Survival strategies for mouse cerebellar Purkinje neurons lacking PMCA2.

Expression of the fast calcium extrusion protein, PMCA2, in the cerebellum is amongst the highest found throughout the central nervous system, and unsurprisingly PMCA2 knockout mice exhibit cerebellar ataxia or loss of controlled movement. The sole output neurons of the cerebellar cortex, Purkinje neurons, are functionally compromised in these knockout mice, yet remarkably these neurons survive. In this mini-review we review and speculate on the importance of multiple PMCA2 dependent actions at cellular and synaptic sites within the cerebellar Purkinje neuron network. We also explore how loss of PMCA2-/- can lead to the ataxic phenotype, but can paradoxically also minimise calcium rises in cerebellar Purkinje neurons, thereby ensuring their resilience and survival.

Effects of Disruption of PMC1 in the tfp1∆/∆ Mutant on Calcium Homeostasis, Oxidative and Osmotic Stress Resistance in Candida albicans.

The vacuolar-type H+-ATPase (V-ATPase) is essential for many cell processes. Our previous study has demonstrated that Tfp1 is a putative subunit of V-ATPase, loss of which causes disorders in calcium homeostasis and decreased resistance to oxidative stress. In this study, we found that further deletion of PMC1, a vacuolar calcium pump, in tfp1∆/∆ mutant led to more severe dysregulation of calcium homeostasis. Besides, the tfp1∆/∆pmc1∆/∆ mutant was more sensitive to H2O2 and had a higher ROS level. As is known, V-ATPase mutants are sensitive to NaCl, and PMC1 mutant is resistant against NaCl. However, the tfp1∆/∆pmc1∆/∆ mutant exhibited sensitivity to NaCl. Mechanism study demonstrated that their sensitivity was associated with reduced osmotic resistance caused by relatively low expression of GPD1. In addition, we first found that NaCl addition significantly declined ROS levels in tfp1∆/∆ and tfp1∆/∆pmc1∆/∆ mutants. In tfp1∆/∆ mutant, decreased ROS levels were relevant to enhanced antioxidant activities. However, in tfp1∆/∆pmc1∆/∆ mutant, reduced ROS resulted from decreased total calcium content, revealing that NaCl affected ROS levels in the two mutants through different mechanisms. Taken together, our data indicated that loss of both TFP1 and PMC1 further affected calcium homeostasis and other cellular processes in Candida albicans and provides a potential antifungal target.

Reduced expression of PMCA1 is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries.

Hypertension is a well-established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1Ht ) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age-matched wild-type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.

Deletion of the intestinal plasma membrane calcium pump, isoform 1, Atp2b1, in mice is associated with decreased bone mineral density and impaired responsiveness to 1, 25-dihydroxyvitamin D3.

The physiological importance of the intestinal plasma membrane calcium pump, isoform 1, (Pmca1, Atp2b1), in calcium absorption and homeostasis has not been previously demonstrated in vivo. Since global germ-line deletion of the Pmca1 in mice is associated with embryonic lethality, we selectively deleted the Pmca1 in intestinal absorptive cells. Mice with loxP sites flanking exon 2 of the Pmca1 gene (Pmca1(fl/fl)) were crossed with mice expressing Cre recombinase in the intestine under control of the villin promoter to give mice in which the Pmca1 had been deleted in the intestine (Pmca1(EKO) mice). Pmca1(EKO) mice were born at a reduced frequency and were small at the time of birth when compared to wild-type (Wt) littermates. At two months of age, Pmca1(EKO) mice fed a 0.81% calcium, 0.34% phosphorus, normal vitamin D diet had reduced whole body bone mineral density (P < 0.037), and reduced femoral bone mineral density (P < 0.015). There was a trend towards lower serum calcium and higher serum parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) concentrations in Pmca1(EKO) mice compared to Wt mice but the changes were not statistically significant. The urinary phosphorus/creatinine ratio was increased in Pmca1(EKO) mice (P < 0.004). Following the administration of 200 ng of 1α,25(OH)2D3 intraperitoneally to Wt mice, active intestinal calcium transport increased ∼2-fold, whereas Pmca1(EKO) mice administered an equal amount of 1α,25(OH)2D3 failed to show an increase in active calcium transport. Deletion of the Pmca1 in the intestine is associated with reduced growth and bone mineralization, and a failure to up-regulate calcium absorption in response to 1α,25(OH)2D3.

Impaired nitric oxide production and increased blood pressure in systemic heterozygous ATP2B1 null mice.

