Plasma Membrane Calcium-Transporting ATPase
The existence of a plasma membrane calcium-transporting ATPase (PMCA) that actively pumps Ca2+ ions out of the cell was first demonstrated in erythrocyte (red blood cell) membranes by Schatzmann (1966). Because of its generally low abundance and difficult biochemical properties, it took over a decade until the PMCA was first isolated in purified form. Crucial for the successful purification was the discovery that the PMCA binds with high affinity, and in a Ca2+-dependent manner, to the Ca2+ sensor protein calmodulin (Niggli et al. 1979). Subsequent work showed that at least one type of plasma membrane Ca2+ ATPase is found in all eukaryotic cells including those from fungi, animals, and plants (Axelsen and Palmgren 1998; Thever and Saier 2009). It is now well established that active Ca2+ expulsion by the PMCAs is an essential component of eukaryotic cellular Ca2+ handling. Although, PMCAs were originally thought to be required mainly for the “housekeeping” function of maintaining and resetting the low intracellular free [Ca2+] levels, recent studies have shown that these pumps are also active participants in global and local Ca2+ signaling (Strehler 2016).
General Structure and Isoforms
The cytosolic loops between transmembrane segments 2 and 3 and between transmembrane segments 4 and 5 are large and contribute the bulk of the mass to the so-called A (actuator) and N/P (nucleotide-binding/catalytic phosphorylation) domains, respectively. In animal PMCAs, the C-terminal tail following the last membrane-spanning segment is about 150 residues long and contains the auto-inhibitory regulatory sequences and the high-affinity calmodulin-binding domain. In contrast, in several plant PMCAs, the calmodulin-binding and auto-inhibitory regulatory domain are instead found in the extended N-terminal tail. The three-dimensional structure of the PMCA has not yet been solved in atomic detail, but based on the high conservation of secondary structure elements (including the 10 transmembrane helices and the functionally important A, N, and P domains), the general structure of the PMCA is thought to be very similar to that of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), for which several high-resolution x-ray structures are available (Toyoshima 2009; Moeller et al. 2010).
Regulation and Functional Properties
Tissue and Subcellular Expression
All tissues and cells express at least one isoform of the PMCA; however, multiple PMCA isoforms and splice variants are often found in the same cell. During (mouse) embryonic development, PMCA1 (splice variant 1x/b) is detected from the earliest time points studied and is expressed in most tissues throughout life. PMCA1 is thus considered a “housekeeping” PMCA isoform, although this only applies to splice variant 1x/b. PMCA1x/a and 1x/c are much more restricted in their expression and are mainly found in differentiated neurons and skeletal muscle cells. PMCA4x/b is also fairly ubiquitous, although there are large differences in PMCA4b expression among different tissues and cell types. By contrast, PMCAs 2 and 3 are almost exclusively found in excitable tissues including brain and muscle, as well as in secretory cells such as insulin-secreting pancreatic β-cells and lactating mammary epithelial cells (Strehler and Zacharias 2001). Some splice variants are highly specific for particular cells: PMCA2w/a, for example, is specifically and abundantly expressed in auditory and vestibular hair cells of the inner ear (Hill et al. 2006). Different PMCAs are often co-expressed in the same cell, where they are specifically targeted to distinct membrane compartments such as the apical or basolateral side of a polarized epithelial cell. This suggests that different PMCA isoforms fulfill distinct roles in global and local Ca2+ handling. In cochlear hair cells, for example, PMCA1x/b is expressed in the basolateral membrane, where it is involved in maintaining the low resting level of Ca2+ in the cell soma. In the same cells, PMCA2w/a is highly concentrated in the apical stereocilia, where it plays an important role in regulating [Ca2+] in the endolymph and modulating the function of the mechanotransduction channels. Some PMCA isoforms are also enriched in membrane domains of specific lipid composition (e.g., caveolae) where they associate with other signaling and transport proteins (Oceandy et al. 2011).
Functions in Health and Disease
In cells such as human erythrocytes, where the PMCA is the sole Ca2+ export system, the pump is obviously essential for normal Ca2+ homeostasis and cell physiology. However, because of its low abundance in most plasma membranes and its low capacity (maximal turnover of about 100 Ca2+ ions per second), the PMCA is generally not well suited for handling high global Ca2+ loads such as those occurring with each heart beat in a cardiac muscle cell. Conversely, due to its very high Ca2+ affinity (Kd < 0.2–0.5 μM) in the activated state, the PMCA is the only Ca2+ export system capable of lowering intracellular [Ca2+] to the very low resting levels (∼100 nM) normally found in most cells. In terms of global cellular Ca2+ homeostasis, the expression of different PMCA isoforms and splice variants allows cells to fine tune and maintain the specific Ca2+ set point optimal for their physiological function.
