- Email: [email protected]
The α1A subunits of rat brain calcium channels are developmentally regulated by alternative RNA splicing
PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 2 1 3 - 0
Neuroscience Vol. 113, No. 3, pp. 509^517, 2002 @ 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
THE K1A SUBUNITS OF RAT BRAIN CALCIUM CHANNELS ARE DEVELOPMENTALLY REGULATED BY ALTERNATIVE RNA SPLICING S. VIGUES,a1 M. GASTALDI,b1 A. MASSACRIER,b P. CAUb and J. VALMIERa a
Inserm U-432, Universite¤ Montpellier II, Place Eugene Bataillon, 34095 Montpellier Cedex 5, France b
Inserm U-491, Faculte¤ de Me¤decine Timone, 13385 Marseille Cedex 5, France
Abstract4Calcium in£ux through voltage-gated calcium channels governs important aspects of CNS development. Multiple alternative splicings of the pore-forming K1 subunits have been evidenced in adult brain but little information about their expression during ontogenesis is presently available. The aim of this study was to focus on the expression of three rat voltage-gated calcium channel K1A splice variants (K1Aa , K1Ab and K1AEFe ) during brain ontogenesis in vivo. Using a reverse transcription-polymerase chain reaction strategy, we found that the three isoforms have di¡erent timings of development throughout the brain: K1Ab is expressed from embryonic to the adult stage, K1AEFe is restricted to the embryonic period whereas K1Aa is expressed only postnatally. In situ hybridization indicated that K1Aa and K1Ab isoforms develop with di¡erent regional and cellular patterns. In hippocampus and cerebellum, K1Ab represented the predominant isoform at all developmental stages. Taken together, these data reveal that alternative RNA splicing may modulate the K1A calcium channel properties during development. @ 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: calcium current, ontogenesis, hippocampus, cerebellar neurons.
electrophysiological and pharmacological properties into T-, L-, N-, P/Q- and R-types (Tsien et al., 1991; Birnbaumer et al., 1994; Ertel et al., 2000). Low-threshold T-type Ca2þ currents are encoded by three di¡erent K1 subunits (K1G , K1H , K1I ) appearing not to require regulatory subunits for functional expression (PerezReyes, 1998). Data obtained from biochemical studies indicate that high-threshold Ca2þ currents are composed of at least three subunits: a pore-forming K1 subunit and the regulatory K2 N and L regulatory subunits (Hofmann et al., 1994; Dunlap et al., 1995). More recently molecular cloning has revealed at least six K1 (K1A K1F ), four L (L1-L4) and four N (N1-N4) gene products expressed in the nervous system (Perez-Reyes et al., 1994; BechHansen et al., 1998; Perez-Reyes, 1998). Although the identity of the channel formed after expression of K1E remains unclear (Soong et al., 1993; Zhang et al., 1993; Schneider et al., 1994; Williams et al., 1994; Bourinet et al., 1996), K1B is thought to encode N-type channels (Williams et al., 1992b; Zhang et al., 1993), and K1C , K1D and K1F are thought to be components of L-type channels (Hui et al., 1991; Singer et al., 1991; Snutch et al., 1991; Williams et al., 1992a; Bech-Hansen et al., 1998; Strom et al., 1998). Further diversity of VGCCs arises through alternative splicing of the K1 (and also L) transcripts (Hofmann et al., 1994; Dunlap et al., 1995). Based on pharmacology (Sather et al., 1993), structure-function experiments (Zhang et al., 1993) and subcellular localization (Wheeler et al., 1994), K1A subunit gene is thought to form the Aga IVA-sensitive P/Q-type
In neurons, calcium in£ux through voltage-gated calcium channels (VGCCs) orchestrates key functions, including neuronal excitability (Kennedy, 1989), neurotransmitter release (Mulkey and Zucker, 1991) and gene expression (Ghosh and Greenberg, 1995). Growing evidence indicates that, during nervous system development, voltagedependent calcium in£ux is also important in the control of cell proliferation and migration and neurite outgrowth (Kocsis et al., 1994) as well as di¡erentiation, synaptogenesis and natural cell death (Schmid and Guenther, 1999). A single embryonic neuron expresses a precise and complex pattern of VGCCs (Desmadryl et al., 1998) that di¡ers depending on the stage of development analyzed. It has also been shown that, during ontogenesis, calcium-dependent events initiated by VGCCs depend on the type of channel through which calcium enters the cell (Bading et al., 1993). The study of the types of VGCCs expressed during speci¢c developmental stages in a distinct neuronal subpopulation is important for an understanding of the role of VGCCs in the di¡erentiation of the nervous system in vivo. VGCCs have been initially classi¢ed based on their
1 Equal contribution. *Corresponding author. Tel. : +33-4-67144976; fax: +33-467143696. E-mail address: [email protected] (J. Valmier). Abbreviations : DG, dentate gyrus; ISH, in situ hybridization; P, postnatal day; RT-PCR, reverse transcription-polymerase chain reaction; VGCC, voltage-gated calcium channels.
509
NSC 5658 23-7-02
510
S. Vigues et al.
Ca2þ channel family that controls neurotransmitter release at the central synapses (Llinas, 1988; Mori et al., 1991; Starr et al., 1991; Sather et al., 1993; Stea et al., 1994; Dunlap et al., 1995). The K1A subunit gene encodes proteins with diverse structures and functions. Evidence from rabbit, rat, mouse and human K1A cDNAs suggest that this diversity is generated, at least in part, by alternative splicing in this single-copy gene (Mori et al., 1991; Starr et al., 1991; Fletcher et al., 1996; Ligon et al., 1998; Bourinet et al., 1999). In rat brain, two variants of K1A subunit (namely K1Aa and K1Ab ) have been well characterized. K1Ab contains a single valine insertion in the I^II linker, an insertion of two residues of asparagine and proline in the extracellular linker between subdomain IVS3 and IVS4 and 10 substituted residues in a stretch of 30 amino acids in the C-terminus domain. Of the 10 substituted residues, six are in the region highly similar to the divalent ion binding domain (EF-hand) of Ca2þ binding protein. The electrophysiological study of the two variants showed that K1Aa and K1Ab di¡er by their biophysical properties, their sensitivity to Aga IVA and their localization in the adult brain (Bourinet et al., 1999). The K1Aa expresses a P-type-like current whereas K1Ab expresses a current with biophysical properties and sensitivity to Aga IV that are close to the Q-type current characteristics. Recent data argue in favor of a role for K1A P/Q-type VGCCs in modulating neuronal ontogenesis. In situ hybridization (ISH) studies have revealed K1A mRNAs in pre- and postnatal brain undergoing active di¡erentiation (Tanaka et al., 1995). Interestingly, a new alternative spliced variant from the gene K1A (called K1AEFe ), which is a truncated channel protein lacking the C-terminus, has been described in the rat hippocampus, only during the embryonic period (Vigues et al., 1998). Many embryonic neurons express functional P/Q-type VGCCs as evidenced by electrophysiological studies (Diochot et al., 1995; Hivert et al., 1995) and their biophysical and pharmacological properties change depending on the stage of development of their expressing neurons (Hilaire et al., 1996). Recently, a role for this channel family has been evidenced in the developing rat hippocampus: Q-type VGCC, a splicing of the K1A subunit gene, regulates the di¡erentiation of a subpopulation of embryonic calbindin-positive neurons, by controlling Ca2þ in£ux (Boukhaddaoui et al., 2000; Boukhaddaoui et al., 2001). Despite the fact that all these data support a signi¢cant role for the K1A Ca2þ channel subunit family in the development of the nervous system, no data are available concerning the speci¢c neuronal expression of the di¡erent K1A isoforms during CNS ontogenesis in vivo. The present study, by using reverse transcription-polymerase chain reaction (RT-PCR) and ISH analysis, investigated the temporal and spatial pattern of expression of three K1A subunit isoforms (K1Aa , K1Ab and K1AEFe ) from embryonic to adult stages in rat brain. Our data suggest that developing neurons use alternative mRNA splicing to modulate the K1A calcium channel properties during development.
EXPERIMENTAL PROCEDURES
RNA isolation and RT-PCR analysis Sprague^Dawley rats (I¡a Credo, France) were used in this study at di¡erent stages of development (n = 4 for each stage). Their care and use conformed to institutional policies and guidelines. Rats were anaesthetized using pentobarbital (50 mg/kg) and killed by decapitation. At each stage of the development, cortex, cerebellum and hippocampus or whole brain were quickly dissected and frozen in liquid nitrogen. Total RNA was isolated using the TRIzol protocol (Gibco BRL, Rockville, MD, USA) derived from the Chomczynski and Sacchi method (Chomczynski and Sacchi, 1987) and treated with DNase (Gibco, 1.5 U, 15 min, 37‡C) to remove any residual genomic DNA. cDNA templates for ampli¢cation by PCR were synthesized by reverse transcription of total RNA (1 Wg) using Moloney’s murine leukemia virus reverse transcriptase (Superscript II, Gibco) with random hexanucleotides primers and dNTP for 50 min at 42‡C in the bu¡er (supplied by the manufacturer). Reverse transcriptase was omitted in control samples to ensure that ampli¢cation products were from mRNA and not from genomic DNA. Three PCRs were carried out using synthetic oligonucleotides as primers (purchased from Eurogentec). The ¢rst PCR strategy used the primers homologous to the rat calcium channel K1Aa (Genbank, sequence M64373) and K1Ab (unpublished observations) sequences to amplify the EF-hand domain localized on the carboxyl terminal region of the protein: Forward primer I was the same for the three PCR (5PTCCAAAAACCAGAGTGTG3P): position 5183 bp; Reverse primer II (5PTTGAAGTGAACGGTGTTG3P) was speci¢c for K1Aa and K1Ab variants: position 5534 bp. The second PCR used the reverse primer speci¢c to the rat calcium channel K1Aa to amplify a part of EF-hand domain: Reverse primer III (5PTATTACTCGCAATAAACTG3P): position 5410 bp. The third PCR used the reverse primer speci¢c to the rat calcium channel K1Ab to amplify a part of EF-hand domain: Reverse primer IV (5PCATGTGTCTCAGCATCTGA3P): position 5410 bp. In preliminary experiments, we have tested other K1A regions by RT-PCR to discriminate K1Aa and K1Ab isoforms (data not shown), located in the I^II linker (valine insertion) and in the subdomain IVS3^IVS4 (asparagine^proline insertion). The same pattern of expression of EF-hand domain has been observed during development. The cDNA mixture was supplemented with 2.5 U taq polymerase (Thermus aquaticus YT1 from Gibco), 25 pmol of each primer, 5 Wl 10U PCR bu¡er (100 mM Tris^HCl pH 8.4, 20 mM MgCl2 , 500 mM KCl), 200 WM dNTP mix and water to bring the ¢nal volume to 50 Wl. The reaction mixture was overlaid with 50 Wl mineral oil (Promega, Madison, WI, USA) and incubated in a thermal cycler (Eppendorf) for 30 cycles each of them consisting of 1 min denaturation at 94‡C, 1 min hybridization at 50‡C, and 45 s polymerization at 72‡C. After ampli¢cation, 10 Wl of PCR product was run on an ethidium bromidestained gel (1.5% agarose). K1AEFe , K1Aa and K1Ab PCR products were cloned into pGEM-T vector (Promega) and sequenced (ACT Gene) to con¢rm the PCR speci¢city. The PCR experiments (n = 4) for each stage of the development are reproducible and one representative experiment is illustrated for each tissue studied. Tissue and probe preparation ^ ISH Brains from Sprague^Dawley rats, adults (n = 3) and at postnatal days (P) 10 (n = 3), P7 (n = 3) and P0 (n = 3), were excised and immediately frozen into liquid nitrogen vapors. Frozen sections (12 Wm) were obtained using a cryostat (Microm, France): coronal (adult brains) or sagittal sections (postnatal brains) were collected onto silanized slides (Sigma, St. Louis, MO, USA) and ¢xed in 4% paraformaldehyde in 0.1 M phosphate bu¡er (pH 7.4) at room temperature for 30 min. Slides were rinsed in the
NSC 5658 23-7-02
Development of K1A calcium channels
511
Fig. 1. Primer location on the K1Aa C terminus domain. Three PCR were carried out using synthetic oligonucleotides as primers (purchased from Eurogentec). The ¢rst PCR strategy used the primers homologous to the rat calcium channel K1Aa (Genbank, sequence M64373) and K1Ab (unpublished) sequences to amplify the EF-hand domain localized on the carboxyl terminal region of the protein: Forward primer I was the same for the three PCR (TCCAAAAACCAGAGTGTG) at position 5183 bp; Reverse primer II (TTGAAGTGAACGGTGTTG) was speci¢c for K1Aa and K1Ab variants at position 5534 bp. The second PCR used the reverse primer speci¢c to the rat calcium channel K1Aa to amplify a part of EF-hand domain : Reverse primer III (TATTACTCGCAATAAACTG) at position 5410 bp. The third PCR used the reverse primer speci¢c to the rat calcium channel K1Ab to amplify a part of EF-hand domain: Reverse primer IV (CATGTGTCTCAGCATCTGA) at position 5410 bp.
