Ortega, Claudia Karina (2007) Functional properties of an unusual isoform of the plasma membrane calcium ATPase: PMCA 2. [Ph.D. thesis]
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The plasma membrane calcium ATP-ase (PMCA) represents a primary system for the specific extrusion of calcium from eukaryotic cells. Together with Na+/Ca2+ exchangers, it is the major plasma membrane transport system responsible for the long-term regulation of the resting intracellular calcium concentration. The PMCAs contain 10 membrane-spanning segments and the NH2 and COOH termini are both located on the cytosolic side of the membrane. The PMCA pump is the product of 4 separate genes, isoform diversity being further increased by a complex pattern of alternative splicing of the primary transcripts. The four basic gene products (PMCA 1-4) and the numerous splice variants vary in expression level during development, have peculiar distribution in tissues and within
cells, and differ with respect to functional parameters, especially those involving regulation properties. PMCA 1 and 4 are ubiquitously expressed while 2 and 3 are mostly found in the central nervous system and, in lesser amounts, in the striated muscle.
Alternative splicing is peculiarly complex in PMCA2 because it involves the insertion of up to three novel exons at site A (variant w) and of two at site C (variant a). The insert at site C creates instead a novel stop codon, leading to the truncation of the pump. The site-C insertions eliminate approximately half of the calmodulin binding domain; those at site A occur next to a domain that binds activatory acidic phospholipids.
Calcium enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph
by an unusual splicing isoform (wa) of plasma-membrane calcium-pump isoform 2 (PMCA2).
Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. The wa variant and their mutations were
overexpressed in Sf9 cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca2+ pulse. The defect is more pronounced
in the dfw mutant than in the G293S (human) mutant. A third mutation was analyzed: Oblivion mouse mutation (where a Serine is replaced by a Phenylalanine at position 877 in TM6), it was slightly more active than the pumps bearing the 283 and 293 mutations but still much lower that of the wild type wa. The other important aspect studied, it is the regulation mechanism of the PMCA 2 wa variant. Microsomal membranes isolated from CHO cells transfected with PMCA 2 zb, PMCA 2 wb, PMCA 2 wa and PMCA 2 wa (Tommy), where a mutation in the ATP binding site occurs in the valine 586, were assayed. Both PMCA 2 zb and PMCA 2 wb had higher basal activity than PMCA 2 wa. PMCA 2 wa (Tommy) only had about 25 per cent of the activity of PMCA 2 wa. In the presence of Calmodulin, PMCA2 zb and wb showed the highest response to it i.e., the activity of these isoforms was over eight times higher than of PMCA 2 wa. PMCA2 wa had lower affinity for calmodulin than PMCA2 zb and wb, but had still higher affinity than the corresponding Tommy mutant.
The mechanism by which different phospholipids activate the PMCAs is not known. However, given the location of the lipid-binding sequences in the pump, one speculated
that the interaction of acidic phospholipids with the calmodulin binding domain leads to some structural rearrangement that weakens the autoinhibitory intramolecular interactions formed by the COOH-terminal tail. The PMCA 2 zb and wb, when
overexpressed in CHO cells, had the same response to this acidic phospholipids implying that the inserts next to the N-terminal phospholipids binding domain (variant w) was not disturbed by the splicing insert. The PMCA wa was absolutely not stimulated
by phosphatidyl serine apparently, then, no activation by acidic phospholipids can occur when the C-terminal binding site is undisturbed. The response of the PMCA 2 b pumps to PS was 4 fold higher than that of the wa variant.
To investigate whether Calmodulin indeed removes its binding domain from the cytosolic loops of the pump, we have introduced a mutation in one of the two sites that interact with the CaM binding domain in the cytosolic loops of the pump. Prior to this,
the PMCA pump was modeled to decide the residue that should have been mutated. From the model and previous works we decided to mutate the methionine 265 in Alanine (M265A). The activity of the PMCA 2 zb and M265A pumps expressed in transfected CHO cells evaluated as a function of Ca2+ in both the presence and absence of calmodulin shows that the M265A mutant has a response to calmodulin which was the same as that of the wild type at saturating concentrations of calmodulin. The activation by CaM was higher in the M265A pump with respect to wild type pump at 1nM, 5nM and 15 nM calmodulin concentrations. This suggests that the autoinhibitory sequence was bound with less affinity to the intramolecular binding site in the Met265 mutant as less calmodulin was required to relieve the inhibition. Evidently, the inhibition of Met 265 decreased the affinity of the C-terminal autoinhibitory domain, making it
easier for calmodulin to remove it from binding site.
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