is highly charged (18 of 27 of the flanking residues are positive or negative). It is difficult to predict the functional significance of the addition of a proline, alanine and arginine to this region. However, given the putative cytoplasmic location of this segment (Tanabe et al., 1990), the differences between rbC-I and rbC-II in this region could reflect distinct cytoplasmic interactions. The third type of difference between rbC-I and rbC-II is a 28 amino acid substitution corresponding to the fourth domain S3 segment. Within this 28 amino acid stretch, 15 residues are conserved between rbC-I and rbC-II, including 4 acidic residues. While most of the 13 substitutions are conservative in nature, an exception is the proline (rbC-I) to alanine (rbC-II) switch at the amino terminus of the S3 segment. If the S3 segment is physically adjacent to the domain IV voltage sensor (S4) in the membrane, it is possiblethat any of the substitutions could affect interactions between amino acid side chains and thus influence channel gating. Testing of this hypothesis will await the functional expression of full-length rbC-I and rbC-II cDNAs in a standard test environment. Cenomic Structure and Alternative Splicing The genomic organization of the rbC gene was determined by a combination of Southern blotting, PCR analysis, and genomic cloning. Together, these studies demonstrate that the rat genome encodes a single rbC gene. In the domain IV S3 segment examined here, we find that the rbC-I and rbC-II variants are 84 bp exons separated by 632 bp in the rat genome. Examination of 19 rbC cDNAs shows that they contain either the rbC-l- or the rbC-II-type S3 segment. While there are a number of possible alternative splicing mechanisms (McKeown, 1990), these results indicate that the S3 segment heterogeneity in rbC clones occurs via a mutually exclusive splicing event. In the 26 kb of genomic DNA examined, the only coding regions are the two 84 bp S3 exons, indicating that the flanking intron regions are quite large. The genomic nature of the 3 amino acid insertion in the domain IIIII loop of rbC-II has not been examined fully. However, PCR analysis of rat genomic DNA shows that there is a 2.1 kb intron flanking this region and suggests the possibility of additional RNA splicing events (data not shown). The general mechanism of alternative splicing to generate further Ca*+ channel diversity is also indi-

cated in a second rat brain Ca2+ channel gene. Partial sequence of a cDNA encoding the rat brain class D Ca*+ channel (rbD) showed that this Ca*+ channel is approximately 75% identical to the cardiac and skeletal muscle DHP-sensitive Ca*+ channels (Snutch et al., 1990). The rbD Ca*+ channel is highly similar to the partial rat brain cDNA RB19 previously reported (Koch et al., 1989). Partial DNA sequence analysis of two different rbD cDNAs (rbD-I and rbD-II) showed that they differ in 11 of 28 amino acids corresponding to the S3 segment of domain IV (data not shown). Significantly, 8 of the 11 amino acid substitutions between rbD-I and rbD-II also occur between rbC-l and rbC-Il. Preliminary data suggest that the rat genome contains a single rbD channel gene (data not shown); therefore it is highly probable that distinct class D Ca*+channels are also generated by an alternative splicing mechanism. Our previous results indicated that a major source of Ca2+ channel heterogeneity is the generation of distinct Ca2+ channel al subunits encoded by distinct genes. The results presented here indicate that in addition to the four main classes of rat brain Ca*+ channel al subunits described (Snutch et al., 199(I), a much larger degree of Ca*+ channel diversity than was previously predicted is generated via alternative splicing events. In this regard, the generation of Ca2+ channel diversity is similar to that of K+ channels. Two distinct mechanisms have been shown to generate K+ channel diversity. In Drosophila, multiple K+ channel transcripts of the Shakergene are generated by alternative splicing of a highly conserved central core region onto variable Sand 3’segments (Kamb et al., 1988; Schwarz et al., 1988). In both Drosophila and mammals, a second mechanism to generate K+ channel diversity is the expression of distinct K+channel genes (Baumann et al., 1988; Butler et al., 1989; Cristie et al., 1989; McKinnon, 1989). Functional Aspects of Cloned Caz+ Channels Electrophysiological and pharmacological criteria have defined three main categories of Ca2+ channel (Carbone and Lux, 1984; Fedulova et al., 1985; Nowycky et al., 1985; Fox et al., 1987): a low threshold, rapidly inactivating channel (T-type); a high threshold, noninactivating channel that is sensitive to DHP agonists and antagonists (L-type); and a high threshold, partially inactivating channel that is insensitive to DHPs and is blocked by o-conotoxin (N-type). Recently, a which is sensitive to fourth type of Ca2+ channel, funnel-web spider venom, has been shown to underlie high threshold Ca*+-dependent spikes in cerebellar Purkinje cells (P-type; Llinis et al., 1989). The PCR data suggest that the rbC gene encodes Ca2+ channels that are expressed in a wide variety of cells types. The rbC transcripts are detected in RNA isolated from electricallyexcitabletissues(brainand heart),from nonexcitable tissues (liver and spleen) and from endocrine glands (pituitary and adrenal). To date,