same bu¡er, then dehydrated in graded ethanols and stored at 380‡C until use. Antisense and sense cRNA probes corresponded to the same fragment of speci¢c EF-hand sequences of K1Aa and K1Ab spliced variants (Bourinet et al., 1999). Speci¢c templates were obtained by RT-PCR during which reverse or direct primers, respectively, were 5P extended by a short sequence corresponding to the T7 RNA polymerase promoter (Young et al., 1991; Vigues et al., 1999). Probes were then synthesized and digoxigenin-labelled by in vitro transcription. ISH was performed according to our previously described procedure (Vigues et al., 1999) with a ¢nal probe concentration of 80 ng/ml in 20 Wl hybridization bu¡er. In each experiment, adult, P10, P7 and P0 samples were concomitantly processed together with negative controls. Negative controls included: (i) predigestion with RNase A before hybridization, (ii) hybridization using sense probes diluted at the concentration giving the same labelling signal intensity in dots as the antisense probes and (iii) hybridization without probes (control for non-speci¢c binding of antibodies). Triplicate experiments gave similar results. One representative experiment is illustrated.
shown), this variant has a 93 bp deletion corresponding to the region of speci¢city of K1Aa and K1Ab . The second PCR strategy (primer I^III) allowed us to speci¢cally amplify the K1Aa variant of 227 bp (Fig. 2b, e) showing that the K1Aa variant was present only during postnatal stages. The third PCR strategy (primer I^IV) (Fig. 2c, f) showed that K1Ab was detected during all stages of development studied. The same pattern of expression was observed in the total brain (data not shown). Figures 3 and 4 show the ISH detection of K1Aa and K1Ab mRNA variants in adult and postnatal brains. The speci¢city of ISH signal obtained with the K1Aa or with the K1Ab antisense probe was con¢rmed by the following controls: RNase pretreatment of tissue sections completely abolished the hybridization signals and sections hybridized with digoxigenin-labelled sense probes did not show any signal (not shown).
RESULTS
Localization of the two mRNA variants in selected areas of the developing CNS
We examined the pattern of expression of the three Ca2þ channel variants during embryonic and postnatal hippocampus development. We used three PCR strategies (Fig. 1) to amplify speci¢c cDNA fragments of the VGCC K1A subunit encoding a region of the channel starting from the domain IVS6 and going to the carboxyl terminal after the divalent ion binding-like domain (EF-hand) (Fig. 1). During all the periods of hippocampus development studied, K1AEF , a 351 bp fragment corresponding to the K1A mRNA was ampli¢ed with the ¢rst PCR strategy (Fig. 2a, d). Only during embryonic stages (Fig. 2a, d) a 257 bp fragment corresponding to K1AEFe was also ampli¢ed and sequenced (data not
The detection of the two mRNA variants in di¡erent developing brain structures is shown in Table 1. At P0, K1Aa variant was not expressed. By P7, K1Aa expression was observed in cerebral cortex, hippocampus, cerebellum, as well as in some large hypothalamic and medulla oblongata neurons. The labelling signal remained thereafter. The thalamus and all regions anterior to the hippocampus did not express K1Aa variant before P10. No signal could be detected in glial cells from the corpus callosum throughout the development of the brain. K1Ab expression was present as early as P0 in cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus, striatum and medulla oblongata. Ependy-
NSC 5658 23-7-02
512
S. Vigues et al.
Fig. 2. In vivo developmental regulation of K1Aa , K1Ab , K1AEF and K1AEFe transcripts in hippocampus (a, b, c) and cerebellum (d, e, f) investigated by PCR on 1.5% agarose gel. The presence of variants is studied at di¡erent stages: In cerebellum at embryonic day (E) 15, E17, E19, P1, P7, P14, 2 months (2M). In hippocampus at E17, E18, E19, E20, E21, P0 (birth), P1, P2, P7, P14 and 2M. K1Ab (c, f) and K1AEF (a, d) PCR products are present respectively at 227 bp and 351 bp whatever the developmental stages studied. K1Aa (b, e) PCR product is present at 227 bp only after birth and K1AEFe PCR product is present at 257 bp only during embryonic development. The same developmental pattern is observed in hippocampus and cerebellum. Marker (Promega) sizes: 1000, 750, 500, 300, 150, 50 bp. Table 1. ISH detection of K1Aa and K1Ab mRNA variants in di¡erent areas of the developing rat CNS P0
Cerebral cortex Hippocampus Thalamus Hypothalamus Striatum Cerebellum Medulla oblongata Ependymal cells
P7
P10
Adult
1A-a
1A-b
1A-a
1A-b
1A-a
1A-b
1A-a
1A-b
3 3 3 3 3 3 3 3
+ + + + + + + T
+ + 3 + 3 + + 3
+ + + + + + + T
+ + + + + + + 3
+ + + + + + + T
+ + + + + + + 3
+ + + + + + + +
3: Absence of labelling detected. +: Labelling signal detected. T : Labelling signal occasionally detected.
NSC 5658 23-7-02
Development of K1A calcium channels
513
Fig. 3. ISH detection of mRNAs encoding K1Aa (left panel, a, c, e) and K1Ab (right panel, b, d, f) in cerebellum from P7 (a, b), P10 (c, d) and adult brains (e, f). The K1Aa variant appears at P7 in Purkinje cells as well as in the external granular layer and at P10 in the internal granular layer while in adult brains it is only detected in Purkinje cells and in some Golgi type II cells (arrowhead). The K1Ab variant is present in Purkinje cells and in both external and internal layers at P7 and P10 while only present in Purkinje cells, molecular layer and some Golgi type II cells (arrowhead) in adult brain. Pu, Purkinje cells, EGL, external granular layer, IGL, internal granular layer, ML, molecular layer, GL, granular layer. Scale bar = 125 Wm.
mal cells were occasionally labelled. The hippocampus was one of those areas which showed the most intense labelling signal in the whole brain. This labelling pattern was observed throughout the di¡erent stages of development studied. It is noteworthy that in all developmental periods and concerning both mRNA variants, the labelling of striatum, if present, was faint when compared to other CNS structures. Taking the developing cerebral cortex as an example, the K1Aa variant was not detected at P0 while the K1Ab variant was observed in the cortical plate and in early-born large pyramidal cells. By P7 all cortical layers expressed the K1Ab variant and the K1Aa variant by P10 (data not shown). Cellular localization of the two mRNA variants in the developing cerebellum (Fig. 3) Using the K1Aa probe, no labelling could be detected at P0 but a weak signal appeared in Purkinje cells and in the external granular layer by P7. At P10, the K1Aa variant was also present in the internal granular layer. In adult rats, the signal was detected only in Purkinje cells and in sparse Golgi type II cells. Conversely, the K1Ab variant was faintly detected in the developing P0 cerebellum. At P7, labelling by the K1Ab probe was high
in Purkinje cells, while it remained moderate and homogeneous in both external and internal granular layers. At P10, the K1Ab isoform was highly detected in Purkinje cells and in some Golgi type II cells while labelling was faint in both granular layers. In adult rats, K1Ab variant was present in Purkinje cells, in the molecular layer and in sparse Golgi type II cells but disappeared from granular cells. It is noteworthy that both K1Aa and K1Ab probes did not produce any labelling in granular cells from adult cerebellum. This observation is in contrast with previous reports (Bourinet et al., 1999) which, using radiolabelled oligoprobes, detected both variants in these cells. A di¡erent ISH sensitivity may account for this discrepancy, inasmuch as radiolabelled probes are known to be more sensitive. Cellular localization of the two mRNA variants in the developing hippocampus (Fig. 4) K1Aa mRNAs were not observed until P7. At this stage, a weak labelling signal was present in pyramidal cells of the Ammon’s horn from CA1 to CA4 but not in granule cells of the dentate gyrus (DG). A weak labelling was apparent in DG granule cells in P10 and adult rats. In addition, in adult brains, granule cells located at the
NSC 5658 23-7-02
514
S. Vigues et al.
Fig. 4. ISH detection of mRNAs encoding K1Aa (left panel, a, c, e, g) and K1Ab (right panel, b, d, f, h) in hippocampus from P0 (a, b), P7 (c, d), P10 (e, f) and adult brains (g, h). The K1Aa variant appears at P7 in pyramidal cells and at P10 in DG granule cells. The K1Ab variant is detected as soon as P0 in both pyramidal cells and DG granule cells. Note in d the labelling of the DG is higher in cells located along the outer border of the layer (arrowhead) and the di¡erence in labelling intensity seen in b, d, f, between pyramidal cells and DG granule cells. Py, pyramidal cells of Ammon’s horn. Scale bar = 250 Wm.
inner border of the DG were particularly labelled and a labelling of interneurons of the CA4 area could be evidenced. Unlike K1Aa , the K1Ab isoform was present in hippocampus as soon as P0. The developing DG was uniformly labelled whereas pyramidal cells were heterogeneously labelled. Throughout all stages of development, the signal detected was higher in pyramidal cells than in granule cells, a discrepancy that was strongly attenuated in adult brains. At P7, labelling of the DG was higher in cells located along the outer border of the layer. Besides this, the pyramidal cells of Ammon’s horn were highly and homogeneously labelled. On sagittal sections, the labelling signal increased from CA1 to CA3 pyramidal cells (a pattern initiated as soon as P0). In addition, a high signal was also observed in CA4 interneurons. By P10, labelling was present in all pyramidal
and DG granule cells. In adult brains, DG granule cells were highly labelled, particularly those located at the inner border of the gyrus.
