entla, a Novel Epileptic and Ataxic Cacna2d2 Mutant of the Mouse

Abstract

entla (ent) is a novel recessive phenotype of mice. The underlying mutation was mapped to chromosome 9 (60.1 centimorgans) and identified as an allele of the Cacna2d2 gene encoding the α2δ-2 subunit of voltage-gated calcium channels. The Cacna2d2entla allele harbors a 38-kb duplication comprising the 117 nucleotides of exon 3. The predicted duplication of 39 amino acid residues near the subunit's N terminus results in the expression of a full-length, membrane-associated protein. Western blot data were consistent with correct cleavage of the α2δ-2entla precursor into α2entla and δ2 proteins but indicated loss of the disulfide linkage between the two proteins. ent/ent mice develop ataxia by postnatal day 13-15, followed by paroxysmal dyskinesia a few days later. Two distinct types of cortical and hippocampal epileptic activity at 2 and 4 Hz were recorded, indicative of absence epilepsy. Homozygotes display reduced size and weight, increased mortality before weaning, and female infertility. No overt neuroanatomical abnormalities were detected. Ca2+ current densities recorded from acutely dissociated Purkinje cells of homozygous entla animals were reduced by 50% compared with wild type. Ligand binding assays using the antiepileptic drug [3H]gabapentin, a specific ligand of the α2δ-1 and α2δ-2 subunits, revealed a >60% reduced maximum binding to cerebellar membranes of ent/ent compared with unaffected littermates. entla is allelic to ducky and ducky2J, representing the third murine Cacna2d2 allele identified and so far the only one encoding an untruncated protein that is incorporated into membranes. entla (ent) is a novel recessive phenotype of mice. The underlying mutation was mapped to chromosome 9 (60.1 centimorgans) and identified as an allele of the Cacna2d2 gene encoding the α2δ-2 subunit of voltage-gated calcium channels. The Cacna2d2entla allele harbors a 38-kb duplication comprising the 117 nucleotides of exon 3. The predicted duplication of 39 amino acid residues near the subunit's N terminus results in the expression of a full-length, membrane-associated protein. Western blot data were consistent with correct cleavage of the α2δ-2entla precursor into α2entla and δ2 proteins but indicated loss of the disulfide linkage between the two proteins. ent/ent mice develop ataxia by postnatal day 13-15, followed by paroxysmal dyskinesia a few days later. Two distinct types of cortical and hippocampal epileptic activity at 2 and 4 Hz were recorded, indicative of absence epilepsy. Homozygotes display reduced size and weight, increased mortality before weaning, and female infertility. No overt neuroanatomical abnormalities were detected. Ca2+ current densities recorded from acutely dissociated Purkinje cells of homozygous entla animals were reduced by 50% compared with wild type. Ligand binding assays using the antiepileptic drug [3H]gabapentin, a specific ligand of the α2δ-1 and α2δ-2 subunits, revealed a >60% reduced maximum binding to cerebellar membranes of ent/ent compared with unaffected littermates. entla is allelic to ducky and ducky2J, representing the third murine Cacna2d2 allele identified and so far the only one encoding an untruncated protein that is incorporated into membranes. Voltage-gated calcium channels (VGCCs) 1The abbreviations used are: VGCC, voltage-gated calcium channel; PBS, phosphate-buffered saline; pF, picofarads. mediate entry of Ca2+ into excitable cells, thereby initiating a multitude of cellular processes including the release of neurotransmitter into the synaptic cleft. Based on their biophysical properties, VGCCs are subdivided into L-, N-, P/Q-, R-, and T-type channels. VGCCs are protein complexes consisting of a channel-forming, voltage-sensing α1 subunit and α2δ, β, and γ auxiliary subunits (1Hofmann F. Lacinova L. Klugbauer N. Rev. Physiol. Biochem. Pharmacol. 1999; 139: 33-87Crossref PubMed Google Scholar, 2Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1958) Google Scholar). Mutations in VGCC genes underlie various human and murine neurological diseases. Familial hemiplegic migraine (OMIM 602481), spinocerebellar ataxia (OMIM 183086, 603516, and 604432), and idiopathic generalized epilepsy (OMIM 600669) result from mutations in the α1A and β4 subunits. In mice, mutations in α1, β, γ, and α2δ subunits have been described (3Felix R. Cell Mol. Neurobiol. 