The type 1 inositol 1,4,5-trisphosphate receptor gene is altered in the opisthotonos mouse

Abstract

The opisthotonos (opt) mutation arose spontaneously in a C57BL/Ks-db2J colony and is the only known, naturally occurring allele of opt. This mutant mouse was first identified based on its ataxic and convulsive phenotype. Genetic and molecular data presented here demonstrate that the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) protein, which serves as an IP3-gated channel to release calcium from intracellular stores, is altered in the opt mutant. A genomic deletion in the IP3R1 gene removes two exons from the IP3R1 mRNA but does not interrupt the translational reading frame. The altered protein is predicted to have lost several modulatory sites and is present at markedly reduced levels in opt homozygotes. Nonetheless, a strong calcium release from intracellular stores can be elicited in cerebellar Purkinje neurons treated with the metabotropic glutamate receptor (mGluR) agonist quisqualate (QA). QA activates Group 1 mGluRs linked to GTP-binding proteins that stimulate phospholipase C and subsequent production of the intracellular messenger IP3, leading to calcium mobilization via the IP3R1 protein. The calcium response in opt homozygotes shows less attenuation to repeated QA application than in control littermates. These data suggest that the convulsions and ataxia observed in opt mice may be caused by the physiological dysregulation of a functional IP3R1 protein.

Fig. 3.

Expression of IP₃R1 mRNA inopt brain. A, IP₃R1 transcript size is reduced in opt homozygote mice. Fromleft to right, lanes 1–5 represent total brain RNA from a P13 C57BL/6J-+/+, P13 Mi^whhomozygote, P11 Mi^wh/optheterozygote, adult Mi^wh/optheterozygote, and P13 opt homozygote mouse. An identically loaded Northern gel was electrophoresed for a shorter duration in parallel to the one shown here to retain smaller size transcripts. This blot was probed with EF 1 α (Xiang and Werner, 1989) to control for lane loading. B, RT-PCR analysis of IP₃R1 mRNA with primers 11F/12R; lanes 1–5as above. C, Primary structure of altered IP₃R1 mRNA species in opt homozygotes generated with primers 11F/12R. Primer 12R is underlinedby an arrow. Primer 11F located at nt 4875–4897 is not shown. The first and second line contain the amino acid (Furuichi et al., 1989b) and nucleotide sequence (Furuichi et al., 1989a) of the cerebellar IP₃R1 cDNA, respectively. The nucleotide sequence is numbered from the 5′ to 3′ direction where +1 was assigned to the first base of the predicted initiation codon. Line three displays a nucleotide sequence of the RT-PCR product fromMi^wh homozygote RNA, and lines 4–7 display the four different products fromopt homozygote RNA, labeled 1–4. Exon borders determined by genomic sequence analysis are indicated byvertical lines. A GKA and PKA phosphorylation site iscircled, and a putative ATP-binding domain isboxed. Asterisks denote amino acids used to generate antibody SP-2A.

Fig. 5.

Genomic analysis of opt locus.A, Comparison of wild-type and mutant genomic structure reveals deletion in opt DNA. Wild-type composite diagram (top line) was constructed using restriction mapping, Southern blot hybridization, sequencing, and PCR-based approaches in B6/CBA-+/+ genomic DNA derived from a λ phage library. Exons are depicted as filled boxes and intervening introns as asolid line. Bases of the mouse cerebellar IP₃R1 cDNA corresponding to the beginning and end of each exon are as follows: A (5074–5142), B (5143–5193), C (5194–5517), and D (5518–5698). 1F, 6R, 7F, and8R are expand PCR primers. Asterisksindicate the location of five PCR primer pairs used to confirm the extent of the genomic deletion in opt. The lower line depicts an opt homozygote genomic DNA map based on restriction analysis of subcloned fragments derived from a custom-made opt homozygote λ phage library. Restriction endonuclease sites are designated as follows:S, SacI; X,XbaI; and N, NsiI. Not all restriction sites are shown. B, The deletion inopt DNA fuses introns 1 and 3. Exon A isboxed, intron 3 sequence isshaded, and PCR primers are designated byarrows. C, Southern blot hybridization analysis demonstrates that a 3.4 kb NsiI fragment is deleted in opt genomic DNA. D, The intron/exon slice boundaries for exons A, B, C, and Dare compared with a consensus splice sequence.

Fig. 1.

Location of the IP₃R1 gene relative to the opt locus. A, An intersubspecific intercross was established segregating for opt.B, Four microsatellites and a novel PCR marker derived from the IP₃R1 gene were mapped in the 56 progeny from the intersubspecific intercross. The number of recombination events observed among the 112 intercross chromosomes analyzed, and genetic distances in centimorgans (±SE) separating adjacent loci, are represented by the numbers to the left of the chromosome. The human homolog of the mouse IP₃R1 gene maps to the short arm of chromosome 3, as noted inparentheses to the right of the mouse gene (Yamada et al., 1994). C, One hundred twelve chromosomes from the 56 intersubspecific intercross progeny were scored for parental C57BL/Ks-opt/opt(open boxes) and CAST/Ei-+/+ DNAs (filled boxes) at five loci. The number of chromosomes for each haplotype is shown below the columns.

Fig. 2.

Heterozygosity at the IP₃R1 locus. A, Maintenance of the opt colony.B, Southern blot hybridization analysis demonstrates an interstrain RFLV with a probe derived from the 3′ untranslated region of the IP₃R1 gene.

Fig. 4.

Expression of IP₃R1 protein inopt cerebellum. Western blot analysis of cerebellar microsomal membrane fractions from a 3-month-old C57BL/6-+/+, P12Mi^wh homozygote, P12Mi^wh/opt heterozygote, P12opt homozygote, and P19 opt homozygote with antibodies SP-3A and SP-2A (Lin, 1995). P12 pups are littermates.

Fig. 6.

Paraffin sections of cerebellum stained with cresyl violet. P4 pups are shown in A–D, P23 mice inE–H. Control C57BL/6J-+/+ mice are shown on theleft, opt homozygote mice on theright. Scale bars: A, B, E, F, 200 μm; C, D, G, H, 50 μm.

Fig. 7.

Physiological analysis of optmutation. A, Examples of experiments in +/+ (left) and opt/opt(right) cerebellar slices showing 340/380 ratios of each of several Purkinje neurons in the field under control conditions and in response to repeated 30 sec 100 μm QA application in Ca²⁺-free Ringer’s solution. Eachcolored trace represents the response from one individual Purkinje neuron. Images were taken at points indicated in traces above where A is the control, andB and C are the responses to the first and second QA application, respectively. Blue bars show periods of Ca²⁺-free Ringer’s solution flow. Higher 340/380 ratios refer to higher Ca²⁺ ion levels on the color scale. B, Histogram of amplitudes of changes in the ratio of 340/380 signals in response to the first QA application compared betweenopt/opt (n = 4 animals), opt/+ (n = 1),opt/Mi^wh(n = 7), and +/+ (n = 2) pups.Numbers above SE bars are the number of neurons. The number of +/+ and opt/+ mice is limited, because they are produced only through recombination in theopt/Mi^wh intercross.C, Comparison of successive QA responses plotted relative to the intensity changes for the first response of each genotype [opt/opt (n= 4), opt/+ (n = 1),opt/Mi^wh(n = 7 and 3 for second and third response, respectively), and +/+ (n = 2)]. All genotypes were significant at >0.01 level compared with opthomozygotes.