Plasma membrane calcium transporting ATPase isoform 2

Plasma membrane calcium ATPase isoform 2 (PMCA2) is one of four mammalian isoforms of the PMCA family. The PMCAs are calcium-transporting P-type ATPases responsible for the extrusion of ionized calcium (Ca2+) from the cytosol to the extracellular milieu. The PMCAs form an obligatory aspartyl-phosphate intermediate during the reaction cycle (hence their classification as P-type ATPases). These pumps are responsible for the resetting and maintenance of resting levels of intracellular free Ca2+ and may be involved in local and dynamic regulation of Ca2+ signaling in diverse tissues and cell types. The PMCAs generally exhibit low activity at low (<50 nM) Ca2+ concentrations and are activated by interaction with Ca2+-calmodulin. PMCA2 has a high basal activity and is one of the fastest pumps with respect to Ca2+-calmodulin activation. PMCA2 is most highly expressed in the brain, where, for example, it is crucial for sensory neuron function in the inner ear. Some alternative splice variants of PMCA2 interact with PDZ domain-containing proteins to form multi-protein calcium signaling complexes anchored to the membrane cytoskeleton. Ser/Thr phosphorylation of PMCA2 has been reported to regulate pump activity in a complex manner.

PMCA2 is regulated (activated) by Ca2+-calmodulin. PMCAs have also been reported to be activated by acidic phospholipids and oligomerization, although this has not yet been rigorously shown for PMCA2. The activity of PMCA2 is also regulated by Ser/Thr phosphorylation via protein kinase C and, possibly, protein kinase A. No specific inhibitors are known for PMCA2, but calmodulin antagonists (e.g., calmidazolium), orthovanadate, (carboxy-)eosin, and lanthanum (La3+) can be used to block pump activity. A synthetic peptide (caloxin 2A1) has been reported to inhibit PMCA activity by interacting with the second extracellular loop of the pump. However, the inhibitory effect of caloxin 2A1 on PMCA2 has not yet been characterized in detail.

PMCA2 interacts with Ca2+-calmodulin with very high affinity. The b splice form of PMCA2 binds via its C-terminal tail to a variety of PDZ domain-containing scaffolding and signaling proteins. These include synapse-associated proteins (SAPs) SAP90/PSD95, SAP93/chapsyn110, and SAP97/hDlg. Other PDZ proteins shown to bind to PMCA2 are the Na+/H+ exchanger regulatory factor-2 (NHERF-2) and the PMCA-interacting single-PDZ protein (PISP). PMCA2 may also interact with ankyrin-B. Interaction sites for acidic phospholipids (phosphatidic acid, phosphatidylinositol, phosphatidylserine) have been reported for the PMCAs, although detailed studies for PMCA2 have not yet been published. As a calcium-transporting ATPase, PMCA2 uses Mg2+-ATP and Ca2+ as major substrate and ligand. An invariant aspartate residue (Asp-454 in PMCA2z) serves as intramolecular acceptor of the gamma-phosphate from ATP to form the obligatory aspartyl-phosphate intermediate in the reaction cycle.

In mouse embryos, PMCA2 transcripts are first detected around day 12 post coitum in the developing nervous system. Throughout embryonic development, PMCA2 expression is essentially confined to the nervous system and increases steadily in the CNS and the dorsal root ganglia. PMCA2 expression is also dramatically (up to 100-fold) upregulated in the mammary tissue of lactating rats. In developing cerebellar granule cells, PMCA2 expression has been shown to be (up)regulated by an increase in intracellular Ca2+ (e.g., upon KCl- (depolarization) or NMDA-induced Ca2+ influx).

PMCA2 is highly expressed in the soma and dendritic membrane of cerebellar Purkinje cells, where it concentrates in dendritic spines. In the sensory cortex, hippocampus, and neostriatum, PMCA2 is predominantly found in a punctate pattern in the neuropil, as well as being associated with the somatic plasma membrane. In auditory and vestibular hair cells, PMCA2 is highly concentrated in the apical stereociliar bundle membranes. PMCA2 is also prominently expressed in the mouse inner retina in rod bipolar cells, amacrine cells, and ganglion cells. In rod bipolar cells, the pump is localized to the synaptic terminals. In lactating mammary gland epithelial cells, PMCA2 is concentrated apically and shed into milk as part of the milk fat globule membrane.