all cloned

full-length

Ca2+ channel

al sub-

units that have been successfully expressed have been shown to encode functional L-type Ca2+ channels (Tanabe et al., 1988; Perez-Reyes et al., 1989; Mikami et al., 1989; Biel et al., 1990). The high degree of sequence identity between the rbC cDNAs and L-type Ca*+ channels suggests that the rat rbC gene is the rat homolog of the rabbit cardiac and lung DHP-sensitive Ca2+channels. This suggestion is reinforced by hybrid depletion data which demonstrate that antisense rbC oligonucleotides can block the expression of rat heart L-type Ca2+ channels in Xenopus oocytes injected with heart RNA. However, we note that rbC antisense oligonucleotides can also block the expression of Ca2+ channels induced in Xenopus oocytes by injection of rat brain RNA (Snutch et al., 1990). Significantly, the brain Ca2+ channel induced in Xenopus oocytes is completely insensitive to DHPs and differs in waveform from the heart current (Leonard et al., 1987). There are three possible explanations for these results. First, it is possible that the single rbC gene encodes an L-type Ca2+ channel expressed in heart and that brain rbC cDNAs encode functionally distinct Ca2+ channels. In this scenario, the structural differences between rbC-I and rbC-II and the cardiac and lung Ca2+ channels would affect the electrophysiological and pharmacological properties of the channel. Second, it is possible that the brain and heart RNAs encode other polypeptides that interact with rbC Ca2+ channel al subunits to alter their functional properties in oocytes. Third, it is possible that the heart DHPsensitive and brain DHP-insensitive Ca2+ channels expressed in oocytes are in fact encoded by distinct genes, but that the hybrid depletion technique does not distinguish between Ca*+ channel isoforms.

flanking sequences. It is entirely possible that the rbC transcripts detected in the other tissues could differ in other regions from the brain transcripts. We note that in the adrenal gland two distinct rbC transcripts are expressed which are shorter in the S3 region than those found in other tissues. Significantly, a recent report by Perez-Reyes and co-workers (1990) demonstrates the existence of a cardiac Ca2+ channel isoform (from hamster, rabbit, and human) that has a 33 bp deletion in the domain IV S3 region. While crossspecies comparisons cannot be made directly, these results suggest the possibility that the single rbC gene may express multiple Ca2+ channel isoforms in a variety of tissues. Given the moderate levels ot expression, the wide distribution of rbC transcripts, and the high degree of similarity to the heart and lung L-type Cal’ channels, it is tempting to speculate that the rbC gene encodes L-type Ca?+ channels in a variety of tissues. Indeed, one might propose that the rbC gene is not a “neuronal” Ca2+channel, but rather encodes vascular smooth muscle L-type Ca’+ channels. However, we believe that this line of reasoning is probably too simplistic for two reasons. First, there are a large number of potential functionally significant amino acid substitutions in rbC transcripts when compared with the muscle Cal+ channels. Second, we detect the expression of rbC transcripts (both domain IV S3 variants) in a variety of homogeneous neuronal and endocrine cell lines, including PC12, GH4, and calcitonin-secreting C cells (T. P. Snutch and J. J. Enyeart, unpublished data). This result indicatesthat rbcgeneexpression is not limited to smooth muscle. Experimental