2002; 22: 103-120Crossref PubMed Scopus (43) Google Scholar). The α1A mutants tottering (4Doyle J Ren X. Lennon G. Stubbs L. Mamm. Genome. 1997; 8: 113-120Crossref PubMed Scopus (115) Google Scholar) and rocker (5Zwingman T.A. Neumann P.E. Noebels J.L. Herrup K. J. Neurosci. 2001; 21: 1169-1178Crossref PubMed Google Scholar), the β4 mutant lethargic (6Burgess D.L. Jones J.M. Meisler M.H. Noebels J.L. Cell. 1997; 88: 385-392Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar), the γ2 mutant stargazer (7Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett II, F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Crossref PubMed Scopus (489) Google Scholar), and the α2δ-2 mutants ducky and ducky2J (8Barclay J. Balaguero N. Mione M. Ackerman S.L. Letts V.A. Brodbeck J. Canti C. Meir A. Page K.M. Kusumi K. Perez-Reyes E. Lander E.S. Frankel W.N. Gardiner R.M. Dolphin A.C. Rees M. J. Neurosci. 2001; 21: 6095-6104Crossref PubMed Google Scholar) all suffer from ataxia, paroxysmal dyskinesia, and epileptic spike wave discharges. Each α2δ subunit is encoded by a single gene and post-translationally cleaved into a long, N-terminal, extracellular α2 protein and a shorter, membrane-anchored δ polypeptide; the α2 and δ proteins are covalently linked by disulfide bonds (9De Jongh K.S. Warner C. Catterall W.A. J. Biol. Chem. 1990; 265: 14738-14741Abstract Full Text PDF PubMed Google Scholar). Upon recombinant coexpression with α1, β, and γ subunits, α2δ subunits modulate channel activity by increasing calcium current density, shifting the voltage dependence of activation to more negative potentials, or increasing steady-state inactivation (10Dolphin A.C. Wyatt C.N. Richards J. Beattie R.E. Craig P. Lee J.H. Cribbs L.L. Volsen S.G. Perez-Reyes E. J. Physiol. 1999; 519: 35-45Crossref PubMed Scopus (116) Google Scholar, 11Hobom M. Dai S. Marais E. Lacinova L. Hofmann F. Klugbauer N. Eur. J. Neurosci. 2000; 12: 1217-1226Crossref PubMed Scopus (104) Google Scholar). The α2δ-1 and α2δ-2 subunits possess high affinity binding sites for the antiepileptic drug gabapentin, which exerts an inhibitory effect on VGCC-mediated Ca2+ currents (12Marais E. Klugbauer N. Hofmann F. Mol. Pharmacol. 2001; 59: 1243-1248Crossref PubMed Scopus (224) Google Scholar, 13Stefani A. Spadoni F. Bernardi G. Neuropharmacology. 1998; 37: 83-91Crossref PubMed Scopus (192) Google Scholar). The spontaneous mouse mutants ducky and ducky2J were recently shown to be functional Cacna2d2 null alleles encoding prematurely truncated α2 and no δ2 protein (8Barclay J. Balaguero N. Mione M. Ackerman S.L. Letts V.A. Brodbeck J. Canti C. Meir A. Page K.M. Kusumi K. Perez-Reyes E. Lander E.S. Frankel W.N. Gardiner R.M. Dolphin A.C. Rees M. J. Neurosci. 2001; 21: 6095-6104Crossref PubMed Google Scholar). ducky homozygotes exhibit ataxia, absence epilepsy, and paroxysmal dyskinesia. Dysgenesis of brainstem and spinal cord as well as myelination defects and altered morphology of Purkinje cell dendrites are anatomical correlates of the phenotype (14Meier H. Acta Neuropathol. (Berl.). 1968; 11: 15-28Crossref PubMed Scopus (32) Google Scholar, 15Brodbeck J. Davies A. Courtney J.M. Meir A. Balaguero N. Canti C. Moss F.J. Page K.M. Pratt W.S. Hunt S.P. Barclay J. Rees M. Dolphin A.C. J. Biol. Chem. 2002; 277: 7684-7693Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Here, we provide a phenotypic, molecular, and functional characterization of a novel spontaneous Cacna2d2 mutant, entla (symbol used here: ent). Animals—Animals were maintained in a certified animal facility according to state legal guidelines and provided a commercial diet and water ad libitum. The entla phenotype was first observed in a heterogeneous genetic background (DBA/2J, CD1). Heterozygous animals were subsequently crossed into a C57BL/6J genetic background for 10 generations. For our studies, N2 to N10 animals were used. For tissue preparation, animals were anesthetized under CO2 and decapitated, and the desired tissue was removed, shock-frozen in liquid nitrogen, and stored at -70 °C until further use. Nucleic Acid Preparation—DNA from tail tip biopsies was isolated according to standard methods. DNA from spleen or liver