BACKGROUND: In the 'Millennium Genome Project', we identified ATP2B1 as a gene responsible for hypertension through single-nucleotide polymorphism analysis. The ATP2B1 gene encodes the plasma membrane calcium ATPase isoform 1, which contributes to the maintenance of intracellular calcium homeostasis by removing calcium ions. METHOD: Since ATP2B1 knockout mice are reported to be embryo-lethal, we generated systemic heterozygous ATP2B1 null (ATP2B1(+/-)) mice, and evaluated the implication of ATP2B1 in blood pressure. RESULTS: ATP2B1(+/-) mice revealed significantly higher SBP as measured by a radiotelemetric method. Phenylephrine-induced vasoconstriction was significantly increased in vascular rings from ATP2B1(+/-) mice, and the difference in this contraction disappeared in the presence of a nitric oxide synthase (NOS) inhibitor. Vasorelaxation to acetylcholine was significantly attenuated in vascular rings from ATP2B1(+/-) mice. In addition, cultured endothelial cells of ATP2B1(+/-) mice showed that the phosphorylation (Ser-1177) level of endothelial NOS protein was significantly lower, and nitric oxide production in endothelial cells and aorta was lower compared with those in control mice. In contrast, neural NOS expression in vascular smooth muscle cells from ATP2B1(+/-) mice and control mice were not significantly different. CONCLUSION: These results suggest that decreased ATP2B1 gene expression is associated with impaired endothelial NOS activity and nitric oxide production, and the ATP2B1 gene plays a crucial role in the regulation of blood pressure.

A new Atp2b2 deafwaddler allele, dfw(i5), interacts strongly with Cdh23 and other auditory modifiers.

Tight regulation of calcium (Ca2+) concentrations in the stereocilia bundles of auditory hair cells of the inner ear is critical to normal auditory transduction. The plasma membrane Ca2+ ATPase 2 (PMCA2), encoded by the Atp2b2 gene, is the primary mechanism for clearance of Ca2+ from auditory stereocilia, keeping intracellular levels low, and also contributes to maintaining adequate levels of extracellular Ca2+ in the endolymph. This study characterizes a novel null Atp2b2 allele, dfw(i5), by examining cochlear anatomy, vestibular function and auditory physiology in mutant mice. Loss of auditory function in PMCA2 mutants can be attributed to dysregulation of intracellular Ca2+ inside the stereocilia bundles. However, extracellular Ca2+ ions surrounding the stereocilia are also required for rigidity of cadherin 23, a component of the stereocilia tip-link encoded by the Cdh23 gene. This study further resolves the interaction between Atp2b2 and Cdh23 in a gene dosage and frequency-dependent manner, and finds that low frequencies are significantly affected by the interaction. In +/dfw(i5) mice, one mutant copy of Cdh23 is sufficient to cause broad frequency hearing impairment. Additionally, we report another modifying interaction with Atp2b2 on auditory sensitivity, possibly caused by an unidentified hearing loss gene in mice.

Enhanced synaptic inhibition in the cerebellar cortex of the ataxic PMCA2(-/-) knockout mouse.

Mice with genetic deletion of a calcium extrusion pump, the plasma membrane calcium ATPase isoform 2, PMCA2, exhibit overt cerebellar ataxia, but the cellular mechanisms are only partially understood. Here, we report an enhanced synaptic GABAergic inhibition within the molecular layer of cerebellar cortex slices from PMCA2 knockout (PMCA2(-/-)) mice, a finding that could contribute to the observed ataxia. Purkinje neurons from PMCA2(-/-) mice exhibited an increased frequency and amplitude of spontaneous inhibitory post-synaptic currents that was accompanied by an enhanced spontaneous firing frequency of molecular layer interneurons (both basket cells and stellate cells). The elevated inhibition was sufficient to reduce the frequency and regularity of spike firing by PMCA2(-/-) Purkinje neurons. Acute pharmacological inhibition of PMCA recapitulated some of these features in wild-type mice indicating that the changes were in part a direct result of PMCA2 removal. However, additional compensatory mechanisms within the PMCA2(-/-) mouse were also a major factor. Indeed, morphological studies revealed an abnormally large number of molecular layer interneurons (basket cells and stellate cells) and GABAergic synapses within the PMCA2(-/-) cerebellar cortex. We conclude that loss of PMCA2 adversely influences the function and organisation of Purkinje neuron synaptic inhibition as a major contributory mechanism to the ataxic phenotype of the PMCA2(-/-) mouse.

ATP2B1 and blood pressure: from associations to pathophysiology.