DISCUSSION
The present study focuses on the developmental expression of three naturally occurring splice variants of the K1A subunit gene (K1Aa , K1Ab and K1Ae ) that di¡er in nucleotide sequences corresponding to the EF-hand domain of the C-terminus. Alternative splicing of the K1A gene results in the expression of multiple VGCC phenotypes (Krovetz et al., 2000). In this regard, the C-terminus region of K1A subunit, between the K1A IVS3^IVS4 linker and the terminal stop codon, includes
NSC 5658 23-7-02
Development of K1A calcium channels
multiple important structural domains regulating functional properties of the channel. The C-terminus region of the human K1A gene is encoded by 12 exons (numbered 36^47), of which four exons (37, 44, 46 and 47) undergo alternative splicings. Of the 16 possible combinations, nine of them have been isolated previously. Each combination creates a channel with di¡erent properties. For example, the inclusion of exon 44 a¡ects the rate of inactivation; exon 37b gives new activation and inactivation properties to the channel and changes the a⁄nity for Aga IV by inserting two amino acids (NP) in the K1A IVS3-IVS4 linker. The C-terminus region is also essential for modulation by G proteins (Kinoshita et al., 2001) (exon 45^46) or the L4 subunit (exon 43^47) that enhances the rate of inactivation (Walker and De Waard, 1998). Polyglutamine tract expansions in the exon 47 increase current density and alter activation and inactivation properties, leading to a variety of human neurological diseases (Lehmann-Horn and Jurkat-Rott, 1999). Functional expression and localization studies in the adult brain indicate that this diversity generated by alternative splicing may be important for ¢ne tuning neuronal function under physiological and pathological conditions. The present results suggest that an alternative splicing mechanism may also contribute to the heterogeneous properties of K1A subunits during neuronal development. RT-PCR studies have de¢ned that, from embryonic stages to adulthood, each K1A isoform develops with speci¢c and restricted timings of expression. For example, K1AEFe is restricted to the embryonic period and K1Aa is expressed only postnatally, whatever the region analyzed. Speci¢c expression of voltage-gated channel alternative splicing during embryonic and early postnatal period has been previously shown for the pore-forming K II and III subunit of sodium channels (Waxman et al., 1994). ISH analysis indicates that, in addition to a di¡erence in the temporal expression of the three di¡erent K1A isoforms during brain ontogenesis, each neuronal subpopulation exhibited a pattern of expression that di¡ers depending on the stage of development. For example, cerebellar Purkinje cells express dominant K1Ab gene subunit during perinatal period whereas K1Aa gene subunit becomes the predominant isoform in adult. By contrast, hippocampal pyramidal neurons express K1Ab as the major isoform throughout its development. Nevertheless, independently of their di¡erent cellular localization, the timing of expression of K1Aa and K1Ab have some common features whatever the brain structure studied: K1Ab is present during embryonic period (until adulthood) and K1Aa is only postnatally expressed. During the ¢rst postnatal week, the latter variant is either not expressed (i.e. granule cells of DG) or when expressed, levels are weak compared to K1Ab . Increment in K1Aa expression parallels cell maturation, especially synaptogenesis as shown for the Ammon’s horn where synaptogenesis and K1Aa expression are concomitant (Jacobson and Sapolsky, 1991). These data suggest that each isoform is involved in speci¢c modulatory functions during development. For the moment, the K1AEFe variant has not been electrophysiologically characterized and its role remains
515
unknown but some K1A mutations mimic the truncation of the K1AEFe variant. For example, tgla (short) mutation (Fletcher et al., 1996) that lacks the C-terminal domain or the type-2 episodic ataxia (Jen, 1999), which has a stop codon in domain IV, induces neurological disorders resulting from signi¢cant modi¢cations in current density and gating behavior (Wakamori et al., 1998). From previous structure-function experiments using mutations of other K1 subunits C-terminus, we can suggest that the K1AEFe variant has speci¢c biophysical and pharmacological properties in comparison with other isoforms (De Leon et al., 1995). However if K1AEFe variant is not a functional channel, it can modulate the activity of the full-length K1A proteins as a decoy (Colotta et al., 1993) or functions as a voltage sensor with its four S4 segments always present. These results show that K1AEFe may have an important role in early brain development by interacting with the variants K1Aa . The cloning of K1AEFe and its expression in oocytes or mammalian cells should be an e¡ective way to test these hypotheses. Before synaptogenesis, neuronal development proceeds through a sequence of events including proliferation of neuronal precursor cells, migration of neuroblasts, neurite extensions and phenotypic di¡erentiation. Present results indicate a role for the K1Ab gene subunit during this early developmental period, since K1Ab mRNAs are expressed during all these developmental stages. In certain areas, K1Ab appears to be mainly present when cells are undergoing proliferation and migration. For instance, this variant is highly expressed in the cortical plate, a proliferative zone of the developing cortex. Moreover, it is noteworthy that in DG, where neuron proliferation persists into adulthood, K1Ab expression remains until this age. The majority of DG granule cells (approximately 80%) are formed postnatally and the cytoarchitecture of the gyrus is not recognizable until near the end of the ¢rst postnatal week (Schlessinger et al., 1975). During the ¢rst two postnatal weeks and in adult brain, although at a slower rate, dentate granule progenitor cells divide at the border of the hilus and migrate to reach their ¢nal position in the granule cell layer (Schlessinger et al., 1975; Rickmann et al., 1987; Cameron et al., 1993; Kuhn et al., 1996). Surprisingly, the expression of K1Ab ¢rst appears along the outer border of the granule cell layer (facing CA1) and extends to the whole layer thereafter. Expression of this variant thus develops in an opposite direction to that taken by the migrating granule cells. It is unlikely that neurons in the hilus do not express K1Ab mRNA before they migrate since, in the developing hippocampus, the labelling that we observed in the hilar region consistently appears to be more intense than in the DG. A delayed migration of the cells that express K1Ab could account for this late appearance of K1Ab mRNA-containing cells. An alternative possibility could be that neurons reach their position at early ages and delay their expression of K1Ab . A birthdating study would be necessary to clarify this point. The possible role of the K1A isoform expression in the development of the hippocampal pyramidal neurons is especially signi¢cant. The K1Ab but not the K1Aa gene
NSC 5658 23-7-02
516
S. Vigues et al.
subunit is present at the time when hippocampal neuroblasts migrate, settle, elaborate axons and dendrites and di¡erentiate. Functional Q-type VGCC have been evidenced during this period and described as a modulator of the acquisition of the calbindin phenotype expressed by an hippocampal neuron subpopulation (Boukhaddaoui et al., 2000). These results suggest that Q-type channels, which have been predominantly associated with neurotransmitter release in adult brain, transiently act to direct early neuronal di¡erentiation of hippocampal neurons before the establishment of their synaptic circuits. Then synaptogenesis begins and develops during the ¢rst weeks after birth, when neurotransmission is ¢rst dominated by N-type VGCC, the role of which declines as their task becomes shared by P/Q-type VGCCs. Remarkably, this phase coincides with the initial detection of the K1Aa P-type gene mRNA subunit whose expression increases during the two postnatal weeks concomitant with a migration of Q-type VGCC out of the hippocampal neuronal somata (probably towards the neurites). Thus, each isoform develops in the same time window as several physiological correlates
of hippocampal maturation, suggesting that neurons use K1A alternative splicing to ¢ne tune Ca2þ in£ux according to their developmental requirements. Activation of voltage-gated Ca2þ channels is thought to be one of the major molecular mechanisms by which calcium in£ux regulates CNS ontogenesis (Ghosh and Greenberg, 1995; Gu and Spitzer, 1995). However, the characterization of the increasingly numerous Ca channel subtypes in the developing brain as well as their pattern of expression are not yet well resolved. Using RT-PCR and ISH analysis, the present results have de¢ned the spatio-temporal expression of three alternative splicing of the K1A gene (namely K1Aa , K1Ab and K1Ae ) during rat brain development in vivo. From embryonic stages to adulthood, each K1A isoform develops with a speci¢c timing and di¡erent regional and cellular patterns. Therefore, our data support a signi¢cant role for the K1A gene alternative splicing mechanism during ontogenesis as a tool for a developing neuron to select Ca2þ channels with properties that are appropriate for its speci¢c needs in Ca2þ in£ux depending on its stage of development.