Distribution and Differential Expression of rbC Transcripts The rbC gene is expressed at moderate levels in the brain, liver, kidney, and pituitary. Its level of expression is highest in the heart and adrenal gland. In the rat brain (Figure 6) and rat heart (T. P. Snutch, unpublished data) the rbC gene encodes two large transcripts of approximately 8 and 12 kb. In the rat brain both of the transcripts are present from early in development through the adult stage (data not shown), suggesting that the rbC gene products mediate electrical activity in both developing and mature cells in the CNS. Currently, we do not know the relationship between the rbC-I and rbC-II cDNAs and the 8 and 12 kb transcripts detected in brain and heart RNAs. The demonstration that two brain Ca2+ channel isoforms are generated by alternative splicing raises the question of whether additional isoforms are also generated from the rbC gene. While we have detected onlythe rbC-I and rbC-II isoforms in rat brain, we have not performed a detailed DNA sequence analysis of all isolated brain cDNAs, and it is possible that additional rbC transcripts exist in the CNS as well as other tissues. We note that the PCR assay utilized here examined only the domain IV S3 segment and immediate

Procedures

Isolation of rbC cDNA Clones and DNA Sequencing The 5’ terminal 2.0 kb EcoRl fragment of rbC-61 (Snutch et al , 1990)was gel isolated, radiolabeled by random priming (Feinberg and Vogelstein, 1983), and used to screen 500,000 pfu of a rat brain cDNA library that was enriched for cDNAs greater than 4 kb in size (Snutch et al., 1990). Final purification resulted in an additional 18 positive i; clones. The insert cDNAs were excised from the b-Zap11 vector using the in viva excision protocol described by the supplier (Stratagene). Sequential deletions of the coding strand of rbC-61 and rbC-30 were constructed using exonuclease III and Sl nuclease (Henikoff, 1984). DNA sequencing utilized the modified dideoxy nucleotide protocol of Biggin et al. (1983) and Sequenase version 2.0 (United States Biochemical Corp.). After determination of the first strand sequence, a set ot oligonucleotide primers was constructed and used to determine the noncoding strand sequence. A second set of primers that represented the coding strand of rbC-I was synthesized, and both sets of primers were then used to sequence rbC-2019 and rbC-2040. Synthetic oligonucleotides were prepared using an Applied Biosystems Model 391 DNA svnthesizer. RNA Isolation and Northern Blot Analysis Total cellular RNA was isolated from adult male rat tissues bv two independent methods. Fresh tissue samples were homogenized in 5 M guanidinium thiocyanate followed by pelleting ot RNA through a CsCl stepgradient (Chirgwin et al., 1979). Alternatively, RNA was isolated by homogenization in 6 M urea, 3 M LiCl using a modification of the method of Auffray and Rougeon (1980). Rat brain regions were dissected according to Paxinos and Watson (1986). Total RNA was stored in aliquots in sterile water at