The major site of expression of PMCA2 is the nervous system. The highest levels of PMCA2 are found in the cerebellum, but all other brain structures and peripheral nerves also express high levels of PMCA2. Locally high levels of PMCA2 are observed in auditory and vestibular hair cells. PMCA2 is also expressed in non-neuronal tissues such as the lactating mammary gland, heart, liver, and distal kidney. Pancreatic beta cells have also been shown to express PMCA2. However, with the exception of the lactating mammary gland that expresses large amounts of PMCA2, it has been difficult to detect PMCA2 protein (by Western blotting) in extraneuronal tissues such as heart, kidney, and liver.

PMCA2-knockout mice (Atp2b2-/-) are deaf and show severe balance deficits. Several spontaneous mouse mutants of Atp2b2 have been described, including the deafwaddler (Atp2b2dfw), deafwaddler-2J (Atp2b2dfw2J), wriggle mouse Sagami (Atp2b2wri), and tommy (Atp2b2tmy). All homozygous mutants are profoundly deaf and exhibit movement disorders. In the dfw mutant, a single A->G nucleotide transition results in a glycine to serine change at residue 283 of PMCA2; in dfw-2J, a 2 bp deletion leads to a frameshift and premature stop codon after residue 471 of PMCA2; in wri, a G->A transition results in a glutamate to lysine change in a highly conserved region in the fourth transmembrane domain of PMCA2; and in tmy, another G->A transition results in a glutamate to lysine change in the ATP binding domain of PMCA2. The balance and movement disorders in dfw mice are less severe than in dfw-2J, wri, or Atp2b2-null mice, which express no functional PMCA2. The dfw mutation leads to a reduction in Ca2+ transport activity to 30% of the wild-type activity of PMCA2. Heterozygote Atp2b2+/- and mutant mice show age-related, noise-induced hearing loss; onset and severity vary with the genetic background. Atp2b2-/--null mice have a decreased number of spinal cord motor neurons, consistent with a role for PMCA2 in the protection of neurons against calcium overload-induced pathology. The calcium content in the milk from lactating Atp2b2-/- mice is reduced by about 60% compared to that from wild-type or heterozygote animals, suggesting an important biological role for PMCA2 in providing (extracellular) milk calcium. Overexpression of PMCA2 has been achieved in stably transfected insulin-secreting BRIN-BD11 cells. PMCA2 overexpression in these cells reduced the K+-stimulated rise in intracellular Ca2+ and suppressed Ca2+ oscillations, without altering basal Ca2+ levels.

Alternative RNA splicing affects two major sites in the PMCA2 (Atp2b2) transcripts, called sites A and C. At site A, three separate exons are optionally included or excluded, giving rise to splice variants w (all exons included), x (third exon included), y (first and second exon included), and z (none of the exons included). These splice variants vary in the length of the first intracellular loop, adding 0 (PMCA2z) to 45 (PMCA2w) residues to a region preceding a phospholipid-sensitive domain. At splice site C, the optional insertion of two separate exons leads to variants a (both exons included), b (both exons excluded), and c (only the first exon included). Splicing at site C affects the calmodulin-binding domain and results in different C-terminal sequences due to a change in reading frame. The combinatorial use of alternative splicing results in the following theoretically possible PMCA2 splice variants: PMCA2w/a, PMCA2w/b, PMCA2w/c, PMCA2x/a, PMCA2x/b, PMCA2x/c, PMCA2y/a, PMCA2y/b, PMCA2y/c, PMCA2z/a, PMCA2z/b, PMCA2z/c.

5F10, a mouse monoclonal antibody raised against erythrocyte PMCA, recognizes all PMCA isoforms, including PMCA2, from a wide variety of species, including mouse, human, rat, chicken, and others. The epitope for this antibody is a conserved stretch of ~20 amino acids in the central cytoplasmic domain.

Rabbit polyclonal antibody 2N was generated against a His-tagged fusion protein of residues 1-96 of the human PMCA2. Rabbit polyclonal antibody NR2 was raised against a synthetic peptide comprising residues 5-19 of rat PMCA2, and rabbit polyclonal antibody CR2A was raised against a synthetic peptide corresponding to the 20 C-terminal residues of the rat PMCA2a splice variant. 2N and NR2 recognize all splice variants of PMCA2, whereas CR2A is specific for the PMCA2a splice variant. These antibodies are not species specific and cross-react with PMCA2 from mouse, rat, human, and probably many other species.

Antibodies 5F10 and NR2 are commercially available from Affinity BioReagents, Inc. (catalog no. MA3-914 and PA1-915, respectively) or Upstate (catalog no. 05-625 and 07-245, respectively). 2N is available from Swant (lot no. 73).