REFERENCES
Bading, H., Ginty, D.D., Greenberg, M.E., 1993. Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science 260, 181^186. Bech-Hansen, N.T., Naylor, M.J., Maybaum, T.A., Pearce, W.G., Koop, B., Fishman, G.A., Mets, M., Musarella, M.A., Boycott, K.M., 1998. Loss-of-function mutations in a calcium-channel alpha1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness. Nat. Genet. 19, 264^267. Birnbaumer, L., Campbell, K.P., Catterall, W.A., Harpold, M.M., Hofmann, F., Horne, W.A., Mori, Y., Schwartz, A., Snutch, T.P., Tanabe, T., 1994. The naming of voltage-gated calcium channels. Neuron 13, 505^506. Boukhaddaoui, H., Sieso, V., Scamps, F., Valmier, J., 2001. An activity-dependent neurotrophin-3 autocrine loop regulates the phenotype of developing hippocampal pyramidal neurons before target contact. J. Neurosci. 21, 8789^8797. Boukhaddaoui, H., Sieso, V., Scamps, F., Vigues, S., Roig, A., Valmier, J., 2000. Q- and L-type calcium channels control the development of calbindin phenotype in hippocampal pyramidal neurons in vitro. Eur. J. Neurosci. 12, 2068^2078. Bourinet, E., Soong, T.W., Sutton, K., Slaymaker, S., Mathews, E., Monteil, A., Zamponi, G.W., Nargeot, J., Snutch, T.P., 1999. Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels. Nat. Neurosci. 2, 407^415. Bourinet, E., Zamponi, G.W., Stea, A., Soong, T.W., Lewis, B.A., Jones, L.P., Yue, D.T., Snutch, T.P., 1996. The alpha 1E calcium channel exhibits permeation properties similar to low-voltage-activated calcium channels. J. Neurosci. 16, 4983^4993. Cameron, H.A., Woolley, C.S., McEwen, B.S., Gould, E., 1993. Di¡erentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56, 337^344. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156^159. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., Giri, J.G., Dower, S.K., Sims, J.E., Mantovani, A., 1993. Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4. Science 261, 472^475. De Leon, M., Wang, Y., Jones, L., Perez-Reyes, E., Wei, X., Soong, T.W., Snutch, T.P., Yue, D.T., 1995. Essential Ca(2+)-binding motif for Ca(2+)-sensitive inactivation of L-type Ca2þ channels. Science 270, 1502^1506. Desmadryl, G., Hilaire, C., Vigues, S., Diochot, S., Valmier, J., 1998. Developmental regulation of T-, N- and L-type calcium currents in mouse embryonic sensory neurones. Eur. J. Neurosci. 10, 545^552. Diochot, S., Richard, S., Valmier, J., 1995. Diversity of voltage-gated calcium currents in large diameter embryonic mouse sensory neurons. Neuroscience 69, 627^641. Dunlap, K., Luebke, J.I., Turner, T.J., 1995. Exocytotic Ca2þ channels in mammalian central neurons. Trends Neurosci. 18, 89^98. Ertel, E.A., Campbell, K.P., Harpold, M.M., Hofmann, F., Mori, Y., Perez-Reyes, E., Schwartz, A., Snutch, T.P., Tanabe, T., Birnbaumer, L., Tsien, R.W., Catterall, W.A., 2000. Nomenclature of voltage-gated calcium channels. Neuron 25, 533^535. Fletcher, C.F., Lutz, C.M., O’Sullivan, T.N., Shaughnessy, J.D., Jr., Hawkes, R., Frankel, W.N., Copeland, N.G., Jenkins, N.A., 1996. Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell 87, 607^617. Ghosh, A., Greenberg, M.E., 1995. Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268, 239^247. Gu, X., Spitzer, N.C., 1995. Distinct aspects of neuronal di¡erentiation encoded by frequency of spontaneous Ca2þ transients. Nature 375, 784^ 787. Hilaire, C., Diochot, S., Desmadryl, G., Baldy-Moulinier, M., Richard, S., Valmier, J., 1996. Opposite developmental regulation of P- and Q-type calcium currents during ontogenesis of large diameter mouse sensory neurons. Neuroscience 75, 1219^1229. Hivert, B., Bouhanna, S., Diochot, S., Camu, W., Dayanithi, G., Henderson, C.E., Valmier, J., 1995. Embryonic rat motoneurons express a functional P-type voltage-dependent calcium channel. Int. J. Dev. Neurosci. 13, 429^436. Hofmann, F., Biel, M., Flockerzi, V., 1994. Molecular basis for Ca2þ channel diversity. Annu. Rev. Neurosci. 17, 399^418. Hui, A., Ellinor, P.T., Krizanova, O., Wang, J.J., Diebold, R.J., Schwartz, A., 1991. Molecular cloning of multiple subtypes of a novel rat brain isoform of the alpha 1 subunit of the voltage-dependent calcium channel. Neuron 7, 35^44. Jacobson, L., Sapolsky, R., 1991. The role of the hippocampus in feedback regulation of the hypothalamic-pituitary-adrenocortical axis. Endocr. Rev. 12, 118^134.
NSC 5658 23-7-02
Development of K1A calcium channels
517
Jen, J., 1999. Calcium channelopathies in the central nervous system. Curr. Opin. Neurobiol. 9, 274^280. Kennedy, M.B., 1989. Regulation of neuronal function by calcium. Trends Neurosci. 12, 417^420. Kinoshita, M., Nukada, T., Asano, T., Mori, Y., Akaike, A., Satoh, M., Kaneko, S., 2001. Binding of G alpha(o) N terminus is responsible for the voltage-resistant inhibition of alpha(1A) (P/Q-type, Ca(v)2.1) Ca(2+) channels. J. Biol. Chem. 276, 28731^28738. Kocsis, J.D., Rand, M.N., Lankford, K.L., Waxman, S.G., 1994. Intracellular calcium mobilization and neurite outgrowth in mammalian neurons. J. Neurobiol. 25, 252^264. Krovetz, H.S., Helton, T.D., Crews, A.L., Horne, W.A., 2000. C-Terminal alternative splicing changes the gating properties of a human spinal cord calcium channel alpha 1A subunit. J. Neurosci. 20, 7564^7570. Kuhn, H.G., Dickinson-Anson, H., Gage, F.H., 1996. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J. Neurosci. 16, 2027^2033. Lehmann-Horn, F., Jurkat-Rott, K., 1999. Voltage-gated ion channels and hereditary disease. Physiol. Rev. 79, 1317^1372. Ligon, B., Boyd, A.E., Dunlap, K., 1998. Class A calcium channel variants in pancreatic islets and their role in insulin secretion. J. Biol. Chem. 273, 13905^13911. Llinas, R.R., 1988. The intrinsic electrophysiological properties of mammalian neurons : insights into central nervous system function. Science 242, 1654^1664. Mori, Y., Friedrich, T., Kim, M.S., Mikami, A., Nakai, J., Ruth, P., Bosse, E., Hofmann, F., Flockerzi, V., Furuichi, T., 1991. Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature 350, 398^402. Mulkey, R.M., Zucker, R.S., 1991. Action potentials must admit calcium to evoke transmitter release. Nature 350, 153^155. Perez-Reyes, E., 1998. Molecular characterization of a novel family of low voltage-activated, T-type, calcium channels. J. Bioenerg. Biomembr. 30, 313^318. Perez-Reyes, E., Yuan, W., Wei, X., Bers, D.M., 1994. Regulation of the cloned L-type cardiac calcium channel by cyclic-AMP-dependent protein kinase. FEBS Lett. 342, 119^123. Rickmann, M., Amaral, D.G., Cowan, W.M., 1987. Organization of radial glial cells during the development of the rat dentate gyrus. J. Comp. Neurol. 264, 449^479. Sather, W.A., Tanabe, T., Zhang, J.F., Mori, Y., Adams, M.E., Tsien, R.W., 1993. Distinctive biophysical and pharmacological properties of class A (BI) calcium channel alpha 1 subunits. Neuron 11, 291^303. Schlessinger, A.R., Cowan, W.M., Gottlieb, D.I., 1975. An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat. J. Comp. Neurol. 159, 149^175. Schmid, S., Guenther, E., 1999. Voltage-activated calcium currents in rat retinal ganglion cells in situ: changes during prenatal and postnatal development. J. Neurosci. 19, 3486^3494. Schneider, T., Wei, X., Olcese, R., Costantin, J.L., Neely, A., Palade, P., Perez-Reyes, E., Qin, N., Zhou, J., Crawford, G.D., 1994. Molecular analysis and functional expression of the human type E neuronal Ca2þ channel alpha 1 subunit. Recept. Channels 2, 255^270. Singer, D., Biel, M., Lotan, I., Flockerzi, V., Hofmann, F., Dascal, N., 1991. The roles of the subunits in the function of the calcium channel. Science 253, 1553^1557. Snutch, T.P., Tomlinson, W.J., Leonard, J.P., Gilbert, M.M., 1991. Distinct calcium channels are generated by alternative splicing and are di¡erentially expressed in the mammalian CNS. Neuron 7, 45^57. Soong, T.W., Stea, A., Hodson, C.D., Dubel, S.J., Vincent, S.R., Snutch, T.P., 1993. Structure and functional expression of a member of the low voltage-activated calcium channel family. Science 260, 1133^1136. Starr, T.V., Prystay, W., Snutch, T.P., 1991. Primary structure of a calcium channel that is highly expressed in the rat cerebellum. Proc. Natl. Acad. Sci. USA 88, 5621^5625. Stea, A., Tomlinson, W.J., Soong, T.W., Bourinet, E., Dubel, S.J., Vincent, S.R., Snutch, T.P., 1994. Localization and functional properties of a rat brain alpha 1A calcium channel re£ect similarities to neuronal Q- and P-type channels. Proc. Natl. Acad. Sci. USA 91, 10576^10580. Strom, T.M., Nyakatura, G., Apfelstedt-Sylla, E., Hellebrand, H., Lorenz, B., Weber, B.H., Wutz, K., Gutwillinger, N., Ruther, K., Drescher, B., Sauer, C., Zrenner, E., Meitinger, T., Rosenthal, A., Meindl, A., 1998. An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness. Nat. Genet. 19, 260^263. Tanaka, O., Sakagami, H., Kondo, H., 1995. Localization of mRNAs of voltage-dependent Ca(2+)-channels: four subtypes of alpha 1- and betasubunits in developing and mature rat brain. Brain Res. Mol. Brain Res. 30, 1^16. Tsien, R.W., Ellinor, P.T., Horn, W.A., 1991. Molecular diversity of voltage-dependent Ca2þ channels. Trends Pharmacol. Sci. 12, 349^354. Vigues, S., Chabret, C., Valentin, S., Valmier, J., 1998. Rat embryonic hippocampal neurons express a new class A calcium channel variant. Neurosci. Lett. 258, 37^40. Vigues, S., Gastaldi, M., Chabret, C., Massacrier, A., Cau, P., Valmier, J., 1999. Regulation of calcium channel alpha(1A) subunit splice variant mRNAs in kainate-induced temporal lobe epilepsy. Neurobiol. Dis. 6, 288^301. Wakamori, M., Yamazaki, K., Matsunodaira, H., Teramoto, T., Tanaka, I., Niidome, T., Sawada, K., Nishizawa, Y., Sekiguchi, N., Mori, E., Mori, Y., Imoto, K., 1998. Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel. J. Biol. Chem. 273, 34857^34867. Walker, D., De Waard, M., 1998. Subunit interaction sites in voltage-dependent Ca2þ channels : role in channel function. Trends Neurosci. 21, 148^154. Waxman, S.G., Kocsis, J.D., Black, J.A., 1994. Type III sodium channel mRNA is expressed in embryonic but not adult spinal sensory neurons, and is reexpressed following axotomy. J. Neurophysiol. 72, 466^470. Wheeler, D.B., Randall, A., Tsien, R.W., 1994. Roles of N-type and Q-type Ca2þ channels in supporting hippocampal synaptic transmission. Science 264, 107^111. Williams, M.E., Brust, P.F., Feldman, D.H., Patthi, S., Simerson, S., Marou¢, A., McCue, A.F., Velicelebi, G., Ellis, S.B., Harpold, M.M., 1992b. Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel. Science 257, 389^395. Williams, M.E., Feldman, D.H., McCue, A.F., Brenner, R., Velicelebi, G., Ellis, S.B., Harpold, M.M., 1992a. Structure and functional expression of alpha 1, alpha 2, and beta subunits of a novel human neuronal calcium channel subtype. Neuron 8, 71^84. Williams, M.E., Marubio, L.M., Deal, C.R., Hans, M., Brust, P.F., Philipson, L.H., Miller, R.J., Johnson, E.C., Harpold, M.M., Ellis, S.B., 1994. Structure and functional characterization of neuronal alpha 1E calcium channel subtypes. J. Biol. Chem. 269, 22347^22357. Young, I.D., Ailles, L., Deugau, K., Kisilevsky, R., 1991. Transcription of cRNA for in situ hybridization from polymerase chain reactionampli¢ed DNA. Lab. Invest. 64, 709^712. Zhang, J.F., Randall, A.D., Ellinor, P.T., Horne, W.A., Sather, W.A., Tanabe, T., Schwarz, T.L., Tsien, R.W., 1993. Distinctive pharmacology and kinetics of cloned neuronal Ca2þ channels and their possible counterparts in mammalian CNS neurons. Neuropharmacology 32, 1075^ 1088. (Accepted 12 April 2002)
NSC 5658 23-7-02
Neuroscience Vol. 113, No. 3, pp. 509^517, 2002 @ 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
THE K1A SUBUNITS OF RAT BRAIN CALCIUM CHANNELS ARE DEVELOPMENTALLY REGULATED BY ALTERNATIVE RNA SPLICING S. VIGUES,a1 M. GASTALDI,b1 A. MASSACRIER,b P. CAUb and J. VALMIERa a
Inserm U-432, Universite¤ Montpellier II, Place Eugene Bataillon, 34095 Montpellier Cedex 5, France b
Inserm U-491, Faculte¤ de Me¤decine Timone, 13385 Marseille Cedex 5, France
Abstract4Calcium in£ux through voltage-gated calcium channels governs important aspects of CNS development. Multiple alternative splicings of the pore-forming K1 subunits have been evidenced in adult brain but little information about their expression during ontogenesis is presently available. The aim of this study was to focus on the expression of three rat voltage-gated calcium channel K1A splice variants (K1Aa , K1Ab and K1AEFe ) during brain ontogenesis in vivo. Using a reverse transcription-polymerase chain reaction strategy, we found that the three isoforms have di¡erent timings of development throughout the brain: K1Ab is expressed from embryonic to the adult stage, K1AEFe is restricted to the embryonic period whereas K1Aa is expressed only postnatally. In situ hybridization indicated that K1Aa and K1Ab isoforms develop with di¡erent regional and cellular patterns. In hippocampus and cerebellum, K1Ab represented the predominant isoform at all developmental stages. Taken together, these data reveal that alternative RNA splicing may modulate the K1A calcium channel properties during development. @ 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: calcium current, ontogenesis, hippocampus, cerebellar neurons.