Differential 55

Calcium

Channel

Expression

-8OOC. Northern blot analysis was performed with total cellular RNA (30 pg per lane) as previously described (Snutch et al., 1990). R~~was electrophoresed through 1.1% agarose gels containing 1.1 M formaldehyde, transferred by capillary blot to Hybond-N nylon membrane (Amersham), and fixed by UV cross-linking. Hybridization was performed in 5x SSPE, 0.3% SDS, 2.5~ Denhardt’s solution, 0.2 mg/ml denatured salmon sperm DNA, 0.1 mg/ml yeast tRNA at 68OC. Blots were washed at 68’C in 0.2~ SSPE. For probes, gel-eluted cDNA fragments were radiolabeled by random priming (Feinberg and Vogelstein, 1983). DNA Isolation and Southern Blot Analysis Genomic DNA was isolated from rat liver by digestion with proteinase K and followed by equilibrium density gradient centrifugation in CsCl (Sambrook et al., 1989). DNA (12 pg) was digested with individual restriction enzymes, electrophoresed through 0.8% agarose gels, and transferred by capillary blot to a Nytran nylon membrane (Schleicher & Schuell). Probes specific for the S3 segments of domain IV of rbC-I and rbC-II cDNAs were prepared using synthetic oligonucleotides. For rbC-I, a sense 51 base oligonucleotide (5’~CCTCATCCT~~GCGAG~T~~GATCTCAnCTCACTGAAACTAAT-3’) and a 15 base complementary oligonucleotide (S-ATrAGmCACTCAC-3’) were prepared against the S3 segment of domain IV of rbC-I. For rbC-II, a 51 base sense oligonucleotide (S-CCClTGATTC~GTCCCTACCAlTGlTGATATAGCAATCACCGAGGTACAC-3’) and a 15 base complementary oligonucleotide (5’GTCTACCTCCCTGAT-3’) were prepared against the S3 segment of domain IV of rbC-Il. For each probe the complementary oligonucleotides (at a I:1 molar ratio of sense:antisense) were heated to 65OC for 5 min in 100 m M NaCI, 20 m M MgC12, 100 m M Tris (pH 8.0) and allowed to anneal by cooling to room temperature for 30 min. The DNA hybridswere then filled in using 3 U of Klenow enzyme, 50 PM dGTP, 50 PM TTP, 50 PCi of [a-32P]dCTP and [a-32P]dATP (3000 Cilmmol) for 30 min at 37OC and then chased with cold dCTP and dATP (50 PM) for 10 min. The reaction was stopped by centrifugation through a 1 ml spin column containing Sephadex G-50 equilibrated in TE buffer. Probes weredenatured by heating to 95OC and then hybridized to Southern blot filters in 5x SSPE, 0.3% SDS, 2.5x Denhardt’s solution, 0.2 mg/ml denatured salmon sperm DNA at 65Y. Blots were washed at 60°C in 0.2x SSPE. The rbC-l- and rbC-II-specific probes share 49% identity and do not cross-hybridize under the above conditions. Polymerase Chain Reaction Rat genomic DNA (250 ng) and cDNA controls (50 pg) were amplified with 1.25 U of Taq DNA polymerase (Perkin Elmer) in a 50 ~1 mix consisting of 10 m M Tris (pH 8.3), 3 m M MgC12, 50 m M KCI, 200 W M each deoxynucleotide triphosphate, and 0.3 FM upstream and downstream oligonucleotide primers. After 32 cycles of amplification (1 min at 9S°C, 30 sat 52Y, 1.5 min at 72OC), 10 pl of the reaction mixture was separated on a 1.2% agarose gel. For RNA analyses, first strand cDNA was synthesized from each RNA sample in a 10 ~1 mix consisting of 2 pg of total RNA, 10 m M Tris (pH 8.3), 5 m M MgCl*, 50 m M KCI, 1 m M each deoxynucleotide triphosphate, 1 U of RNAsin, 2.5 U of AMV reverse transcriptase, and 1.5 PM downstream oligonucleotide primer (#8; 5’AGGAC‘TTCATCAACCTCC-3’). After incubation for 60 min at 42OC, the entire 10 pl mix was amplified in a 50 ~1 reaction mix that included 0.3 PM upstream primer (#14, S-CTATCCTGTCCAGATCTC-3’) and downstream primer (#8) as described above (32 cycles). The reaction products were separated on a 1.2 % agarose gel, and the DNA was transferred to a Nytran nylon membranes (Schleicher & Schuell). For examination of differential expression, the blots were probed with the rbC-I and rbC-II domain IV S3-specific oligonucleotide probes described above. Results similar to those shown in Figure 7 were obtained when the the PCR amplification was performed for only 25 cycles (data not shown). As a control for PCR amplification, 1 pg of in vitro synthesized sense rbC-61 RNA and IO pg of synthetic sense rbC-2019 RNA were mixed, and first strand cDNAwas synthesized as described above. The mixture of rbC-I and rbC-II cDNAs was amplified for