In tissues involved in transcellular Ca2+ transport, the PMCA (specifically, PMCA1b) plays an essential role in normal physiology. In kidney and intestinal epithelial cells, PMCA1b in the basolateral membrane is important for the vectorial transport of Ca2+ from the apical (lumenal) to the basal (blood) compartment and thus for Ca2+ (re)absorption. As may be expected, in these tissues the expression and localization of the PMCA are under hormonal control by vitamin D3 (Strehler 2016). Similarly, in the lactating mammary gland, the PMCA (isoform PMCA2w/b) is concentrated in the apical membrane of secretory epithelial cells and is essential for the export of Ca2+ into the (milk) lumen. Accordingly, female mice lacking PMCA2 produce Ca2+ deficient milk and their offspring are underweight (Reinhardt et al. 2004).
In addition to their roles in bulk vectorial Ca2+ transport, PMCAs are involved in the spatiotemporal control of Ca2+ signaling. Specific PMCA isoforms and splice variants are targeted to plasma membrane sub-compartments where they form multiprotein signaling complexes with adaptor proteins and other transporters and receptors to control local Ca2+ signals. By influencing the amplitude and recovery time of locally evoked Ca2+ spikes, the PMCAs contribute to the decoding of Ca2+ signals and affect the frequency of Ca2+ oscillations (Pászty et al. 2015). By reducing the spread of a local increase in Ca2+, they help suppress signaling “noise,” thereby increasing the fidelity and spatial resolution of Ca2+ signaling. Because of the pronounced differences in their kinetic and regulatory properties, different PMCA isoforms are adapted to handle very different Ca2+ signals ranging from slow, solitary Ca2+ waves to highly localized, high-frequency Ca2+ spikes such as those elicited at neuronal synapses. For example, specific PMCA2 splice variants play important roles in pre- and postsynaptic function such as in the regulation of excitatory synaptic transmission at hippocampal CA3 synapses or in short-term plasticity in cerebellar parallel fiber to Purkinje neuron connections. Deficiency of the corresponding PMCA leads to functional deficits such as impaired motor coordination (Huang et al. 2010; Brini et al. 2016). In general, other PMCA isoforms are unable to compensate for the deficiency in a specific isoform, supporting the notion of the highly defined functions that specific PMCAs assume in Ca2+ signaling.
Given its early expression during development and ubiquitous presence in all tissues, it is not surprising that PMCA1 ablation is embryonic lethal (Prasad et al. 2007). Heterozygous mice lacking one copy of the PMCA1 gene appear phenotypically normal, although they do show physiological differences from the wild type such as an increased stimulated peak tension of bladder smooth muscles and reduced bone mineral mass. Mutations in the human ATP2B1 (PMCA1) gene lead to specific disease phenotypes: Genome-wide association studies revealed a highly significant association of specific single-nucleotide polymorphisms (SNPs) in ATP2B1 with high systolic blood pressure and cardiovascular disease risk in humans.
Deletion of PMCA2 leads to profound deafness, ataxia, and various other deficiencies such as a decrease of milk calcium (as already mentioned above), reduced visual responses from retinal bipolar cells, and spinal cord pathology (Prasad et al. 2007). Even single point mutations affecting the function of PMCA2 can cause significant hearing impairment, underscoring the importance of the unique role of PMCA2w/a in auditory hair cell function. Mutations in the PMCA2 (ATP2B2) gene have been linked to human hearing loss; in the heterozygous state, they generally manifest in a more complex digenic inheritance pattern (Brini et al. 2016).
Loss of PMCA3 in mice was shown to be associated with increased sleep duration due to altered Ca2+-dependent hyperpolarization in neurons involved in the regulation of sleep homeostasis. In contrast, missense mutations in human ATP2B3 (PMCA3) have been linked to X-linked congenital cerebellar ataxia and to developmental delay, hypotonia, and ataxia in a case with concomitant mutations in the laminin 1α gene. These findings are in agreement with the predominant expression of PMCA3 in the brain and an important function of this isoform in cerebellar neurons. Somatic PMCA3 mutations resulting in impaired pump function have also been detected in several cases of human aldosterone-producing adenomas (and secondary hypertension), pointing to an important role for this pump in the regulation of membrane polarization (Brini et al. 2016).