electrophysiological and pharmacological properties into T-, L-, N-, P/Q- and R-types (Tsien et al., 1991; Birnbaumer et al., 1994; Ertel et al., 2000). Low-threshold T-type Ca2þ currents are encoded by three di¡erent K1 subunits (K1G , K1H , K1I ) appearing not to require regulatory subunits for functional expression (PerezReyes, 1998). Data obtained from biochemical studies indicate that high-threshold Ca2þ currents are composed of at least three subunits: a pore-forming K1 subunit and the regulatory K2 N and L regulatory subunits (Hofmann et al., 1994; Dunlap et al., 1995). More recently molecular cloning has revealed at least six K1 (K1A K1F ), four L (L1-L4) and four N (N1-N4) gene products expressed in the nervous system (Perez-Reyes et al., 1994; BechHansen et al., 1998; Perez-Reyes, 1998). Although the identity of the channel formed after expression of K1E remains unclear (Soong et al., 1993; Zhang et al., 1993; Schneider et al., 1994; Williams et al., 1994; Bourinet et al., 1996), K1B is thought to encode N-type channels (Williams et al., 1992b; Zhang et al., 1993), and K1C , K1D and K1F are thought to be components of L-type channels (Hui et al., 1991; Singer et al., 1991; Snutch et al., 1991; Williams et al., 1992a; Bech-Hansen et al., 1998; Strom et al., 1998). Further diversity of VGCCs arises through alternative splicing of the K1 (and also L) transcripts (Hofmann et al., 1994; Dunlap et al., 1995). Based on pharmacology (Sather et al., 1993), structure-function experiments (Zhang et al., 1993) and subcellular localization (Wheeler et al., 1994), K1A subunit gene is thought to form the Aga IVA-sensitive P/Q-type
In neurons, calcium in£ux through voltage-gated calcium channels (VGCCs) orchestrates key functions, including neuronal excitability (Kennedy, 1989), neurotransmitter release (Mulkey and Zucker, 1991) and gene expression (Ghosh and Greenberg, 1995). Growing evidence indicates that, during nervous system development, voltagedependent calcium in£ux is also important in the control of cell proliferation and migration and neurite outgrowth (Kocsis et al., 1994) as well as di¡erentiation, synaptogenesis and natural cell death (Schmid and Guenther, 1999). A single embryonic neuron expresses a precise and complex pattern of VGCCs (Desmadryl et al., 1998) that di¡ers depending on the stage of development analyzed. It has also been shown that, during ontogenesis, calcium-dependent events initiated by VGCCs depend on the type of channel through which calcium enters the cell (Bading et al., 1993). The study of the types of VGCCs expressed during speci¢c developmental stages in a distinct neuronal subpopulation is important for an understanding of the role of VGCCs in the di¡erentiation of the nervous system in vivo. VGCCs have been initially classi¢ed based on their
1 Equal contribution. *Corresponding author. Tel. : +33-4-67144976; fax: +33-467143696. E-mail address: [email protected] (J. Valmier). Abbreviations : DG, dentate gyrus; ISH, in situ hybridization; P, postnatal day; RT-PCR, reverse transcription-polymerase chain reaction; VGCC, voltage-gated calcium channels.
509
NSC 5658 23-7-02
510
S. Vigues et al.
Ca2þ channel family that controls neurotransmitter release at the central synapses (Llinas, 1988; Mori et al., 1991; Starr et al., 1991; Sather et al., 1993; Stea et al., 1994; Dunlap et al., 1995). The K1A subunit gene encodes proteins with diverse structures and functions. Evidence from rabbit, rat, mouse and human K1A cDNAs suggest that this diversity is generated, at least in part, by alternative splicing in this single-copy gene (Mori et al., 1991; Starr et al., 1991; Fletcher et al., 1996; Ligon et al., 1998; Bourinet et al., 1999). In rat brain, two variants of K1A subunit (namely K1Aa and K1Ab ) have been well characterized. K1Ab contains a single valine insertion in the I^II linker, an insertion of two residues of asparagine and proline in the extracellular linker between subdomain IVS3 and IVS4 and 10 substituted residues in a stretch of 30 amino acids in the C-terminus domain. Of the 10 substituted residues, six are in the region highly similar to the divalent ion binding domain (EF-hand) of Ca2þ binding protein. The electrophysiological study of the two variants showed that K1Aa and K1Ab di¡er by their biophysical properties, their sensitivity to Aga IVA and their localization in the adult brain (Bourinet et al., 1999). The K1Aa expresses a P-type-like current whereas K1Ab expresses a current with biophysical properties and sensitivity to Aga IV that are close to the Q-type current characteristics. Recent data argue in favor of a role for K1A P/Q-type VGCCs in modulating neuronal ontogenesis. In situ hybridization (ISH) studies have revealed K1A mRNAs in pre- and postnatal brain undergoing active di¡erentiation (Tanaka et al., 1995). Interestingly, a new alternative spliced variant from the gene K1A (called K1AEFe ), which is a truncated channel protein lacking the C-terminus, has been described in the rat hippocampus, only during the embryonic period (Vigues et al., 1998). Many embryonic neurons express functional P/Q-type VGCCs as evidenced by electrophysiological studies (Diochot et al., 1995; Hivert et al., 1995) and their biophysical and pharmacological properties change depending on the stage of development of their expressing neurons (Hilaire et al., 1996). Recently, a role for this channel family has been evidenced in the developing rat hippocampus: Q-type VGCC, a splicing of the K1A subunit gene, regulates the di¡erentiation of a subpopulation of embryonic calbindin-positive neurons, by controlling Ca2þ in£ux (Boukhaddaoui et al., 2000; Boukhaddaoui et al., 2001). Despite the fact that all these data support a signi¢cant role for the K1A Ca2þ channel subunit family in the development of the nervous system, no data are available concerning the speci¢c neuronal expression of the di¡erent K1A isoforms during CNS ontogenesis in vivo. The present study, by using reverse transcription-polymerase chain reaction (RT-PCR) and ISH analysis, investigated the temporal and spatial pattern of expression of three K1A subunit isoforms (K1Aa , K1Ab and K1AEFe ) from embryonic to adult stages in rat brain. Our data suggest that developing neurons use alternative mRNA splicing to modulate the K1A calcium channel properties during development.
EXPERIMENTAL PROCEDURES
RNA isolation and RT-PCR analysis Sprague^Dawley rats (I¡a Credo, France) were used in this study at di¡erent stages of development (n = 4 for each stage). Their care and use conformed to institutional policies and guidelines. Rats were anaesthetized using pentobarbital (50 mg/kg) and killed by decapitation. At each stage of the development, cortex, cerebellum and hippocampus or whole brain were quickly dissected and frozen in liquid nitrogen. Total RNA was isolated using the TRIzol protocol (Gibco BRL, Rockville, MD, USA) derived from the Chomczynski and Sacchi method (Chomczynski and Sacchi, 1987) and treated with DNase (Gibco, 1.5 U, 15 min, 37‡C) to remove any residual genomic DNA. cDNA templates for ampli¢cation by PCR were synthesized by reverse transcription of total RNA (1 Wg) using Moloney’s murine leukemia virus reverse transcriptase (Superscript II, Gibco) with random hexanucleotides primers and dNTP for 50 min at 42‡C in the bu¡er (supplied by the manufacturer). Reverse transcriptase was omitted in control samples to ensure that ampli¢cation products were from mRNA and not from genomic DNA. Three PCRs were carried out using synthetic oligonucleotides as primers (purchased from Eurogentec). The ¢rst PCR strategy used the primers homologous to the rat calcium channel K1Aa (Genbank, sequence M64373) and K1Ab (unpublished observations) sequences to amplify the EF-hand domain localized on the carboxyl terminal region of the protein: Forward primer I was the same for the three PCR (5PTCCAAAAACCAGAGTGTG3P): position 5183 bp; Reverse primer II (5PTTGAAGTGAACGGTGTTG3P) was speci¢c for K1Aa and K1Ab variants: position 5534 bp. The second PCR used the reverse primer speci¢c to the rat calcium channel K1Aa to amplify a part of EF-hand domain: Reverse primer III (5PTATTACTCGCAATAAACTG3P): position 5410 bp. The third PCR used the reverse primer speci¢c to the rat calcium channel K1Ab to amplify a part of EF-hand domain: Reverse primer IV (5PCATGTGTCTCAGCATCTGA3P): position 5410 bp. In preliminary experiments, we have tested other K1A regions by RT-PCR to discriminate K1Aa and K1Ab isoforms (data not shown), located in the I^II linker (valine insertion) and in the subdomain IVS3^IVS4 (asparagine^proline insertion). The same pattern of expression of EF-hand domain has been observed during development. The cDNA mixture was supplemented with 2.5 U taq polymerase (Thermus aquaticus YT1 from Gibco), 25 pmol of each primer, 5 Wl 10U PCR bu¡er (100 mM Tris^HCl pH 8.4, 20 mM MgCl2 , 500 mM KCl), 200 WM dNTP mix and water to bring the ¢nal volume to 50 Wl. The reaction mixture was overlaid with 50 Wl mineral oil (Promega, Madison, WI, USA) and incubated in a thermal cycler (Eppendorf) for 30 cycles each of them consisting of 1 min denaturation at 94‡C, 1 min hybridization at 50‡C, and 45 s polymerization at 72‡C. After ampli¢cation, 10 Wl of PCR product was run on an ethidium bromidestained gel (1.5% agarose). K1AEFe , K1Aa and K1Ab PCR products were cloned into pGEM-T vector (Promega) and sequenced (ACT Gene) to con¢rm the PCR speci¢city. The PCR experiments (n = 4) for each stage of the development are reproducible and one representative experiment is illustrated for each tissue studied. Tissue and probe preparation ^ ISH Brains from Sprague^Dawley rats, adults (n = 3) and at postnatal days (P) 10 (n = 3), P7 (n = 3) and P0 (n = 3), were excised and immediately frozen into liquid nitrogen vapors. Frozen sections (12 Wm) were obtained using a cryostat (Microm, France): coronal (adult brains) or sagittal sections (postnatal brains) were collected onto silanized slides (Sigma, St. Louis, MO, USA) and ¢xed in 4% paraformaldehyde in 0.1 M phosphate bu¡er (pH 7.4) at room temperature for 30 min. Slides were rinsed in the
NSC 5658 23-7-02
Development of K1A calcium channels
511
Fig. 1. Primer location on the K1Aa C terminus domain. Three PCR were carried out using synthetic oligonucleotides as primers (purchased from Eurogentec). The ¢rst PCR strategy used the primers homologous to the rat calcium channel K1Aa (Genbank, sequence M64373) and K1Ab (unpublished) sequences to amplify the EF-hand domain localized on the carboxyl terminal region of the protein: Forward primer I was the same for the three PCR (TCCAAAAACCAGAGTGTG) at position 5183 bp; Reverse primer II (TTGAAGTGAACGGTGTTG) was speci¢c for K1Aa and K1Ab variants at position 5534 bp. The second PCR used the reverse primer speci¢c to the rat calcium channel K1Aa to amplify a part of EF-hand domain : Reverse primer III (TATTACTCGCAATAAACTG) at position 5410 bp. The third PCR used the reverse primer speci¢c to the rat calcium channel K1Ab to amplify a part of EF-hand domain: Reverse primer IV (CATGTGTCTCAGCATCTGA) at position 5410 bp.