22-36 cycles, and the products were analyzed by agarose gel electrophoresis followed by blotting and hybridization to the rbC-l- and rbC-II-specific probes. The ratio of rbC.-I and rbC-II products detected was proportional to the amount of starting sense RNA and was independent of amplification cycle number (22-36 cycles), indicating that no selective amplification of rbC-I and rbC-II RNAs occurred (data not shown). Hybrid Depletion and Oocyte Electrophysiology Xenopus oocytes were dissected from gravid adult females (XenopusOne,AnnArbor,MI).Surroundingovarian tissueandfollicular cells were removed by treatment with 2 mglml collagenase (type IA; Sigma) in OR-2 (82.5 m M NaCI, 2 m M KCI, 1 m M MgCI,, 5 m M HEPES [pH 7.51). Oocytes were microinjected with 60 nl of heart RNA (2 mg/ml total RNA) and incubated in standard oocyte saline (SOS: 100 m M NaCI, 2 m M KCI, 1.8 m M CaC&, 1 m M MgCI,, 5 m M HEPES [pH 7.61, supplemented with 2.S m M sodium pyruvate and 50 pglml gentamycin) for 2-4 days prior to recording. For hybrid depletion experiments, total rat heart RNA (4 mglml) was heated to 6S°C for 5 min, diluted I:1 with a solution containing 1.6pg/pl oligonucleotideand 100 m M NaCI, and incubated at 42OC for 10 min prior to injection into oocytes. The sequence of the rbC antisense oligonucleotide was STTCTCCTCTCCACTCATA-3’ (located in domain IV in the S3-S4 loop); the sequence of the rbC sense was S-TClTCACGGTGGACATCA-3’. Electrophysiological recording employed a standard two-microelectrode voltage clamp (Dagan 8500) and microelectrodes filled with 3 M KCI (resistances were 0.5-2.0 MO). In each oocyte, K’ current was first measured in SOS without supplements. The bath was then changed to the following composition: 40 m M BaCI,, 36 m M TEA, 5 m M haminopyridine, 2 m M KCI, 5 m M HEPES (pH 7.6), 150 PM niflumic acid. This bath allows measurement of the Ca*+ channel activity that is undetectable in SOS. The pulse protocol for the measurement of K+current consisted of a series of 35 ms, 10 mV step depolarizations (-70 to +40 mV) from a holding potential of -100 mV. K’ current was measured at +40 mV in SOS, a condition that produces negligible contamination by inward current. The protocol for measurement of Ba2+ current in Ba2+ saline consisted of 300 ms steps to -70, -60, -50, 0, +lO, and f20 mV from a holding potential of -80 mV. Stimulation and analysis programs utilized pClamp software (Axon Instruments). All currents were leak subtracted. Acknowledgments We are grateful to Steve Dubel and Ward Prystay for excellent technical assistance. We also thank Drs. Terry Starr, Donald Moerman, and Michael White for critical reading of the manuscript. We aregrateful to Dr. SteveVincent for help in dissection of the rat CNS for RNA preparation. This work was supported by grants from the Medical Research Council of Carlada and the B. C. Health Care Research Foundation (to T. P. S.) and by National Institutes of Health Grant NS-26432 (to J. P. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

January

16, 1991; revised

April

11, 1991.

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