Plasma Membrane Calcium ATPase Isoform Research Articles

Plasma membrane calcium ATPase deficiency causes neuronal pathology in the spinal cord: a potential mechanism for neurodegeneration in multiple sclerosis and spinal cord injury

Dysfunction and death of spinal cord neurons are critical determinants of neurological deficits in various pathological conditions, including multiple sclerosis (MS) and spinal cord injury. Yet, the molecular mechanisms underlying neuronal/axonal damage remain undefined. Our previous studies raised the possibility that a decrease in the levels of plasma membrane calcium ATPase isoform 2 (PMCA2), a major pump extruding calcium from neurons, promotes neuronal pathology in the spinal cord during experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and after spinal cord trauma. However, the causal relationship between alterations in PMCA2 levels and neuronal injury was not well established. We now report that inhibition of PMCA activity in purified spinal cord neuronal cultures delays calcium clearance, increases the number of nonphosphorylated neurofilament H (SMI-32) immunoreactive cells, and induces swelling and beading of SMI-32-positive neurites. These changes are followed by activation of caspase-3 and neuronal loss. Importantly, the number of spinal cord motor neurons is significantly decreased in PMCA2-deficient mice and the deafwaddler(2J), a mouse with a functionally null mutation in the PMCA2 gene. Our findings suggest that a reduction in PMCA2 level or activity leading to delays in calcium clearance may cause neuronal damage and loss in the spinal cord.

Loss of Autoinhibition of the Plasma Membrane Ca2+ Pump by Substitution of Aspartic 170 by Asparagine: ACTIVATION OF PLASMA MEMBRANE CALCIUM ATPase 4 WITHOUT DISRUPTION OF THE INTERACTION BETWEEN THE CATALYTIC CORE AND THE C-TERMINAL REGULATORY DOMAIN

The plasma membrane calcium ATPase (PMCA) actively transports Ca(2+) from the cytosol to the extra cellular space. The C-terminal segment of the PMCA functions as an inhibitory domain by interacting with the catalytic core. Ca(2+)-calmodulin binds to the C-terminal segment and stops inhibition. Here we showed that residue Asp(170), in the putative "A" domain of human PMCA isoform 4xb, plays a critical role in autoinhibition. In the absence of calmodulin a PMCA containing a site-specific mutation of D170N had 80% of the maximum activity of the calmodulin-activated PMCA and a similar high affinity for Ca(2+). The mutation did not change the activation of the PMCA by ATP. Deletion of the C-terminal segment further downstream of the calmodulin-binding site led to an additional increase in the maximal activity of the mutant, which suggests that the mutation did not affect the inhibition because of this portion of the C-terminal segment. The calmodulin-activated PMCA was more sensitive to vanadate inhibition than the autoinhibited enzyme. In contrast, inhibition of the D170N mutant required higher concentrations of vanadate and was not affected by calmodulin. Despite its higher basal activity, the mutant had an apparent affinity for calmodulin similar to that of the wild type enzyme, and its rate of proteolysis at the C-terminal segment was still calmodulin-dependent. Altogether these results suggest that activation by mutation D170N does not involve the displacement of the calmodulin-binding autoinhibitory domain from the catalytic core and may arise directly from changes in the accessibility to the calcium-binding residues of the pump.

Expression of calcium transporters in the retina of the tiger salamander (Ambystoma tigrinum).