same bu¡er, then dehydrated in graded ethanols and stored at 380‡C until use. Antisense and sense cRNA probes corresponded to the same fragment of speci¢c EF-hand sequences of K1Aa and K1Ab spliced variants (Bourinet et al., 1999). Speci¢c templates were obtained by RT-PCR during which reverse or direct primers, respectively, were 5P extended by a short sequence corresponding to the T7 RNA polymerase promoter (Young et al., 1991; Vigues et al., 1999). Probes were then synthesized and digoxigenin-labelled by in vitro transcription. ISH was performed according to our previously described procedure (Vigues et al., 1999) with a ¢nal probe concentration of 80 ng/ml in 20 Wl hybridization bu¡er. In each experiment, adult, P10, P7 and P0 samples were concomitantly processed together with negative controls. Negative controls included: (i) predigestion with RNase A before hybridization, (ii) hybridization using sense probes diluted at the concentration giving the same labelling signal intensity in dots as the antisense probes and (iii) hybridization without probes (control for non-speci¢c binding of antibodies). Triplicate experiments gave similar results. One representative experiment is illustrated.
shown), this variant has a 93 bp deletion corresponding to the region of speci¢city of K1Aa and K1Ab . The second PCR strategy (primer I^III) allowed us to speci¢cally amplify the K1Aa variant of 227 bp (Fig. 2b, e) showing that the K1Aa variant was present only during postnatal stages. The third PCR strategy (primer I^IV) (Fig. 2c, f) showed that K1Ab was detected during all stages of development studied. The same pattern of expression was observed in the total brain (data not shown). Figures 3 and 4 show the ISH detection of K1Aa and K1Ab mRNA variants in adult and postnatal brains. The speci¢city of ISH signal obtained with the K1Aa or with the K1Ab antisense probe was con¢rmed by the following controls: RNase pretreatment of tissue sections completely abolished the hybridization signals and sections hybridized with digoxigenin-labelled sense probes did not show any signal (not shown).
RESULTS
Localization of the two mRNA variants in selected areas of the developing CNS
We examined the pattern of expression of the three Ca2þ channel variants during embryonic and postnatal hippocampus development. We used three PCR strategies (Fig. 1) to amplify speci¢c cDNA fragments of the VGCC K1A subunit encoding a region of the channel starting from the domain IVS6 and going to the carboxyl terminal after the divalent ion binding-like domain (EF-hand) (Fig. 1). During all the periods of hippocampus development studied, K1AEF , a 351 bp fragment corresponding to the K1A mRNA was ampli¢ed with the ¢rst PCR strategy (Fig. 2a, d). Only during embryonic stages (Fig. 2a, d) a 257 bp fragment corresponding to K1AEFe was also ampli¢ed and sequenced (data not
The detection of the two mRNA variants in di¡erent developing brain structures is shown in Table 1. At P0, K1Aa variant was not expressed. By P7, K1Aa expression was observed in cerebral cortex, hippocampus, cerebellum, as well as in some large hypothalamic and medulla oblongata neurons. The labelling signal remained thereafter. The thalamus and all regions anterior to the hippocampus did not express K1Aa variant before P10. No signal could be detected in glial cells from the corpus callosum throughout the development of the brain. K1Ab expression was present as early as P0 in cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus, striatum and medulla oblongata. Ependy-
NSC 5658 23-7-02
512
S. Vigues et al.
Fig. 2. In vivo developmental regulation of K1Aa , K1Ab , K1AEF and K1AEFe transcripts in hippocampus (a, b, c) and cerebellum (d, e, f) investigated by PCR on 1.5% agarose gel. The presence of variants is studied at di¡erent stages: In cerebellum at embryonic day (E) 15, E17, E19, P1, P7, P14, 2 months (2M). In hippocampus at E17, E18, E19, E20, E21, P0 (birth), P1, P2, P7, P14 and 2M. K1Ab (c, f) and K1AEF (a, d) PCR products are present respectively at 227 bp and 351 bp whatever the developmental stages studied. K1Aa (b, e) PCR product is present at 227 bp only after birth and K1AEFe PCR product is present at 257 bp only during embryonic development. The same developmental pattern is observed in hippocampus and cerebellum. Marker (Promega) sizes: 1000, 750, 500, 300, 150, 50 bp. Table 1. ISH detection of K1Aa and K1Ab mRNA variants in di¡erent areas of the developing rat CNS P0
Cerebral cortex Hippocampus Thalamus Hypothalamus Striatum Cerebellum Medulla oblongata Ependymal cells
P7
P10
Adult
1A-a
1A-b
1A-a
1A-b
1A-a
1A-b
1A-a
1A-b
3 3 3 3 3 3 3 3
+ + + + + + + T
+ + 3 + 3 + + 3
+ + + + + + + T
+ + + + + + + 3
+ + + + + + + T
+ + + + + + + 3
+ + + + + + + +
3: Absence of labelling detected. +: Labelling signal detected. T : Labelling signal occasionally detected.
NSC 5658 23-7-02
Development of K1A calcium channels
513
Fig. 3. ISH detection of mRNAs encoding K1Aa (left panel, a, c, e) and K1Ab (right panel, b, d, f) in cerebellum from P7 (a, b), P10 (c, d) and adult brains (e, f). The K1Aa variant appears at P7 in Purkinje cells as well as in the external granular layer and at P10 in the internal granular layer while in adult brains it is only detected in Purkinje cells and in some Golgi type II cells (arrowhead). The K1Ab variant is present in Purkinje cells and in both external and internal layers at P7 and P10 while only present in Purkinje cells, molecular layer and some Golgi type II cells (arrowhead) in adult brain. Pu, Purkinje cells, EGL, external granular layer, IGL, internal granular layer, ML, molecular layer, GL, granular layer. Scale bar = 125 Wm.
mal cells were occasionally labelled. The hippocampus was one of those areas which showed the most intense labelling signal in the whole brain. This labelling pattern was observed throughout the di¡erent stages of development studied. It is noteworthy that in all developmental periods and concerning both mRNA variants, the labelling of striatum, if present, was faint when compared to other CNS structures. Taking the developing cerebral cortex as an example, the K1Aa variant was not detected at P0 while the K1Ab variant was observed in the cortical plate and in early-born large pyramidal cells. By P7 all cortical layers expressed the K1Ab variant and the K1Aa variant by P10 (data not shown). Cellular localization of the two mRNA variants in the developing cerebellum (Fig. 3) Using the K1Aa probe, no labelling could be detected at P0 but a weak signal appeared in Purkinje cells and in the external granular layer by P7. At P10, the K1Aa variant was also present in the internal granular layer. In adult rats, the signal was detected only in Purkinje cells and in sparse Golgi type II cells. Conversely, the K1Ab variant was faintly detected in the developing P0 cerebellum. At P7, labelling by the K1Ab probe was high
in Purkinje cells, while it remained moderate and homogeneous in both external and internal granular layers. At P10, the K1Ab isoform was highly detected in Purkinje cells and in some Golgi type II cells while labelling was faint in both granular layers. In adult rats, K1Ab variant was present in Purkinje cells, in the molecular layer and in sparse Golgi type II cells but disappeared from granular cells. It is noteworthy that both K1Aa and K1Ab probes did not produce any labelling in granular cells from adult cerebellum. This observation is in contrast with previous reports (Bourinet et al., 1999) which, using radiolabelled oligoprobes, detected both variants in these cells. A di¡erent ISH sensitivity may account for this discrepancy, inasmuch as radiolabelled probes are known to be more sensitive. Cellular localization of the two mRNA variants in the developing hippocampus (Fig. 4) K1Aa mRNAs were not observed until P7. At this stage, a weak labelling signal was present in pyramidal cells of the Ammon’s horn from CA1 to CA4 but not in granule cells of the dentate gyrus (DG). A weak labelling was apparent in DG granule cells in P10 and adult rats. In addition, in adult brains, granule cells located at the
NSC 5658 23-7-02
514
S. Vigues et al.
Fig. 4. ISH detection of mRNAs encoding K1Aa (left panel, a, c, e, g) and K1Ab (right panel, b, d, f, h) in hippocampus from P0 (a, b), P7 (c, d), P10 (e, f) and adult brains (g, h). The K1Aa variant appears at P7 in pyramidal cells and at P10 in DG granule cells. The K1Ab variant is detected as soon as P0 in both pyramidal cells and DG granule cells. Note in d the labelling of the DG is higher in cells located along the outer border of the layer (arrowhead) and the di¡erence in labelling intensity seen in b, d, f, between pyramidal cells and DG granule cells. Py, pyramidal cells of Ammon’s horn. Scale bar = 250 Wm.
inner border of the DG were particularly labelled and a labelling of interneurons of the CA4 area could be evidenced. Unlike K1Aa , the K1Ab isoform was present in hippocampus as soon as P0. The developing DG was uniformly labelled whereas pyramidal cells were heterogeneously labelled. Throughout all stages of development, the signal detected was higher in pyramidal cells than in granule cells, a discrepancy that was strongly attenuated in adult brains. At P7, labelling of the DG was higher in cells located along the outer border of the layer. Besides this, the pyramidal cells of Ammon’s horn were highly and homogeneously labelled. On sagittal sections, the labelling signal increased from CA1 to CA3 pyramidal cells (a pattern initiated as soon as P0). In addition, a high signal was also observed in CA4 interneurons. By P10, labelling was present in all pyramidal
and DG granule cells. In adult brains, DG granule cells were highly labelled, particularly those located at the inner border of the gyrus.
