Mutation of plasma membrane Ca2+ ATPase isoform 3 in a family with X-linked congenital cerebellar ataxia impairs Ca2+ homeostasis

Abstract

Ca(2+) in neurons is vital to processes such as neurotransmission, neurotoxicity, synaptic development, and gene expression. Disruption of Ca(2+) homeostasis occurs in brain aging and in neurodegenerative disorders. Membrane transporters, among them the calmodulin (CaM)-activated plasma membrane Ca(2+) ATPases (PMCAs) that extrude Ca(2+) from the cell, play a key role in neuronal Ca(2+) homeostasis. Using X-exome sequencing we have identified a missense mutation (G1107D) in the CaM-binding domain of isoform 3 of the PMCAs in a family with X-linked congenital cerebellar ataxia. PMCA3 is highly expressed in the cerebellum, particularly in the presynaptic terminals of parallel fibers-Purkinje neurons. To study the effects of the mutation on Ca(2+) extrusion by the pump, model cells (HeLa) were cotransfected with expression plasmids encoding its mutant or wild-type (wt) variants and with the Ca(2+)-sensing probe aequorin. The mutation reduced the ability of the PMCA3 pump to control the cellular homeostasis of Ca(2+). It significantly slowed the return to baseline of the Ca(2+) transient induced by an inositol-trisphosphate (InsP(3))-linked plasma membrane agonist. It also compromised the ability of the pump to oppose the influx of Ca(2+) through the plasma membrane capacitative channels.

Fig. 1.

Pedigree of the X-linked congenital ataxia family and mutation in ATP2B3. (A) Pedigree of the family with two affected males in different sibships suggesting X-linked inheritance. (B) Partial ATP2B3 Sanger sequence chromatograms are shown for a normal control (Bottom), the index patient (III:1) (Middle), and his mother (II:2) (Top). The mutated amino acid change is shown on top. The mutated nucleotide in the index patient is marked by an arrow. ATPB3 cDNA mutation is annotated according to human sequence GenBank accession no. NM_001001344, where +1 corresponds to the A of the ATG translation initiation codon.

Fig. 2.

Alternative splicing mechanism of PMCA3 at site C and analysis of the expression of exogenous PMCA3 variants in HeLa cells. (A) The numbers 1 and 2 indicate the splicing positions. The insertion of an exon generates a premature stop codon, leading to the formation of truncated isoform a. In 2, the pale blue exon is removed originating the full-length b isoform. The sequences of the two alternative C-terminal CaM binding domains generated by the splicing are shown in the right inbox of A. (B and C) Immunocytochemistry (B) and Western blot (C) analysis of PMCA3 distribution and expression level in HeLa cells expressing wt and mutant PMCA3 constructs. The PMCA3 was revealed by the 5F10 and the anti-PMCA3–specific antibody.

Fig. 3.

Cytosolic Ca²⁺ measurements in HeLa cells overexpressing the wt and the mutant PMCA3 isoforms. Cells were cotransfected with cytAEQ and the expression plasmid for the a (A) or the b (B) wt, as well as mutated, PMCA3 variants. Cytosolic Ca²⁺ transients (A and B) were recorded following histamine stimulation, and the average peak values (C and D), as well as the mean slope of Ca²⁺ transients, were calculated at the half time of the peak decay (E and F). Insets in E and F show histograms representing the half time of the peak decay of Ca²⁺ transients. Bars in C and D represent mean [Ca²⁺] values upon stimulation (μM ± SEM). Bars in the Insets represent mean time for half-peak (seconds ± SEM). The traces and the histograms were obtained by averaging 15 independent experiments for each condition. ***P < 0.001; **P < 0.01; *P ≤ 0.05.

Fig. 4.

Effect of the truncated (A and B) and full-length (C and D) wt and mutant pump on the ER Ca²⁺ mobilization and on the Ca²⁺ influx from the extracellular ambient. HeLa cells were cotransfected with cytAEQ and the PMCA3 constructs or transfected with cytAEQ only, perfused in KRB/EGTA buffer, and stimulated with histamine to release Ca²⁺ from the intracellular stores (first peak). Then, the perfusion medium was switched to KRB/Ca²⁺ (in the continuous presence of histamine) to stimulate Ca²⁺ entry from the extracellular ambient (second peak). The Ca²⁺ transients in A are those for the PMCA3a isoform, and the Ca²⁺ transients in C are those for the b variant. They are representative of at least six independent experiments. B and D show the averaged peak [Ca²⁺] values obtained upon stimulation. Bars represent means ± SEM obtained by averaging the values obtained in six independent experiments for each condition. **P < 0.01; *P ≤ 0.05. Ns., not significant.

Fig. 5.

Protein sequence alignment of human PMCA3 with its orthologs and molecular modeling of the interaction of the wt or mutated CaM-binding domain of PMCA and CaM. (A) Only the sequence around the variants p.G1107D in PMCA3 (highlighted in red) is shown. The p.G1107 residue is evolutionarily conserved throughout species. (B) The NMR structure of human PMCA4 CaM binding domain (PDB code 2KNE), which shares 100% sequence identity to the CaM-binding domain of human PMCA3, was used as a template (cyan helix). The p.G1096 residue, corresponding to p.G1107 of PMCA3, is highlighted in magenta. (C) Molecular model of mutated CaM-binding domain with substitution of glycine (G) with aspartic acid (D). As calculated by UCSF Chimera, all rotamers of the aspartic side (D) chain create atomic clashes [i.e., unfavorable interactions, with CaM (gray helix), in particular with residues L112 and E114 (in green)].