Changes in intracellular calcium concentration, [Ca2+]i, modulate the flow of visual signals across all stages of processing in the retina, yet the identities of Ca2+ transporters responsible for these changes are still largely unknown. In the current study, the distribution of plasma membrane and intracellular Ca2+ transporters in the retina of tiger salamander, a model system for physiological studies of retinal function, was determined. Plasma membrane calcium ATPases (PMCAs), responsible for high-affinity Ca2+ extrusion, were highly expressed in the salamander retina. PMCA isoforms 1, 2, and 4 were localized to photoreceptors, whereas the inner retina expressed all four isoforms. PMCA3 was expressed in a sparse population of amacrine and ganglion neurons, whereas PMCA2 was expressed in most amacrine and ganglion cells. Na+/Ca2+ exchangers, a high-capacity Ca2+ extrusion system, were expressed in the outer plexiform layer and in a subset of inner nuclear and ganglion layer cells. Intracellular Ca2+ store transporters were also represented prominently. SERCA2a, a splice variant of the sarcoplasmic-endoplasmic Ca2+ ATPase, was found mostly in photoreceptors, whereas SERCA2b was found in the majority of retinal neurons and in glial cells. The predominant endoplasmic reticulum (ER) Ca2+ channels in the salamander retina are represented by the isoform 2 of the IP3 receptor family and the isoform 2 of the ryanodine receptor family. These results indicate that Ca2+ transporters in the salamander retina are expressed in a cell type-specific manner.

Haplo-insufficiency revealed in deafwaddler mice when tested for hearing loss and ataxia

The auditory and vestibular systems rely on the plasma membrane calcium ATPase, isoform 2 (PMCA2) to extrude calcium that enters the stereocilia during transduction. Mutations in the gene encoding this protein result in recessive sensorineural deafness and ataxia in the deafwaddler mouse. In this study, we report the identification of a new allele of deafwaddler, dfw 3 J . This allele contains a 4-nucleotide deletion resulting in a frame-shift and predicted truncation of PMCA2. No protein is detected in dfw 3 J homozygotes. To examine the dependence of auditory and vestibular function on PMCA2 activity, we compared dfw 3 J with another functional null allele, dfw 2 J , and the partial loss-of-function allele, dfw. All mice studied were in the good-hearing CBA/CaJ background. Heterozygotes of either functional null allele displayed highly significant hearing loss by auditory-evoked brainstem responses relative to controls ( P<0.0001), particularly at high frequencies (>24 kHz). Ataxia was also apparent in these mice on an accelerating rotarod ( P<0.05). In contrast, +/ dfw mice were not measurably different from controls in either behavioral test. dfw/dfw mice were deaf, but showed less ataxia than dfw 2 J / dfw 2 J or dfw 3 J / dfw 3 J mice. These results demonstrate that hearing loss and ataxia are dependent on gene dosage and PMCA2 dysfunction.

Plasma membrane Ca2+-ATPase mRNA expression in murine hepatocarcinoma and regenerating liver cells.

The plasma membrane calcium ATPase (PMCA) is an ubiquitous enzyme that extrudes calcium from the cytoplasm to the extracellular space. Four PMCA genes through alternative splicing produce a large diversity of isoforms of this enzyme. We reported previously that the PMCA contained in AS-30D hepatocarcinoma cells showed significant differences in activity in comparison to normal and regenerating liver. In the present study we investigate if the difference in PMCA activity could be related to differential expression of mRNAs encoding different isoforms of PMCA. Using RT-PCR we found that variants 1b, 1x, and 4b are expressed in all liver samples. The hepatoma AS-30 and liver at 2 days of regeneration express low amounts of isoforms 2w, 4b and 4x, and do not express isoforms 4a, 4d and 4z. Fetal and neonatal liver do not express variants 4a and 4d, but they do express variants 4x and 4z. Immunoblot analysis showed a higher ratio ATPase/total protein in the hepatoma AS-30D in comparison to normal liver. Our results suggest that the Ca2+-ATPase kinetic pattern previously observed by us in the AS-30D cells, could be at least partially explained by changes in the mRNA expression of several of the PMCA isoforms expressed in the liver.

Cell-specific expression of plasma membrane calcium ATPase isoforms in retinal neurons.