DISCUSSION
The present study focuses on the developmental expression of three naturally occurring splice variants of the K1A subunit gene (K1Aa , K1Ab and K1Ae ) that di¡er in nucleotide sequences corresponding to the EF-hand domain of the C-terminus. Alternative splicing of the K1A gene results in the expression of multiple VGCC phenotypes (Krovetz et al., 2000). In this regard, the C-terminus region of K1A subunit, between the K1A IVS3^IVS4 linker and the terminal stop codon, includes
NSC 5658 23-7-02
Development of K1A calcium channels
multiple important structural domains regulating functional properties of the channel. The C-terminus region of the human K1A gene is encoded by 12 exons (numbered 36^47), of which four exons (37, 44, 46 and 47) undergo alternative splicings. Of the 16 possible combinations, nine of them have been isolated previously. Each combination creates a channel with di¡erent properties. For example, the inclusion of exon 44 a¡ects the rate of inactivation; exon 37b gives new activation and inactivation properties to the channel and changes the a⁄nity for Aga IV by inserting two amino acids (NP) in the K1A IVS3-IVS4 linker. The C-terminus region is also essential for modulation by G proteins (Kinoshita et al., 2001) (exon 45^46) or the L4 subunit (exon 43^47) that enhances the rate of inactivation (Walker and De Waard, 1998). Polyglutamine tract expansions in the exon 47 increase current density and alter activation and inactivation properties, leading to a variety of human neurological diseases (Lehmann-Horn and Jurkat-Rott, 1999). Functional expression and localization studies in the adult brain indicate that this diversity generated by alternative splicing may be important for ¢ne tuning neuronal function under physiological and pathological conditions. The present results suggest that an alternative splicing mechanism may also contribute to the heterogeneous properties of K1A subunits during neuronal development. RT-PCR studies have de¢ned that, from embryonic stages to adulthood, each K1A isoform develops with speci¢c and restricted timings of expression. For example, K1AEFe is restricted to the embryonic period and K1Aa is expressed only postnatally, whatever the region analyzed. Speci¢c expression of voltage-gated channel alternative splicing during embryonic and early postnatal period has been previously shown for the pore-forming K II and III subunit of sodium channels (Waxman et al., 1994). ISH analysis indicates that, in addition to a di¡erence in the temporal expression of the three di¡erent K1A isoforms during brain ontogenesis, each neuronal subpopulation exhibited a pattern of expression that di¡ers depending on the stage of development. For example, cerebellar Purkinje cells express dominant K1Ab gene subunit during perinatal period whereas K1Aa gene subunit becomes the predominant isoform in adult. By contrast, hippocampal pyramidal neurons express K1Ab as the major isoform throughout its development. Nevertheless, independently of their di¡erent cellular localization, the timing of expression of K1Aa and K1Ab have some common features whatever the brain structure studied: K1Ab is present during embryonic period (until adulthood) and K1Aa is only postnatally expressed. During the ¢rst postnatal week, the latter variant is either not expressed (i.e. granule cells of DG) or when expressed, levels are weak compared to K1Ab . Increment in K1Aa expression parallels cell maturation, especially synaptogenesis as shown for the Ammon’s horn where synaptogenesis and K1Aa expression are concomitant (Jacobson and Sapolsky, 1991). These data suggest that each isoform is involved in speci¢c modulatory functions during development. For the moment, the K1AEFe variant has not been electrophysiologically characterized and its role remains
515
unknown but some K1A mutations mimic the truncation of the K1AEFe variant. For example, tgla (short) mutation (Fletcher et al., 1996) that lacks the C-terminal domain or the type-2 episodic ataxia (Jen, 1999), which has a stop codon in domain IV, induces neurological disorders resulting from signi¢cant modi¢cations in current density and gating behavior (Wakamori et al., 1998). From previous structure-function experiments using mutations of other K1 subunits C-terminus, we can suggest that the K1AEFe variant has speci¢c biophysical and pharmacological properties in comparison with other isoforms (De Leon et al., 1995). However if K1AEFe variant is not a functional channel, it can modulate the activity of the full-length K1A proteins as a decoy (Colotta et al., 1993) or functions as a voltage sensor with its four S4 segments always present. These results show that K1AEFe may have an important role in early brain development by interacting with the variants K1Aa . The cloning of K1AEFe and its expression in oocytes or mammalian cells should be an e¡ective way to test these hypotheses. Before synaptogenesis, neuronal development proceeds through a sequence of events including proliferation of neuronal precursor cells, migration of neuroblasts, neurite extensions and phenotypic di¡erentiation. Present results indicate a role for the K1Ab gene subunit during this early developmental period, since K1Ab mRNAs are expressed during all these developmental stages. In certain areas, K1Ab appears to be mainly present when cells are undergoing proliferation and migration. For instance, this variant is highly expressed in the cortical plate, a proliferative zone of the developing cortex. Moreover, it is noteworthy that in DG, where neuron proliferation persists into adulthood, K1Ab expression remains until this age. The majority of DG granule cells (approximately 80%) are formed postnatally and the cytoarchitecture of the gyrus is not recognizable until near the end of the ¢rst postnatal week (Schlessinger et al., 1975). During the ¢rst two postnatal weeks and in adult brain, although at a slower rate, dentate granule progenitor cells divide at the border of the hilus and migrate to reach their ¢nal position in the granule cell layer (Schlessinger et al., 1975; Rickmann et al., 1987; Cameron et al., 1993; Kuhn et al., 1996). Surprisingly, the expression of K1Ab ¢rst appears along the outer border of the granule cell layer (facing CA1) and extends to the whole layer thereafter. Expression of this variant thus develops in an opposite direction to that taken by the migrating granule cells. It is unlikely that neurons in the hilus do not express K1Ab mRNA before they migrate since, in the developing hippocampus, the labelling that we observed in the hilar region consistently appears to be more intense than in the DG. A delayed migration of the cells that express K1Ab could account for this late appearance of K1Ab mRNA-containing cells. An alternative possibility could be that neurons reach their position at early ages and delay their expression of K1Ab . A birthdating study would be necessary to clarify this point. The possible role of the K1A isoform expression in the development of the hippocampal pyramidal neurons is especially signi¢cant. The K1Ab but not the K1Aa gene
NSC 5658 23-7-02
516
S. Vigues et al.
subunit is present at the time when hippocampal neuroblasts migrate, settle, elaborate axons and dendrites and di¡erentiate. Functional Q-type VGCC have been evidenced during this period and described as a modulator of the acquisition of the calbindin phenotype expressed by an hippocampal neuron subpopulation (Boukhaddaoui et al., 2000). These results suggest that Q-type channels, which have been predominantly associated with neurotransmitter release in adult brain, transiently act to direct early neuronal di¡erentiation of hippocampal neurons before the establishment of their synaptic circuits. Then synaptogenesis begins and develops during the ¢rst weeks after birth, when neurotransmission is ¢rst dominated by N-type VGCC, the role of which declines as their task becomes shared by P/Q-type VGCCs. Remarkably, this phase coincides with the initial detection of the K1Aa P-type gene mRNA subunit whose expression increases during the two postnatal weeks concomitant with a migration of Q-type VGCC out of the hippocampal neuronal somata (probably towards the neurites). Thus, each isoform develops in the same time window as several physiological correlates
of hippocampal maturation, suggesting that neurons use K1A alternative splicing to ¢ne tune Ca2þ in£ux according to their developmental requirements. Activation of voltage-gated Ca2þ channels is thought to be one of the major molecular mechanisms by which calcium in£ux regulates CNS ontogenesis (Ghosh and Greenberg, 1995; Gu and Spitzer, 1995). However, the characterization of the increasingly numerous Ca channel subtypes in the developing brain as well as their pattern of expression are not yet well resolved. Using RT-PCR and ISH analysis, the present results have de¢ned the spatio-temporal expression of three alternative splicing of the K1A gene (namely K1Aa , K1Ab and K1Ae ) during rat brain development in vivo. From embryonic stages to adulthood, each K1A isoform develops with a speci¢c timing and di¡erent regional and cellular patterns. Therefore, our data support a signi¢cant role for the K1A gene alternative splicing mechanism during ontogenesis as a tool for a developing neuron to select Ca2þ channels with properties that are appropriate for its speci¢c needs in Ca2þ in£ux depending on its stage of development.