was isolated using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) or by standard phenol extraction. Total RNA was isolated using the peqGold RNAPure system (PeqLab, Erlangen, Germany). cDNA was assembled by reverse transcription of 1-1.5 μg of RNA using random hexamers or nonamers (Invitrogen). Candidate Gene Analysis—The Cacna2d2 coding region was PCR-amplified from cerebellar cDNA in four portions using the following indicated to were using the system with of the of the coding region was using the system for the reverse and an of of the was with spleen DNA using the in before the at or before the at with the reverse system Germany) for and an of were into the and on an and homozygotes were identified at 2 of by their Heterozygous and wild animals were identified or using tail tip DNA and that the reverse and ent/ent animals were by of Cacna2d2 from exon to exon using the and the reverse were using and an of For μg of DNA were with the on an and were using the Kit (Invitrogen). and were according to standard were on a was using and J. R. R. L. N. PubMed Scopus Google Scholar) with tail tip DNA to and was PCR-amplified as described by or in and a and an were in of the and the cDNA was from wild C57BL/6J animals using the and the reverse a and a The were into (Invitrogen). Cacna2d2 wild and described by M. Dai S. Marais E. Lacinova L. Hofmann F. Klugbauer N. Eur. J. Neurosci. 2000; 12: 1217-1226Crossref PubMed Scopus (104) Google Scholar) were used. Cacna2d2 the exon as well as the first nucleotides of exon and Western protein was by and in and the protein was according to the of as described H. G. H. H. Full Text PDF PubMed Scopus Google Scholar). Western were using M. S. Eur. J. Neurosci. 1998; PubMed Scopus Google Scholar) the described by Marais (12Marais E. Klugbauer N. Hofmann F. Mol. Pharmacol. 2001; 59: 1243-1248Crossref PubMed Scopus (224) Google Scholar) with protein on or as described S. K. M. J. 2000; PubMed Scopus Google Scholar). were on a and and were to °C and into on a were stored on Germany) at -70 was at were in and for and with a of in for 2 followed by Germany) in for were under a Germany). For the was according to E. Klugbauer N. Hofmann F. Mol. Pharmacol. 2001; 59: 1243-1248Crossref PubMed Scopus (224) Google in were calcium into according to standard cells were in for and in for in for with of to in for 2 and subsequently with for were in and under a system Germany). binding of to was using of protein and of in a using membranes F. H. PubMed Scopus Google Scholar). were with protein for at binding was by binding observed in the of 10 of from were in and to liquid data were to the following using specific binding as is the of binding is the binding and is the of 4 mice were with a into the and two the In a was the The was of two between the to a at the using the following as The were of the on a were in the so that only the tip the The were to the with and The mice were to from before in the of animals in a were recorded using a system of the a of was for the of the animals to the Each animal was recorded for a of the of the animals to of were with to and cells from animals were acutely and currents were using from Full Text PDF PubMed Scopus Google Scholar). the region of the was into and for at °C in the in and one in and cells were dissociated by using a The cell was and on in were using an extracellular 10 10 and in 10 M. Dai S. Marais E. Lacinova L. Hofmann F. Klugbauer N. Eur. J. Neurosci. 2000; 12: 1217-1226Crossref PubMed Scopus (104) Google Scholar). currents were by to the extracellular cell voltage were from a of according to the following voltage from to in with a between voltage were at a of °C using an and the and Germany). of were using of one entla phenotype was observed in a heterogeneous and an recessive of The mutation was crossed into a C57BL/6J genetic background for 10 and no of the phenotype was In homozygous between postnatal days and and animals a of the of the animals was altered as with a or an 2 days the of ataxia, ent/ent animals to of paroxysmal dyskinesia day and which to and were by to or ent/ent animals were to on a but were to of paroxysmal dyskinesia. animals were their from postnatal day 10 on and before in homozygous animals was but a were with a homozygous and with a reduced the