At the beginning, life on earth had consisted only of single cells that were self sufficient. Their interplay with other cells was essentially limited to the competition for nutrients . 600-700 million years ago, possibly even earlier, multicellular life emerged: it had evidently become advantageous for cells to work together rather than to compete with each other: competition was replaced by cooperation. The cooperation demanded the development of agents that could exchange messages between cells calcium, the third most abundant element in nature soon emerged as the most universal and versatile carrier of messages. The choice of calcium (Ca2+ ) was dictated by its peculiar coordination chemistry, that permitted its easy and reversible complexation by the irregular binding sites offered by biological molecules, essentially proteins: as is self evident, the concentration of messengers that regulate the function of cells must be allowed to change easily and reversibly, and this was uniquely possible in the case of Ca2+. Systems were thus developed in the course of evolution to control cellular Ca2+, and its movements, with the necessary utmost precision.
The control of cell Ca2+
Cellular Ca2+ is controlled by the reversible complexation by soluble proteins (Ca2+sensor proteins), e.g., calmodulin , that interact with targets and transmit to them the Ca2+ message (Fig.1).
Since the amount of these proteins in cells is finite, the quantitatively most important role in the control of cellular Ca2+ is performed by membrane proteins that transport it across the plasma membrane and the membrane of organelles. The cartoon of Figure 2 depicts the various systems that move Ca2+ across membrane boundaries. The acronyms in the cartoon indicate messengers that act on membrane intrinsic Ca2+ binding proteins that move Ca2+ in and out of the cell and of the organelles, The Figure also mentions the soluble Ca2+ sensor proteins (top left).
The early work of the Group in the time when the VIMM had not yet been created, had concentrated on the uptake and release of Ca2+ by mitochondria (ref. 5). Later on, the Group concentrated on the Ca2+ transport ATPases, particularly those of the plasma membrane (PMCA pumps), on which the Group has contributed a large portrion of the information presently available (ref. 4). Four basic gene products of the PMCA pumps exist in mammalian cells, the expression of two of them, isoforms 2 and 3, being restricted to a limited number of tissues, with particular abundance the nervous tissue. The PMCA pumps belong to the superfamily of P-type pumps, and thus share with the other members of the superfamily, including the other Ca2+ ATPases, the essential catalytic properties. However, they have a number of distinctive properties that are not directly related to the catalytic mechanism: one is the wealth of partners, the most important being calmodulin, that regulate their activity; another is the mechanism of autoinhibition, in which the activity of the pumps in the Ca2+-free resting state is repressed by the binding of their long cytosolic tail to sites next to the catalytic center. Activity is then restored when Ca2+-saturated calmodulin removes the autoinhibitory tail from its binding sites.Figure 3 shows the process: CaM is calmodulin. “D” and “K” are the catalytic aspartic acid and the Lysine that is part of the ATP binding site.
The PMCA pumps are qualitatively of minor importance in the regulation of the global cellular Ca2+ homeostasis, as other systems are more powerful and/or more abundantly expressed (the SERCA pumps of the endo/sarcoplasmic reticulum and the Na+-Ca2+ exchangers of the plasma membrane). But the PMCA pumps are uniquely important in the regulation of Ca2+ signaling in specific sub-plasma membrane microdomains in which essential Ca2+ sensitive enzymes reside (ref. 8One Important properties of Ca2+ as a signaling agent is its ambivalence: while essential to the life of cells, as it regulates most of their functions, Ca2+ becomes a conveyor of doom when its regulation within cells fails. Not surprisingly, then, genetic defects that impair the functioning of the PMCA pumps generate pathological phenotypes Given the special importance of Ca2+ signaling in neurons, the neuropathology phenotypes associated to defects of PMCA 2 and PMCA 3 have recently become more frequent (refs. 1, 3, 4, 6). The most recent work of the Group has focused on the genetic defects of the PMCA pumps and their role in neuropathology.
Defects of the PMCA 2 pump in hereditary deafness
A number of genetic defects of the PMCA2 pump have been found to be involved in the origin of a mouse deafness phenotype linked to the malfunctioning of the stereocilia of the outer hair cell of the Corti organ of the inner ear, in which the PMCA2 pump is the sole system that ejects Ca2+ to the endolymph. Basically, the defect of the pump decreases its ability to eject Ca2+ from the cell: the protocol that has permitted the molecular analysis of the defect consists in the cloning of the mutated pump, its expression in model cells together with a suitable Ca2+ indicator, and in the analysis of the ability of the pump to control the increase of Ca2+ induced by the stimulation of the cells with an agonist that promotes the liberation of Ca2+ in the cell. Our Group has identified a human case of hereditary deafness (ref. 2) in which a mutation of the PMCA2 pump (a G293S replacement) generates the same Ca2+ control deficiency found in mice. Figure 4 shows the molecular analysis of the pump expressed in model cell together with the Ca2+ indicator aequorin. Cells were stimulated with a purinergic receptor agonist (ATP) which promotes the formation of Inositol-tris-phosphate (InsP3) that liberates Ca2+ from the endoplasmic reticulum (a mouse PMCA2 pump that carried a similar mutation –G283S- is also shown). The mutated human pump (and the mutated mouse pump) failed to return to baseline the peak of Ca2+ induced by InsP3.
Interestingly, the phenotype resulted by the digenic cooperation of the PMCA 2 pump mutation and a mutation of cadherin 23, a Ca2+ dependent protein that is also essential in the control of the function of the stereocilia. Digenicity seems to be the rule in the defects of the PMCA 2 pump that lead to deafness: in a second human case discovered by others a different mutation of cadherin 23 had also been found, and that has also been the case for the several mice mutations that have been described.
Defects of the PMCA 3 pump in cerebellar ataxias
The least well known of the PMCA pumps is isoform 3. Although also expressed in other tissues, e.g., the skeletal muscle, it is peculiarly abundant in the cerebellum, and several reports have appeared in the last two or three years of its mutations in human X-linked congenital cerebellar ataxias. Our Group has reported two (refs.7 and 10) and has the analysis of several more under way: it is becoming clear that , somehow, the PMCA 3 pump has a particular tendency to mutate. The analysis of the function of the mutated pump using the same protocol of the work on the PMCA 2 pump has shown that also the mutated PMCA 3 pump had impaired ability to clear Ca2+ from the cytosol. In the case of the G1107D replacement the mutation impaired the ability of the pump to interact with calmodulin and affected both its ability to optimally transport Ca2+ in the activated state, and its autoinhibition mechanism in the resting state (ref. 10). Going back to the digenic mechanism, our Group has recently found that another mutation of The PMCA3 pump in an ataxic patient (G733R) is associated with two function-impairing mutations in phosphomannose mutase 2 (PMM2), an enzyme that has recently been found to be inhibited by Ca2+: possibly, the increase I of cellular Ca2+ induced by the PMCA 3 pump mutation could exhacerbate the inhibition of the mutated PMM2. Figure 5 shows a Surface Plasmon Resonance experiment on the interplay between calmodulin (CaM) and a synthetic peptide corresponding to the C-terminal calmodulin –binding domain of the wild type PMCA3 pump and of the G1107D mutated pump. The sensograms (time course of the surface plasmon resonance signal) show the interaction of calmodulin with the biinding peptides, obtained by injecting calmodulin in solution over the peptides immobilized on the surface of a chip, flowing over the surface at the concentrations shown. While the association phase of calmodulin with the peptide was only marginally affected by the mutation (the rising part of the curves), the dissociation phase (the decay part of the curves) of calmodulin from fro mutated peptide was considerably faster.