Ca(2+) extrusion by high-affinity plasma membrane calcium ATPases (PMCAs) is a principal mechanism for the clearance of Ca(2+) from the cytosol. The PMCA family consists of four isoforms (PMCA1-4). Little is known about the selective expression of these isoforms in brain tissues or about the physiological function conferred upon neurons by any given isoform. We investigated the cellular and subcellular distribution of PMCA isoforms in a mammalian retina. Mouse photoreceptors, cone bipolar cells and horizontal cells, which respond to light with a graded polarization, express isoform 1 (PMCA1) of the PMCA family. PMCA2 is localized to rod bipolar cells, horizontal cells, amacrine cells, and ganglion cells, and PMCA3 is predominantly expressed in spiking neurons, including both amacrine and ganglion cells but is also found in horizontal cells. PMCA4 was found to be selectively expressed in both synaptic layers. Optical measurements of Ca(2+) clearance showed that PMCAs mediate Ca(2+) extrusion in both rod and cone bipolar cells. In addition, we found that rod bipolar cells, but not cone bipolar cells possess a prominent Na(+)/Ca(2+) exchange mechanism. We conclude that PMCA isoforms are selectively expressed in retinal neurons and that processes of Ca(2+) clearance are different in rod and cone bipolar cells.

Deficiency in plasma membrane calcium ATPase isoform 2 increases susceptibility to noise-induced hearing loss in mice

Susceptibility to noise-induced hearing loss (NIHL) is poorly understood at the genetic level. Mice homozygous for a null mutation in the plasma membrane Ca 2+-ATPase isoform 2 (PMCA2) gene are deaf (Kozel et al., 1998). PMCA2 is expressed on outer hair cell stereocilia (Furuta et al., 1998). Fridberger et al. (1998) observed that the outer hair cell cytoplasmic Ca 2+ concentration rises following acoustic overstimulation. We hypothesized that Pmca2 +/− mice may be more susceptible to NIHL. Since the auditory brainstem response (ABR) thresholds of Pmca2 +/− mice vary with the presence of a modifier locus (Noben-Trauth et al., 1997), Pmca2 +/− mice were outcrossed to normal hearing CAST/Ei mice. The pre-exposure ABR thresholds of the resulting Pmca2 +/+ and Pmca2 +/− siblings were indistinguishable. Groups of these mice were exposed to varying intensities of broadband noise, and ABR threshold shifts were calculated. Fifteen days following an 8 h, 113 dB noise exposure, the Pmca2 +/− mice displayed significant ( P≤0.0007) permanent threshold shifts at 16 and 32 kHz that were 15 or 25 dB greater than those observed in Pmca2 +/+ littermates. Pmca2 may be the first gene with a known mutated protein product that confers increased susceptibility to NIHL.

Plasma membrane calcium ATPase plays a role in reducing Ca(2+)-mediated cytotoxicity in PC12 cells.

In many cell types, cell death induced by a variety of insults is accompanied by an increase in intracellular calcium. The Ca(2+) homeostatic mechanisms affected by such insults, however, have not been fully determined. Recent evidence indicates that kainic acid-induced seizures alter plasma membrane calcium ATPase mRNA expression within vulnerable hippocampal cell populations before the onset of cell death. We examined the effects of altering plasma membrane calcium ATPase expression on cell vulnerability in rat pheochromocytoma 12 cells. Pheochromocytoma 12 cells are vulnerable to Ca(2+) overload induced by the Ca(2+) ionophore A23187. Reverse transcriptase-PCR and Western blot data indicated that plasma membrane calcium ATPase isoform 4b constitutes a major calcium pump isoform in the pheochromocytoma 12 cells. Therefore, permanently transfected pheochromocytoma 12-derived cell lines were established that either over-expressed plasma membrane calcium ATPase isoform 4b, or suppressed the expression of the endogenous plasma membrane calcium ATPase isoform 4. Over-expressing clones were less vulnerable to Ca(2+)-mediated cell death induced by A23187 whereas "antisense" clones were considerably more susceptible. These data indicate that regulation of plasma membrane calcium ATPase expression may be critical to cellular survival when cells are exposed to pathological increases in intracellular calcium.