In mice, knockout of the PMCA4 (Atp2b4) gene results in viable animals without overt gross abnormalities. However, males are infertile due to a defect in sperm hyperactivated motility, which is apparently dependent on PMCA4 normally concentrated in the sperm tail (Prasad et al. 2007). Lack or altered expression of PMCA4 also leads to changes in cardiac physiology, such as altered response to beta-adrenergic stimulation and change in blood pressure, reinforcing the notion of the PMCA4 as an important signaling molecule rather than as general “sump pump” to remove global Ca2+ (Holton et al. 2010). A missense mutation in the human PMCA4 (ATP2B4) gene has been shown to be a causative for a form of autosomal dominant familial spastic paraplegia in a Chinese family pedigree, suggesting that similar to PMCA2 and PMCA3, PMCA4 also fulfills specialized functions in distinct neuronal cells (Li et al. 2014).
Considering the universal importance of Ca2+ signaling, it is no surprise that numerous diseases are characterized by altered expression and/or impaired function of specific PMCAs. Systemic disease as well as tissue-specific disorders can be caused by deficiency of a single PMCA isoform. Because the PMCAs are functionally integrated in multi-protein signaling complexes, any disturbance of such complexes can have disease-causing consequences. Major diseases that show distinct changes in PMCA isoform expression and function include diverse cancers, neurodegenerative disorders such as Alzheimer’s and Huntington’s disease, and diabetes (Lehotsky et al. 2002; Brini and Carafoli 2009). However, in many cases, it is not clear whether aberrant PMCA expression and/or function is causative or reflects a secondary (potentially compensatory) reaction to a different primary insult.
Plasma membrane Ca2+-transporting ATPases (PMCAs) are essential components of the calcium signaling toolkit of eukaryotic cells. These membrane-embedded transporters couple the expulsion of 1 Ca2+ ion to the hydrolysis of 1 ATP and attain maximal rates of about 100 Ca2+ transported per second. PMCAs are the major high-affinity Ca2+ export system dedicated exclusively to the export of Ca2+ from cells and are essential for the maintenance of the steep [Ca2+] concentration gradient that is a prerequisite for the high specificity and fidelity of Ca2+ signaling. In mammals, four genes encode PMCAs 1–4; alternative RNA splicing augments the number of distinct isoforms to over 30. The PMCAs are highly regulated by multiple mechanisms including Ca2+-calmodulin, acidic phospholipids, phosphorylation, oligomerization, and interactions with numerous signaling, targeting, and anchoring proteins. PMCA isoforms and splice variants show developmental, tissue- and cell type-specific expression, and are targeted to specific membrane compartments where they contribute to local Ca2+ handling. PMCA isoforms show characteristic differences in kinetic and regulatory properties, providing cells with many options to deploy specific PMCAs to distinct plasma membrane compartments with different Ca2+ signaling needs. Consistent with a role in local Ca2+ handling, deletion, mutation, or altered expression of specific PMCAs causes characteristic cellular defects. Deletion of the ubiquitously expressed PMCA1 results in embryonic lethality, whereas mutations in the PMCA1 gene are associated with systolic hypertension and cardiovascular disease risk. Mice lacking PMCA4 are superficially normal but show altered cardiac stress responses and are male-infertile due to impaired sperm motility; in humans a PMCA4 missense mutation leads to autosomal dominant familial spastic paraplegia. Deletion or mutation of PMCA2 results in hearing loss, balance and vision defects, ataxia, spinal cord pathology, as well as reduced milk calcium. Cerebellar ataxia and altered sleep regulation are among the consequences of mutation or the lack of expression of PMCA3. PMCAs are now being recognized as major players in spatiotemporal Ca2+ signaling with specific involvement in diverse cell functions, and are potential targets for intervention in the treatment of various diseases linked to abnormal Ca2+ handling.
- Brini M, Carafoli E, Cali T. The plasma membrane calcium pumps: focus on the role in (neuro) pathology. Biochem Biophys Res Commun. 2016; pii: S0006-291X(16)31239-6. doi: 10.1016/j.bbrc.2016.07.117. Available online 29 July 2016.Google Scholar
- Strehler EE. The ATP2B plasma membrane Ca2+ ATPase family: regulation in response to changing demands of cellular calcium transport. In: Chakraborti S, Dhalla NS, editors. Regulation of Ca2+-ATPases, V-ATPases and F-ATPases, Advances in Biochemistry in Health and Disease14 Cham: Springer; 2016. p. 63–80.CrossRefGoogle Scholar