Plasma membrane calcium pump: structure, function and relationships

The plasma membrane Ca-pump (134 kDa) is stimulated by calmodulin and by other treatments (exposure to acidic phospholipids, treatments with proteases, phosphorylation by protein kinases A or C, self-association to form oligomers). It is the product of four genes (in humans), but additional isoforms originate through alternative mRNA spicing. Most of the pump mass protrudes into the cytoplasm with three main units. The calmodulin binding domain is located in the C-terminal protruding unit. The domain is a positively charged segment of about 25 residues. The calcium-activated protease calpain activates the pump by removing its calmodulin binding domain and the portion C-terminal to it. The-resulting 124 KDa fragment has been used to test the suggestion of an autoinhibitory function of the calmodulin binding domain. The latter interacts with two domains of the pump, one located close to the active site in the mid-cytoplasmic protruding unit, the other in the first (N-terminal) protruding unit. The isoforms of the pump show variations in the regulatory domains, e.g., alternative mRNA splicing can eliminate the domain phosphorylated by protein kinase A, or alter the sensitivity of the pump to calmodulin. This occurs by inserting sequences rich in. His between calmodulin binding subdomains A and B. The inserted domain(s) confer pH sensitivity to the binding of calmodulin. Calcium binding sites have been found in acidic regions preceding and following the calmodulin binding domai

The plasma membrane calcium pump in the hearing process: physiology and pathology

Mammalian cells express four different plasma membrane Ca2+ ATPases. Two of them (PMCA1 and PMCA4) are expressed ubiquitously, and are considered housekeeping isoforms. Two (PMCA2 and PMCA4) have tissue restricted distribution. They are abundantly expressed in the brain and in nervous tissue-derived cell types. The primary transcripts of all PMCAs undergo alternative splicing, generating a large number of additional isoforms. Splicing occurs at site A, in the N-terminal moiety of the pump, and at site C, within the C-terminal calmodulin binding domain: The pumps are canonical targets of calmodulin stimulation. The site C insertion leads to a truncation of the pump about 50 residues short of the original C-terminal. One of the pumps (PMCA2) has special properties: It displays high activity even in the absence of the natural activator calmodulin, and has a particularly complex pattern of alternative splicing at both sites A and C. A variant of the PMCA2 pump containing an insert at site A and truncated C-terminally is the resident isoform of the pump in the stereocilia of hair cells of the inner ear. It exports Ca2+ to the endolymph that bathes the stereocilia less efficiently than the full length, non-inserted PMCA2 pump. The proper functioning of hair cells demands the precise maintenance of the Ca2+ balance between hair cells and the endolymph. Disturbances in the balance affect the process of mechano-electrical transduction, which depends on the ability of the stereociliar bundle to deflect in response to sound waves. The tip links that organize the bundle are formed by the Ca2+ binding protein cadherin 23 and by protocadherin 15: Disturbances of the Ca2+ binding by cadherin 23 and/or of the ability of the PMCA2 variant of the stereocilia to export Ca2+ to the endolymph generate hearing loss phenotypes. Such phenotypes have now been described in mice and humans. In some cases they are linked to mutations of both cadherin 23 and the PMCA2 pump, but in other cases they may be generated by mutations of particular severity in only one of the two proteins. The PMCA2 defect that leads to deafness has now been analyzed molecularly: It affects the long range, unstimulated ability of PMCA2 to export Ca2+

Calcineurin Controls the Transcription of Na+/Ca2+ Exchanger Isoforms in Developing Cerebellar Neurons

The Na(+)/Ca(2+) exchanger (NCX) and the plasma membrane Ca(2+)-ATPase export Ca(2+) from the cytosol to the extracellular space. Three NCX genes (NCX1, NCX2, and NCX3), encoding proteins with very similar properties, are expressed at different levels in tissues. Essentially, no information is available on the mechanisms that regulate their expression. Specific antibodies have been prepared and used to explore the expression of NCX1 and NCX2 in rat cerebellum. The expression of NCX2 became strongly up-regulated during development, whereas comparatively minor effects were seen for NCX1. This was also observed in cultured granule cells induced to mature in physiological concentrations of potassium. By contrast, higher K(+) concentrations, which induce partial depolarization of the plasma membrane and promote the influx of Ca(2+), caused the complete disappearance of NCX2. Reverse transcription-polymerase chain reaction analysis showed that the process occurred at the transcriptional level and depended on the activation of the Ca(2+) calmodulin-dependent protein phosphatase, calcineurin. The NCX1 and NCX3 genes were also affected by the depolarizing treatment: the transcription of the latter became up-regulated, and the pattern of expression of the splice variants of the former changed. The effects on the NCX1 and NCX3 genes were calcineurin-independent

Choice of profile for the wings of an airplane. Part I

The choice of the profile for the wings of an airplane is a problem which should be solved by a scientific method based on data obtained by systematic experimentation. The problem, in its present form, may be stated as follows: "To find a profile which has certain required aerodynamic characteristics and which encloses the spars, whose number, dimensions and separating distance are likewise determined by structural considerations." At present, the static test, corresponding to the case of accelerated flight at limited speed, requires the knowledge of the moment of the aerodynamic resultant at the angle of zero lift, and the possibility of controlling the magnitude of the corresponding absolute coefficient within more or less extensive limits

Choice of profile for the wings of an airplane. Part II

This report gives a general method for drawing airplane profiles. This method is useful, but it leads to a somewhat laborious drawing which becomes quite complicated when we take a transformation function having terms of a high degree

Expression, purification, and characterization of isoform 1 of the plasma membrane Ca2+ pump: focus on calpain sensitivity.

The plasma membrane Ca2+ ATPase isoform 1(PMCA1) is ubiquitously distributed in tissues and cells, but only scarce information is available on its properties. The isoform was overexpressed in Sf9 cells, purified on calmodulin columns, and characterized functionally. The level of expression was very low, but sufficient amounts of the protein could be isolated for biochemical characterization. The affinity of PMCA1 for calmodulin was similar to that of PMCA4, the other ubiquitous PMCA isoform. The affinity of PMCA1 for ATP, evaluated by the formation of the phosphorylated intermediate, was higher than that of the PMCA4 pump. The recombinant PMCA1 pump was a much better substrate for the cAMP-dependent protein kinase than the PMCA2 and PMCA4 isoforms. Pulse and chase experiments on Sf9 cells overexpressing the PMCA pumps showed that PMCA1 was much less stable than the PMCA4 and PMCA2 isoforms, i.e. PMCA1 had a much higher sensitivity to degradation by calpain. The effect of calpain was not the result of a general higher susceptibility of the PMCA1 to proteolytic degradation, because the pattern of degradation by trypsin was the same in the three isoforms