animals into the C57BL/6J we the entla phenotype to be linked to by the allele of PubMed Scopus Google Scholar). mice, a background in which the mutation was first the a on chromosome 9 near the DNA from one N2 and entla mice were for between C57BL/6J and at from the on chromosome 9 The mutation was mapped to the region between the and at with the VGCC subunit gene Cacna2d2 as a of entla Cacna2d2 and of the Cacna2d2 coding region cDNA revealed an duplication of 117 to exon 3. the region encoded by we an of from cDNA of wild the a was from cDNA from ent/ent to the wild the of cDNA from animals No in expression or be the entla gene as an allelic of that entla is allelic to ducky and Based on the duplication observed at the that the entla allele a duplication exon of the Cacna2d2 using a to the region exon to to exon revealed a in to the wild in and ent/ent DNA and In we no in in and ent/ent DNA using to the exon 2 to to exon exon 4 to to exon or an between the sites at and data no were observed using the on DNA that the in 2 between the at and the at Two were in between the at and the at or between the sites at and using DNA from ent/ent and animals with of the sites at or at in and a reverse of the at in 2 only in mutant DNA and we were to of using the and of using the from ent/ent DNA but from DNA of the revealed that of of and 2 with a The to at in 2 to exon and in 3. 38-kb duplication between have a consisting of the first of followed by the of 2 between two of exon are by of Genet. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The in 2 and the sites no of 2 and revealed of which were identified as and The α2δ-2 in ent/ent mutation was predicted to the unaffected and a duplication of 39 amino acid Western blot of proteins under using an of that mutant protein was and incorporated into the at to wild type. The of for wild and for entla protein indicated that the α2δ-2entla subunit correct cleavage into α2entla and δ proteins. α2entla a the observed in the for the 39 amino acid The of is to be to The α2 protein is (12Marais E. Klugbauer N. Hofmann F. Mol. Pharmacol. 2001; 59: 1243-1248Crossref PubMed Scopus (224) Google Scholar), with the 39 amino acid residues encoded by exon wild α2δ subunits are under the disulfide bonds α2 and δ in a to (12Marais E. Klugbauer N. Hofmann F. Mol. Pharmacol. 2001; 59: 1243-1248Crossref PubMed Scopus (224) Google Scholar). was for wild α2δ-2 of entla under result in a a of the as the α2entla protein under was observed was that the α2entla protein is covalently linked to the δ protein disulfide expression of Cacna2d2entla cDNA in cells with the α1A and β4 subunits as in calcium α2δ-2entla was in the and the an in cells on the extracellular and of the entla subunit were from wild and α2δ-2entla was calcium channel subunits, was more the cell with in the that with calcium channel subunits α2δ-2entla to to the system of entla homozygotes In to ducky animals (14Meier H. Acta Neuropathol. (Berl.). 1968; 11: 15-28Crossref PubMed Scopus (32) Google Scholar, 15Brodbeck J. Davies A. Courtney J.M. Meir A. Balaguero N. Canti C. Moss F.J. Page K.M. Pratt W.S. Hunt S.P. Barclay J. Rees M. Dolphin A.C. J. Biol. Chem. 2002; 277: 7684-7693Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, H. Google Scholar), no system no and no Purkinje cell morphology be cerebellar morphology as from in and of entla from homozygous entla mice revealed a of spike wave with an of Hz 10 and of activity was observed in from the as well as from the cortical a generalized and were for the cortical and hippocampal and were and followed by hippocampal and cortical activity and with paroxysmal dyskinesia. The spike wave were by of the animal and by and In to the described in 4 of a with a of Hz 10 was observed of were in cortical hippocampal revealed of were by of the and No activity be recorded from unaffected animals types of are for human and murine absence epilepsy (3Felix R. Cell Mol. Neurobiol. 2002; 22: 103-120Crossref PubMed Scopus (43) Google Scholar, J.L. X. C. 1990; PubMed Scopus Google Scholar, Neurosci. 