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line

The plasma membrane Ca 2+ pump is a key regulator of cytosolic free Ca 2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca 2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca 2+ ATPase (PMCA1) isoform of the plasma membrane Ca 2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously.

Selective decrease of mRNAs encoding plasma membrane calcium pump isoforms 2 and 3 in rat kidney

Although the existence of multiple isoforms of plasma membrane calcium ATPase (PMCA) is now well documented, their biological functions are not yet known. In this study, we set out to investigate the potential role of PMCA isoforms, previously identified in renal cortical tissue, in tubular reabsorption of calcium (Ca2+). With use of reverse transcription-polymerase chain reaction analysis, we determined levels of mRNAs encoding isoforms of PMCA1 through PMCA4 in renal cortex, liver, and brain of rats with hypercalciuria induced by feeding with a low-phosphate diet (LPD) as compared with Ca2+-retaining rats that were fed a high-phosphate diet (HPD). We observed that in hypercalciuric LPD-fed rats, the mRNAs encoding isoforms PMCA2b and PMCA3(a + c) are significantly lower (Delta approximately-50%) than in HPD-fed hypocalciuric rats, whereas no changes in mRNAs encoding isoforms PMCA1b and PMCA4 were observed, and mRNA encoding calbindin 28 kDa was increased. On the other hand, the content of mRNAs encoding PMCA2b and PMCA3(a + c) in liver and brain, respectively, was not changed. These findings are evidence that expression of PMCA isoforms in the kidney can be selectively modulated in response to pathophysiologic stimuli. The association of a decrease in mRNA encoding PMCA2b and PMCA3(a + c) with hypercalciuria suggests that the two PMCA isoforms may be operant in tubular reabsorption of Ca2+ and its regulation.

Plasma membrane calcium ATPases as critical regulators of calcium homeostasis during neuronal cell function.

The plasma membrane calcium ATPases (PMCAs) are ubiquitously expressed proteins that couple the extrusion of calcium across the plasma membrane with the hydrolysis of ATP. In mammals, four separate genes encode distinct PMCA isoforms. Complex patterns of alternative RNA splicing generate additional isoform variability. Functionally, the PMCAs were originally assigned the role of maintaining basal levels of intracellular calcium. Recent evidence, however, is expanding the role of the PMCAs as important participants in dynamic Ca2+ regulation and as crucial players of Ca2+ export during normal and pathological conditions. This review highlights recent advances made on the biology of the PMCAs within the context of neuronal development, cellular responses to external stimuli and cell survival. Particular emphasis is placed on the role of the PMCAs in vestibular and auditory functions, localized calcium signaling in photoreceptor synaptic terminals and calcium-mediated cell death.

The significance of the isoforms of plasma membrane calcium ATPase.

The plasma membrane calcium ATPase (PMCA) or Ca2+ pump transports Ca2+ ions out of the cells, by using the energy stored in ATP. It is essential in the control of Ca2+ concentration in the cytosol. The plasma membrane Ca2+ pump has been found in all mammalian cells and is encoded by four independent genes. The number of possible isoforms is further increased by alternative splicing at two independent sites; transcripts for more than 20 isoforms have been detected. The PMCA isoforms, in particular some of their alternatively spliced isoforms, have been shown to bind calmodulin with different affinity. The activity of these alternatively spliced pumps is possibly differently regulated by kinase-mediated phosphorylation. A short summary of recent work on the properties of the PMCA isoforms is presented here.

Seizure-induced alterations of plasma membrane calcium ATPase isoforms 1, 2 and 3 mRNA and protein in rat hippocampus.

