Very mild presentation in adult with classical cellular phenotype of ataxia telangiectasia.

BACKGROUND: The major clinical feature of ataxia telangiectasia (A-T) is severe progressive neurodegeneration with onset in infancy. This classical A-T phenotype is caused by biallelic null mutations in the ATM gene, leading to the absence of ATM protein and increased cellular radiosensitivity. We report an unusual case of A-T in a 41-year-old mother, A-T210, who had very mild neurological symptoms despite complete loss of ATM protein. METHODS: A neurological examination was performed, cellular radiosensitivity was assessed, and the ATM gene was sequenced. Skin fibroblasts and a lymphoblastoid cell line (LCL) were assayed for ATM protein expression and kinase activity. RESULTS: Patient A-T210 showed mild chorea, dystonia, and gait ataxia, walked independently, and drove a car. LCL and skin fibroblasts were radiosensitive and did not express ATM protein. Two ATM-null mutations were identified. CONCLUSIONS: The severe neurodegeneration resulting from loss of ATM can be mitigated in some circumstances.

A plethora of interacting organellar Ca2+ stores.

The endoplasmic reticulum is not the only major agonist-releasable Ca2+ store within cells; it is now clear that virtually all organelles so far studied have the ability to act as mobilizable Ca2+ stores. From recent findings with regard to Ca2+ transportation and Ca2+ homeostasis within a variety of cell organelles such as the mitochondria, nucleus, Golgi and lysosomes, it emerges that many of these organellar Ca2+ stores appear to interact with each other, adding a further level of complexity to Ca2+ signalling events.

The expression, activity and localisation of the secretory pathway Ca2+ -ATPase (SPCA1) in different mammalian tissues.

The distribution of the secretory pathway Ca2+ -ATPase (SPCA1) was investigated at both the mRNA and protein level in a variety of tissues. The mRNA and the protein for SPCA1 were relatively abundant in rat brain, testis and testicular derived cells (myoid cells, germ cells, primary Sertoli cells and TM4 cells; a mouse Sertoli cell line) and epididymal fat pads. Lower levels were found in aorta (rat and porcine), heart, liver, lung and kidney. SPCA activities from a number of tissues were measured and shown to be particularly high in brain, aorta, heart, fat pads and testis. As the proportion of SPCA activity compared to total Ca2+ ATPase activity in brain, aorta, fat pads and testis were relatively high, this suggests that SPCA1 plays a major role in Ca2+ storage within these tissues. The subcellular localisation of SPCA1 was shown to be predominantly around the Golgi in both human aortic smooth muscle cells and TM4 cells.

Endocrine disrupting alkylphenols: structural requirements for their adverse effects on Ca2+ pumps, Ca2+ homeostasis & Sertoli TM4 cell viability.

Alkylphenols such as nonylphenol are pollutants that are widely dispersed within our environment. They bio-accumulate within man, with levels in the muM concentration range reported in human tissues. These chemicals act as endocrine disruptors, having xenoestrogenic activity. More recently alkylphenols have also been shown to affect Ca2+ signalling pathways. Here we show that alkylphenols are potent inhibitors of sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) activity. For linear chain alkylphenols the potency of inhibition is related to chain length, with the IC50 values for inhibition ranging from 8 microM for 4-n-nonylphenol (C9) to 1.3 mM for 4-n-propylphenol (C3). Branched chain alkylphenols generally had lower potencies than their linear chain counterparts, however, good correlations for all alkylphenols were observed between their Ca2+ pump inhibition and hydrophobicity, molecular volume and flexibility, indicating that these parameters are all important factors. Alkylphenols cause abnormal elevations of intracellular [Ca2+] within TM4 Sertoli cells (cells involved in sperm maturation) depolarise their mitochondria and induce cell death in these cells, in an alkyl chain size-dependent manner.

The effects of the phenylalanine 256 to valine mutation on the sensitivity of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) Ca2+ pump isoforms 1, 2, and 3 to thapsigargin and other inhibitors.