REFERENCES
Bading, H., Ginty, D.D., Greenberg, M.E., 1993. Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science 260, 181^186. Bech-Hansen, N.T., Naylor, M.J., Maybaum, T.A., Pearce, W.G., Koop, B., Fishman, G.A., Mets, M., Musarella, M.A., Boycott, K.M., 1998. Loss-of-function mutations in a calcium-channel alpha1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness. Nat. Genet. 19, 264^267. Birnbaumer, L., Campbell, K.P., Catterall, W.A., Harpold, M.M., Hofmann, F., Horne, W.A., Mori, Y., Schwartz, A., Snutch, T.P., Tanabe, T., 1994. The naming of voltage-gated calcium channels. Neuron 13, 505^506. Boukhaddaoui, H., Sieso, V., Scamps, F., Valmier, J., 2001. An activity-dependent neurotrophin-3 autocrine loop regulates the phenotype of developing hippocampal pyramidal neurons before target contact. J. Neurosci. 21, 8789^8797. Boukhaddaoui, H., Sieso, V., Scamps, F., Vigues, S., Roig, A., Valmier, J., 2000. Q- and L-type calcium channels control the development of calbindin phenotype in hippocampal pyramidal neurons in vitro. Eur. J. Neurosci. 12, 2068^2078. Bourinet, E., Soong, T.W., Sutton, K., Slaymaker, S., Mathews, E., Monteil, A., Zamponi, G.W., Nargeot, J., Snutch, T.P., 1999. Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels. Nat. Neurosci. 2, 407^415. Bourinet, E., Zamponi, G.W., Stea, A., Soong, T.W., Lewis, B.A., Jones, L.P., Yue, D.T., Snutch, T.P., 1996. The alpha 1E calcium channel exhibits permeation properties similar to low-voltage-activated calcium channels. J. Neurosci. 16, 4983^4993. Cameron, H.A., Woolley, C.S., McEwen, B.S., Gould, E., 1993. Di¡erentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56, 337^344. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156^159. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., Giri, J.G., Dower, S.K., Sims, J.E., Mantovani, A., 1993. Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4. Science 261, 472^475. De Leon, M., Wang, Y., Jones, L., Perez-Reyes, E., Wei, X., Soong, T.W., Snutch, T.P., Yue, D.T., 1995. Essential Ca(2+)-binding motif for Ca(2+)-sensitive inactivation of L-type Ca2þ channels. Science 270, 1502^1506. Desmadryl, G., Hilaire, C., Vigues, S., Diochot, S., Valmier, J., 1998. Developmental regulation of T-, N- and L-type calcium currents in mouse embryonic sensory neurones. Eur. J. Neurosci. 10, 545^552. Diochot, S., Richard, S., Valmier, J., 1995. Diversity of voltage-gated calcium currents in large diameter embryonic mouse sensory neurons. Neuroscience 69, 627^641. Dunlap, K., Luebke, J.I., Turner, T.J., 1995. Exocytotic Ca2þ channels in mammalian central neurons. Trends Neurosci. 18, 89^98. Ertel, E.A., Campbell, K.P., Harpold, M.M., Hofmann, F., Mori, Y., Perez-Reyes, E., Schwartz, A., Snutch, T.P., Tanabe, T., Birnbaumer, L., Tsien, R.W., Catterall, W.A., 2000. Nomenclature of voltage-gated calcium channels. Neuron 25, 533^535. Fletcher, C.F., Lutz, C.M., O’Sullivan, T.N., Shaughnessy, J.D., Jr., Hawkes, R., Frankel, W.N., Copeland, N.G., Jenkins, N.A., 1996. Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell 87, 607^617. Ghosh, A., Greenberg, M.E., 1995. Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268, 239^247. Gu, X., Spitzer, N.C., 1995. Distinct aspects of neuronal di¡erentiation encoded by frequency of spontaneous Ca2þ transients. Nature 375, 784^ 787. Hilaire, C., Diochot, S., Desmadryl, G., Baldy-Moulinier, M., Richard, S., Valmier, J., 1996. Opposite developmental regulation of P- and Q-type calcium currents during ontogenesis of large diameter mouse sensory neurons. Neuroscience 75, 1219^1229. Hivert, B., Bouhanna, S., Diochot, S., Camu, W., Dayanithi, G., Henderson, C.E., Valmier, J., 1995. Embryonic rat motoneurons express a functional P-type voltage-dependent calcium channel. Int. J. Dev. Neurosci. 13, 429^436. Hofmann, F., Biel, M., Flockerzi, V., 1994. Molecular basis for Ca2þ channel diversity. Annu. Rev. Neurosci. 17, 399^418. Hui, A., Ellinor, P.T., Krizanova, O., Wang, J.J., Diebold, R.J., Schwartz, A., 1991. Molecular cloning of multiple subtypes of a novel rat brain isoform of the alpha 1 subunit of the voltage-dependent calcium channel. Neuron 7, 35^44. Jacobson, L., Sapolsky, R., 1991. The role of the hippocampus in feedback regulation of the hypothalamic-pituitary-adrenocortical axis. Endocr. Rev. 12, 118^134.
NSC 5658 23-7-02
Development of K1A calcium channels
517
Jen, J., 1999. Calcium channelopathies in the central nervous system. Curr. Opin. Neurobiol. 9, 274^280. Kennedy, M.B., 1989. Regulation of neuronal function by calcium. Trends Neurosci. 12, 417^420. Kinoshita, M., Nukada, T., Asano, T., Mori, Y., Akaike, A., Satoh, M., Kaneko, S., 2001. Binding of G alpha(o) N terminus is responsible for the voltage-resistant inhibition of alpha(1A) (P/Q-type, Ca(v)2.1) Ca(2+) channels. J. Biol. Chem. 276, 28731^28738. Kocsis, J.D., Rand, M.N., Lankford, K.L., Waxman, S.G., 1994. Intracellular calcium mobilization and neurite outgrowth in mammalian neurons. J. Neurobiol. 25, 252^264. Krovetz, H.S., Helton, T.D., Crews, A.L., Horne, W.A., 2000. C-Terminal alternative splicing changes the gating properties of a human spinal cord calcium channel alpha 1A subunit. J. Neurosci. 20, 7564^7570. Kuhn, H.G., Dickinson-Anson, H., Gage, F.H., 1996. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J. Neurosci. 16, 2027^2033. Lehmann-Horn, F., Jurkat-Rott, K., 1999. Voltage-gated ion channels and hereditary disease. Physiol. Rev. 79, 1317^1372. Ligon, B., Boyd, A.E., Dunlap, K., 1998. Class A calcium channel variants in pancreatic islets and their role in insulin secretion. J. Biol. Chem. 273, 13905^13911. Llinas, R.R., 1988. The intrinsic electrophysiological properties of mammalian neurons : insights into central nervous system function. Science 242, 1654^1664. Mori, Y., Friedrich, T., Kim, M.S., Mikami, A., Nakai, J., Ruth, P., Bosse, E., Hofmann, F., Flockerzi, V., Furuichi, T., 1991. Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature 350, 398^402. Mulkey, R.M., Zucker, R.S., 1991. Action potentials must admit calcium to evoke transmitter release. Nature 350, 153^155. Perez-Reyes, E., 1998. Molecular characterization of a novel family of low voltage-activated, T-type, calcium channels. J. Bioenerg. Biomembr. 30, 313^318. Perez-Reyes, E., Yuan, W., Wei, X., Bers, D.M., 1994. Regulation of the cloned L-type cardiac calcium channel by cyclic-AMP-dependent protein kinase. FEBS Lett. 342, 119^123. Rickmann, M., Amaral, D.G., Cowan, W.M., 1987. Organization of radial glial cells during the development of the rat dentate gyrus. J. Comp. Neurol. 264, 449^479. Sather, W.A., Tanabe, T., Zhang, J.F., Mori, Y., Adams, M.E., Tsien, R.W., 1993. Distinctive biophysical and pharmacological properties of class A (BI) calcium channel alpha 1 subunits. Neuron 11, 291^303. Schlessinger, A.R., Cowan, W.M., Gottlieb, D.I., 1975. An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat. J. Comp. Neurol. 159, 149^175. Schmid, S., Guenther, E., 1999. Voltage-activated calcium currents in rat retinal ganglion cells in situ: changes during prenatal and postnatal development. J. Neurosci. 19, 3486^3494. Schneider, T., Wei, X., Olcese, R., Costantin, J.L., Neely, A., Palade, P., Perez-Reyes, E., Qin, N., Zhou, J., Crawford, G.D., 1994. Molecular analysis and functional expression of the human type E neuronal Ca2þ channel alpha 1 subunit. Recept. Channels 2, 255^270. Singer, D., Biel, M., Lotan, I., Flockerzi, V., Hofmann, F., Dascal, N., 1991. The roles of the subunits in the function of the calcium channel. Science 253, 1553^1557. Snutch, T.P., Tomlinson, W.J., Leonard, J.P., Gilbert, M.M., 1991. Distinct calcium channels are generated by alternative splicing and are di¡erentially expressed in the mammalian CNS. Neuron 7, 45^57. Soong, T.W., Stea, A., Hodson, C.D., Dubel, S.J., Vincent, S.R., Snutch, T.P., 1993. Structure and functional expression of a member of the low voltage-activated calcium channel family. Science 260, 1133^1136. Starr, T.V., Prystay, W., Snutch, T.P., 1991. Primary structure of a calcium channel that is highly expressed in the rat cerebellum. Proc. Natl. Acad. Sci. USA 88, 5621^5625. Stea, A., Tomlinson, W.J., Soong, T.W., Bourinet, E., Dubel, S.J., Vincent, S.R., Snutch, T.P., 1994. Localization and functional properties of a rat brain alpha 1A calcium channel re£ect similarities to neuronal Q- and P-type channels. Proc. Natl. Acad. Sci. USA 91, 10576^10580. Strom, T.M., Nyakatura, G., Apfelstedt-Sylla, E., Hellebrand, H., Lorenz, B., Weber, B.H., Wutz, K., Gutwillinger, N., Ruther, K., Drescher, B., Sauer, C., Zrenner, E., Meitinger, T., Rosenthal, A., Meindl, A., 1998. An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness. Nat. Genet. 19, 260^263. Tanaka, O., Sakagami, H., Kondo, H., 1995. Localization of mRNAs of voltage-dependent Ca(2+)-channels: four subtypes of alpha 1- and betasubunits in developing and mature rat brain. Brain Res. Mol. Brain Res. 30, 1^16. Tsien, R.W., Ellinor, P.T., Horn, W.A., 1991. Molecular diversity of voltage-dependent Ca2þ channels. Trends Pharmacol. Sci. 12, 349^354. Vigues, S., Chabret, C., Valentin, S., Valmier, J., 1998. Rat embryonic hippocampal neurons express a new class A calcium channel variant. Neurosci. Lett. 258, 37^40. Vigues, S., Gastaldi, M., Chabret, C., Massacrier, A., Cau, P., Valmier, J., 1999. Regulation of calcium channel alpha(1A) subunit splice variant mRNAs in kainate-induced temporal lobe epilepsy. Neurobiol. Dis. 6, 288^301. Wakamori, M., Yamazaki, K., Matsunodaira, H., Teramoto, T., Tanaka, I., Niidome, T., Sawada, K., Nishizawa, Y., Sekiguchi, N., Mori, E., Mori, Y., Imoto, K., 1998. Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel. J. Biol. Chem. 273, 34857^34867. Walker, D., De Waard, M., 1998. Subunit interaction sites in voltage-dependent Ca2þ channels : role in channel function. Trends Neurosci. 21, 148^154. Waxman, S.G., Kocsis, J.D., Black, J.A., 1994. Type III sodium channel mRNA is expressed in embryonic but not adult spinal sensory neurons, and is reexpressed following axotomy. J. Neurophysiol. 72, 466^470. Wheeler, D.B., Randall, A., Tsien, R.W., 1994. Roles of N-type and Q-type Ca2þ channels in supporting hippocampal synaptic transmission. Science 264, 107^111. Williams, M.E., Brust, P.F., Feldman, D.H., Patthi, S., Simerson, S., Marou¢, A., McCue, A.F., Velicelebi, G., Ellis, S.B., Harpold, M.M., 1992b. Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel. Science 257, 389^395. Williams, M.E., Feldman, D.H., McCue, A.F., Brenner, R., Velicelebi, G., Ellis, S.B., Harpold, M.M., 1992a. Structure and functional expression of alpha 1, alpha 2, and beta subunits of a novel human neuronal calcium channel subtype. Neuron 8, 71^84. Williams, M.E., Marubio, L.M., Deal, C.R., Hans, M., Brust, P.F., Philipson, L.H., Miller, R.J., Johnson, E.C., Harpold, M.M., Ellis, S.B., 1994. Structure and functional characterization of neuronal alpha 1E calcium channel subtypes. J. Biol. Chem. 269, 22347^22357. Young, I.D., Ailles, L., Deugau, K., Kisilevsky, R., 1991. Transcription of cRNA for in situ hybridization from polymerase chain reactionampli¢ed DNA. Lab. Invest. 64, 709^712. Zhang, J.F., Randall, A.D., Ellinor, P.T., Horne, W.A., Sather, W.A., Tanabe, T., Schwarz, T.L., Tsien, R.W., 1993. Distinctive pharmacology and kinetics of cloned neuronal Ca2þ channels and their possible counterparts in mammalian CNS neurons. Neuropharmacology 32, 1075^ 1088. (Accepted 12 April 2002)
NSC 5658 23-7-02