Improper intracellular regulation of the ubiquitous second messenger, calcium, has been linked to several pathological conditions. The plasma membrane calcium ATPase (PMCA) is one of the primary systems for translocating calcium from the cytosol to the extracellular milieu. As an initial assessment of the possible involvement of PMCAs in kainate (KA)-induced neurodegeneration, we have determined the effect of KA-induced seizures upon PMCA mRNA and protein. In situ hybridization was performed on tissue from adult male Sprague-Dawley rats sacrificed at various time points following i.p. injection of KA. KA altered the expression within the hippocampal subfields for mRNAs of PMCA isoforms 1 and 2. PMCA 1 and 2 mRNAs exhibited hybridization below control levels 12-48 h post-injection within CA1 and CA3. Within the dentate gyrus, PMCA 2 mRNA hybridized below control levels 4 h post-injection, but recovered to control levels by 24 h post-injection. Alterations in combined PMCA protein levels occurred at all time points examined post-injection. These observations provide evidence that KA-induced seizures alter the PMCAs at the mRNA and protein levels, suggesting a possible role for this calcium efflux system in the neuronal degeneration inherent to this paradigm.

Primary structure of human plasma membrane Ca 2+-ATPase isoform 3

The complete coding sequence of the human plasma membrane calcium ATPase (PMCA) isoform 3 was determined from overlapping genomic and cDNA clones. The cDNAs for the two major alternative splice variants 3a (3CII) and 3b (3CI) code for proteins of 1173 and 1220 amino-acid residues, respectively, which show 98% identity with the corresponding rat isoforms. On a multiple human tissue Northern blot, a major PMCA3 transcript of about 7 kb was detected exclusively in the brain, demonstrating the highly restricted pattern of expression of this isoform to human neuronal tissues. With the elucidation of the human PMCA3 primary structure, complete sequence information is now available for the entire family of human PMCA isoforms.

Colocalization of the dihydropyridine receptor, the plasma-membrane calcium ATPase isoform 1 and the sodium/calcium exchanger to the junctional-membrane domain of transverse tubules of rabbit skeletal muscle.

The subcellular distribution of the calmodulin-stimulated plasma-membrane Ca(2+)-ATPase (PMCA) has been studied in rat and rabbit skeletal muscle cells by indirect (calmodulin gel overlays) and direct (Western blotting with specific antibodies) methods. It has also been studied in situ in immunocytochemistry experiments. The distribution of PMCA has been compared with that of the NA+/Ca2+ exchanger and of the dihydropyridine receptor, which has been studied by Western blotting with specific antibodies. Both PMCA and the Na+/Ca2+ exchanger had a dual localization, i.e., they were found in the plasma membrane and in the transverse-tubule fractions of the two main types of skeletal muscles studied. The pump and the exchanger were not diffusely distributed in the transverse-tubule-membrane system, but specifically confined to the membrane domain where the dihydropyridine receptor was also localized, i.e., the junctional membrane. Experiments with isoform-specific antibodies have shown that the pump isoform expressed in skeletal muscle is PMCA 1.

Structure of the gene encoding the human plasma membrane calcium pump isoform 1.

The complete structure of the gene for the human plasma membrane calcium ATPase isoform 1 (hPMCA1) has been elucidated. The protein is encoded by 21 exons present on overlapping clones covering more than 100 kilobases (kb) of DNA. An intron of over 35 kb separates the 5'-untranslated exon 1 from the exon containing the translational start codon. The entire putative promoter and 5'-flanking region is embedded in a CpG island and is characterized by the presence of numerous Sp1 factor-binding sequences and by the absence of a TATA box. In accordance with the ubiquitous tissue distribution of its mRNA these results suggest that the hPMCA1 gene is of the housekeeping type. No alternative splicing comparable to that identified in PMCA2 RNAs at site "A" and in PMCA3 RNAs close to site "C" seems to occur in hPMCA1 transcripts; however, a region in intron 6 shows significant resemblance to the site "A" alternatively spliced exons in PMCA2 and may represent a pseudoexon or a functional exon not yet detected in any PMCA1 mRNA. At six positions, intron interruptions in the hPMCA1 gene correlate with the boundaries of putative transmembrane domains in the protein, whereas most of the remaining intron positions do not show an obvious correlation with the proposed pump domain structure. The limited conservation of intron positions in different P-type pump genes indicates their early separation from a common ancestor.