Three isoforms of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) are known to exist in mammalian cells. This study investigated the effects of thapsigargin and a variety of commonly used hydrophobic inhibitors on these SERCA isoforms (i.e. SERCA1b, SERCA2b, and SERCA3a), which were transiently expressed in COS-7 cells. In addition, the study assessed whether the introduction of the phenylalanine to valine mutation at position 256 (F256V), known to reduce the potency of thapsigargin inhibition in avian SERCA1, affects the other SERCA isoforms in a similar manner and whether this mutation also affects the inhibition by other inhibitors. This study has shown that the sensitivity to thapsigargin is different for the SERCA isoforms (apparent K(i) values being 0.21, 1.3, and 12 nm for SERCA1b, SERCA2b, and SERCA3a, respectively). The reduction in thapsigargin sensitivity caused by the F256V mutation was also different for the three isoforms, with SERCA2b only being modestly affected by this mutation. Although some of the other inhibitors investigated (i.e. cyclopiazonic acid and curcumin) showed some differences in their sensitivity toward the SERCA isoforms, most were little affected by the F256V mutation, indicating that they inhibit the Ca(2+)-ATPase by binding to sites on SERCA distinct from that of thapsigargin.

The inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase by macrocyclic lactones and cyclosporin A.

The pharmacology of macrocyclic lactones is varied, with many beneficial effects in treating disease processes. FK-506, rapamycin and ascomycin have been utilized as immunosuppressant agents. Ivermectin is typically used to treat parasitic worm infections in mammals. Another immunosuppressant, cyclosporin A, is a cyclic oligotide that has similar immunosuppressant properties to those exerted by macrocyclic lactones. Here we report on the inhibition by these compounds of sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) Ca(2+) pumps. Ivermectin, cyclosporin A and rapamycin all inhibited the skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1). In addition, although ivermectin inhibited brain microsomal endoplasmic reticulum (type 2b) Ca(2+)-ATPase, cyclosporin A and rapamycin did not. As cyclosporin A also did not inhibit cardiac Ca(2+)-ATPase activity, this would suggest that it could be an isoform-specific inhibitor. Ivermectin was shown to be the most potent Ca(2+)-ATPase inhibitor of the macrocyclic lactones (IC(50)=7 microM). It appears to show a 'competitive' inhibition with respect to high concentrations of ATP by increasing the regulatory binding site K(m) but without affecting the catalytic site K(m). In addition, ivermectin stabilizes the ATPase in an E1 conformational state, and inhibits Ca(2+) release from the enzyme during turnover. This would suggest that ivermectin inhibits Ca(2+) release from the luminal binding sites of the phosphoenzyme intermediate, a step that is known to be accelerated by high [ATP].

The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by paxilline.

Paxilline, an indole alkaloid mycotoxin from Penicillium paxilli, is an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). Paxilline inhibited differing isoforms of SERCA with IC50s between 5 and 50 microM. It inhibited more potently the purified Ca2+ ATPase activity from skeletal muscle with an IC50 of 5 microM. Detailed effects of this inhibitor on the Ca2+ and ATP dependence upon activity indicate that it affects the high-affinity Ca2+-binding (E1) form of the ATPase. In addition, paxilline is a "competitive" inhibitor with respect to high concentrations of ATP, increasing the regulatory binding site K(m), without affecting the catalytic binding site K(m). At higher concentrations, paxilline inhibits phosphoenzyme formation from ATP and inorganic phosphate, without affecting nucleotide binding. We therefore suggest that paxilline has two effects on the Ca2+ ATPase. At lower concentrations (5-10 microM), paxilline inhibits the ATP-dependent acceleration of Ca2+ release from the phosphoenzyme and/or phosphoenzyme decay. At higher concentrations, paxilline inhibits phosphoenzyme formation.

Inhibition of SERCA Ca2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca2+-binding sites.

2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca2+ channels and store-operated Ca2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca2+ signalling experiments. The inhibition of 2-APB on the SERCA Ca2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 micro m, respectively, measured at pH 7.2). The Ca2+-ATPase is also more potently inhibited at lower pH (IC50 = 70 micro m for SERCA1A at pH 6). 2-APB decreases the affinity for Ca2+ binding to the ATPase by more than 20-fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca2+-ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 (L6-7), and reduce access of Ca2+ to their binding sites.