PURPOSE OF REVIEW: Recent genome-wide association studies (GWAS) have revealed that the ATP2B1 gene is associated with hypertension not only in people of European origin, but also in Japanese, Chinese, and Koreans. However, ATP2B1 has never been considered to be a candidate gene for essential hypertension. Thus, this review summarizes the findings obtained in GWAS regarding the role of the ATP2B1 gene in essential hypertension, as well as recent suggestions about the mechanisms responsible for the effects of the ATP2B1 gene on calcium homeostasis. We also review the findings of studies involving spontaneously hypertensive rats and tissue-specific ATP2B1 knockout mice examining the effects of ATP2B1 on hypertension. RECENT FINDINGS: The ATP2B1 gene has been revealed to be a hypertension-susceptibility gene in large-scale GWAS studies. Meta-analysis of the ATP2B1 gene polymorphisms associated with hypertension confirmed that ATP2B1 is significantly associated with hypertension in East Asians. Moreover, vascular smooth muscle cell ATP2B1 knockout mice exhibited high blood pressure in radio telemetry-based experiments. SUMMARY: The ATP2B1 gene has been demonstrated to have a strong influence on blood pressure. Detailed analysis of tissue-specific knockout mice is expected to further confirm the role of ATP2B1 in the near future.

Plasma membrane calcium ATPases and related disorders.

The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia.

Mice lacking hypertension candidate gene ATP2B1 in vascular smooth muscle cells show significant blood pressure elevation.

We reported previously that ATP2B1 was one of the genes for hypertension receptivity in a large-scale Japanese population, which has been replicated recently in Europeans and Koreans. ATP2B1 encodes the plasma membrane calcium ATPase isoform 1, which plays a critical role in intracellular calcium homeostasis. In addition, it is suggested that ATP2B1 plays a major role in vascular smooth muscle contraction. Because the ATP2B1 knockout (KO) mouse is embryo-lethal, we generated mice with vascular smooth muscle cell-specific KO of ATP2B1 using the Cre-loxP system to clarify the relationship between ATP2B1 and hypertension. The KO mice expressed significantly lower levels of ATP2B1 mRNA and protein in the aorta compared with control mice. KO mice showed significantly higher systolic blood pressure as measured by tail-cuff method and radiotelemetric method. Similar to ATP2B1, the expression of the Na(+)-Ca(2+) exchanger isoform 1 mRNA was decreased in vascular smooth muscle cells of KO mice. However, ATP2B4 expression was increased in KO mice. The cultured vascular smooth muscle cells of KO mice showed increased intracellular calcium concentration not only in basal condition but also in phenylephrine-stimulated condition. Furthermore, phenylephrine-induced vasoconstriction was significantly increased in vascular rings of the femoral artery of KO mice. These results suggest that ATP2B1 plays important roles in the regulation of blood pressure through alteration of calcium handling and vasoconstriction in vascular smooth muscle cells.

The role of the calcium transporter protein plasma membrane calcium ATPase PMCA2 in cerebellar Purkinje neuron function.

Genetic deletion of the plasma membrane calcium ATPase type 2 (PMCA2), a calcium transporter protein, is associated with an overtly ataxic phenotype in mice. PMCA2 is expressed at high levels in cerebellar Purkinje neurons (PNs) where functional integrity is essential for normal cerebellar function. Indeed, loss of PN function accompanies cerebellar ataxia in humans and mouse models. In the ataxic PMCA2 knockout (PMCA2-/-) mouse the ability of the PNs to control their cytosolic calcium levels was severely impaired; basal calcium levels were high and calcium recovery kinetics slow. Whole cell patch clamp recordings from PMCA2-/- PNs revealed that they possessed hyperpolarised membrane potentials, reduced frequency and increased irregularity of spontaneous action potential firing, curtailed complex spikes and sustained calcium-dependent outward K+ currents. We propose that these alterations limit pathological excursions in PN cytosolic calcium as an aid to survival but that they are insufficient to prevent loss of functional cerebellar output.

Plasma membrane Ca2+ ATPase 2 contributes to short-term synapse plasticity at the parallel fiber to Purkinje neuron synapse.

Plasma membrane Ca2+ ATPase 2 (PMCA2) is a fast, highly effective mechanism to control resting cytosolic Ca2+ and Ca2+ excursions in neurons and other excitable cells. The strong expression of PMCA2 in the cerebellum and the cerebellar behavioral deficits presented by PMCA2-/- knock-out mice all point to its importance for cerebellar circuit dynamics. Here, we provide direct functional evidence for the influence of presynaptic PMCA2-mediated Ca2+ extrusion for short-term plasticity at cerebellar parallel fiber to Purkinje neuron synapses. Dramatic structural alterations to the Purkinje neurons in the absence of PMCA2 also suggest a strong influence of this fast PMCA2 isoform for development and maintenance of cerebellar function.