1990; PubMed Scopus Google of cortical of hippocampal Hz Hz 4 in a in entla Purkinje currents in Purkinje cells from the cerebellar region of and ent/ent animals days were recorded in the of The were from thereby that the is in animals their are a of in current in entla homozygotes for ent/ent and ent/ent current densities in Purkinje cells of animals from wild current densities at for the were as dependence of activation between the was to be Purkinje cell size by cell between the of to entla α2δ-1 and α2δ-2 subunits high affinity binding sites for In recombinant expression α2δ-1 a affinity compared with α2δ-2 (12Marais E. Klugbauer N. Hofmann F. Mol. Pharmacol. 2001; 59: 1243-1248Crossref PubMed Scopus (224) Google Scholar, J. L. J. Biol. 2001; PubMed Scopus Google Scholar). binding were to binding to cerebellar α2δ-2 is at binding of to cerebellar membranes of ent/ent animals was reduced by compared with membranes from wild animals binding to cerebellar membranes from wild for for for for ent/ent and for The were to and to for α2δ-2 9 that entla membranes possess no altered binding to cerebellar in a In we identified the novel neurological mouse phenotype and the underlying mutation as an allele of the gene encoding the α2δ-2 subunit of The entla phenotype ataxia, paroxysmal dyskinesia, and absence that of VGCC mouse in the allelic Cacna2d2 mutants ducky and In to no anatomical abnormalities of the system be in ent/ent mice. entla are and have the VGCC female a of α2δ-2 for VGCC mouse two distinct types of generalized spike wave were in The with to and with a at a of Hz and at a of 2 The of the and channels in two types of further our into the of absence epilepsy. The entla allele of Cacna2d2 a two of exon 3. The mutant is at with no of of one of the two exon that all for wild are in the are by of Genet. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), from to Genet. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the entla duplication is no was in the of the entla of the were 2 and of the sites 2 and consistent with a a the ducky allele a the of a the murine Cacna2d2 in exon have been for to of all in and P. Mol. Genet. 2002; 11: PubMed Scopus Google Scholar). exon are our entla is the only mouse mutant identified a single The Cacna2d2entla allele to the and of a protein with a acid duplication near the N terminus of the α2 protein. α2 of be in wild and no in protein Western blot data were further consistent with a correct cleavage of the α2δ-2 precursor into α2entla and δ proteins but indicated a of disulfide between the be by in the of α2entla with of disulfide bonds at the with the calcium channel subunits α1A and β4 in cells α2δ-2entla to the in an from The α2δ-2entla subunit to be incorporated into calcium channels. The in maximum binding in ent/ent cerebellar membranes is consistent with the of two binding sites in the wild H. Pharmacol. Full Text PDF PubMed Scopus Google Scholar), the α2δ-1 and α2δ-2 subunits. In the α2δ-1 the amino acid residues to in the α2δ-2entla subunit to the α2 or δ proteins by J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. D.L. Biochem. J. 1999; PubMed Scopus Google Scholar). is the ligand binding of the α2δ-2 to α2entla with binding be that the binding in ent/ent membranes be to the α2δ-1 observed no of or the α2δ-1 subunit in homozygous entla mice the the the α2δ-2 subunit to binding in wild the in between the α2δ-1 and α2δ-2 subunits in recombinant expression and of data of subunits with α1 and subunits, of the in affinity between wild and entla membranes to of α2δ-1 and α2δ-2 subunits to binding in further is used for or of and generalized in absence epilepsy be 2002; 8: Scopus Google Scholar). calcium current densities A. Spadoni F. Bernardi G. Neuropharmacology. 1998; 37: 83-91Crossref PubMed Scopus (192) Google Scholar) and an effect to that of the entla mutation been that in in or absence ataxia is one of the of E. 2001; PubMed Scopus Google Scholar). that α2δ subunit be a for to In to a of the entla voltage-gated calcium currents were in from ent/ent mice and littermates. ataxia is a of cerebellar cerebellar Purkinje cells, which are to the α2δ-2 subunit (8Barclay J. Balaguero N. Mione M. Ackerman S.L. Letts V.A. Brodbeck J. Canti C. Meir A. Page K.M. Kusumi K. Perez-Reyes E. Lander E.S. Frankel W.N. Gardiner R.M. Dolphin A.C. Rees M. J. Neurosci. 2001; 21: 6095-6104Crossref PubMed Google Scholar, 11Hobom M. Dai S. Marais E. Lacinova L. Hofmann F. Klugbauer N. Eur. J. 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