3p-- syndrome defines a hearing loss locus in 3p25.3.

Deletions affecting the terminal end of chromosome 3p result in a characteristic set of clinical features termed 3p-- syndrome. Bilateral, sensorineural hearing loss (SNHL) has been found in some but not all cases, suggesting the possibility that it is due to loss of a critical gene in band 3p25. To date, no genetic locus in this region has been shown to cause human hearing loss. However, the ATP2B2 gene is located in 3p25.3, and haploinsufficiency of the mouse homolog results in SNHL with similar severity. We compared auditory test results with fine deletion mapping in seven previously unreported 3p-- syndrome patients and identified a 1.38Mb region in 3p25.3 in which deletions were associated with moderate to severe, bilateral SNHL. This novel hearing loss locus contains 18 genes, including ATP2B2. ATP2B2 encodes the plasma membrane calcium pump PMCA2. We used immunohistochemistry in human cochlear sections to show that PMCA2 is located in the stereocilia of hair cells, suggesting its function in the auditory system is conserved between humans and mice. Although other genes in this region remain candidates, we conclude that haploinsufficiency of ATP2B2 is the most likely cause of SNHL in 3p-- syndrome.

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice.

We previously showed that plasma membrane Ca(2+)-ATPase (PMCA) activity accounted for 25-30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca(2+) ([Ca(2+)](i)) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1(+/-), Pmca4(+/-), Pmca4(-/-), and Pmca1(+/-)Pmca4(-/-) mice. There were no differences in basal [Ca(2+)](i) values between bladder preparations. KCl (80 mM) elicited both larger forces (150-190%) and increases in [Ca(2+)](i) (130-180%) in smooth muscle from Pmca1(+/-) and Pmca1(+/-)Pmca4(-/-) bladders than those in WT or Pmca4(-/-). The responses to carbachol (CCh: 10 muM) were also greater in Pmca1(+/-) (120-150%) than in WT bladders. In contrast, the responses in Pmca4(-/-) and Pmca1(+/-)Pmca4(-/-) bladders to CCh were significantly smaller (40-50%) than WT. The rise in half-times of force and [Ca(2+)](i) increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4(-/-) (130-190%) and Pmca1(+/-)Pmca4(-/-) (120-250%) bladders, but not in Pmca1(+/-) bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca(2+) clearance, while PMCA4 is essential for the [Ca(2+)](i) increase and contractile response to the CCh receptor-mediated signal transduction pathway.

Molecular alterations in the cerebellum of the plasma membrane calcium ATPase 2 (PMCA2)-null mouse indicate abnormalities in Purkinje neurons.

PMCA2, a major calcium pump, is expressed at particularly high levels in Purkinje neurons. Accordingly, PMCA2-null mice exhibit ataxia suggesting cerebellar pathology. It is not yet known how changes in PMCA2 expression or activity affect molecular pathways in Purkinje neurons. We now report that the levels of metabotropic glutamate receptor 1 (mGluR1), which plays essential roles in motor coordination, synaptic plasticity, and associative learning, are reduced in the cerebellum of PMCA2-null mice as compared to wild type littermates. The levels of inositol 1,4,5-triphosphate receptor type 1 (IP3R1), an effector downstream to mGluR1, which mediates intracellular calcium signaling, and the expression of Homer 1b/c and Homer 3, scaffold proteins that couple mGluR1 to IP3R1, are also reduced in somata and dendrites of some Purkinje cell subpopulations. In contrast, no alterations occur in the levels of mGluR1 and its downstream effectors in the hippocampus, indicating that the changes are region specific. The reduction in cerebellar mGluR1, IP3R1 and Homer 3 levels are neither due to a generic decrease in Purkinje proteins nor extensive dendritic loss as immunoreactivity to total and non-phosphorylated neurofilament H (NFH) is increased in Purkinje dendrites and microtubule associated protein 2 (MAP2) staining reveals a dense dendritic network in the molecular layer of the PMCA2-null mouse cerebellum. PMCA2 coimmunoprecipitates with mGluR1, Homer 3 and IP3R1, suggesting that the calcium pump is a constituent of the mGluR1 signaling complex. Our results suggest that the decrease in the expression of mGluR1 and its downstream effectors and perturbations in the mGluR1 signaling complex in the absence of PMCA2 may cumulatively result in aberrant metabotropic glutamate receptor signaling in Purkinje neurons leading to cerebellar deficits in the PMCA2-null mouse.