Calpain-3-mediated regulation of the Na(+)-Ca(2+) exchanger isoform 3.

Ca(2+) disturbances are observed when Ca(2+)-dependent cysteine proteases malfunction, causing muscle weakness and wasting. For example, loss of calpain-3 (CAPN3) activity leads to limb-girdle muscular dystrophy 2A (LGMD2A). In neuronal excitotoxicity, the cleavage of the Na(+)-Ca(2+) exchanger isoform 3 (NCX3) has been associated with an increase in activity and elevation of the Ca(2+) content in the endoplasmic reticulum (ER). Since NCX3 is expressed in skeletal muscle, we evaluated the cleavage of different NCX3 splice variants by CAPN1 and CAPN3. Using Fura-2-based cellular Ca(2+) imaging, we showed for the first time that CAPN3 increases NCX3 activity and that only NCX3-AC, the variant predominantly expressed in skeletal muscle, is sensitive to calpain. The silencing of the endogenous CAPN1 and the expression of the inactive form of CAPN3 (C129S CAPN3) confirmed the specificity for CAPN1 and CAPN3. Functional studies revealed that cellular Ca(2+) uptake through the reverse mode of NCX3 was significantly increased independently of the mode of activation of the exchanger by either a rise in intracellular Ca(2+) ([Ca(2+)]i) or Na(+) ([Na(+)]i). Subsequently, the sensitivity to CAPN1 and CAPN3 could be abrogated by removal of the six residues coded in exon C of NCX3-AC. Additionally, mutation of the Leu-600 and Leu-601 suggested the presence of a cleavage site at Leu-602. The increased Ca(2+) uptake of NCX3 might participate in the Ca(2+) refilling of the sarcoplasmic reticulum (SR) after the excitation-contraction uncoupling following exercise and therefore be implicated in the impaired reticular Ca(2+) storage observed in LGMD2A

Function and regulation of the Na+-Ca2+ exchanger NCX3 splice variants in brain and skeletal muscle

Contains fulltext : 134050.pdf (publisher's version ) (Open Access)Isoform 3 of the Na(+)-Ca(2+) exchanger (NCX3) is crucial for maintaining intracellular calcium ([Ca(2+)]i) homeostasis in excitable tissues. In this sense NCX3 plays a key role in neuronal excitotoxicity and Ca(2+) extrusion during skeletal muscle relaxation. Alternative splicing generates two variants (NCX3-AC and NCX3-B). Here, we demonstrated that NCX3 variants display a tissue-specific distribution in mice, with NCX3-B as mostly expressed in brain and NCX-AC as predominant in skeletal muscle. Using Fura-2-based Ca(2+) imaging, we measured the capacity and regulation of the two variants during Ca(2+) extrusion and uptake in different conditions. Functional studies revealed that, although both variants are activated by intracellular sodium ([Na(+)]i), NCX3-AC has a higher [Na(+)]i sensitivity, as Ca(2+) influx is observed in the presence of extracellular Na(+). This effect could be partially mimicked for NCX3-B by mutating several glutamate residues in its cytoplasmic loop. In addition, NCX3-AC displayed a higher capacity of both Ca(2+) extrusion and uptake compared with NCX3-B, together with an increased sensitivity to intracellular Ca(2+). Strikingly, substitution of Glu(580) in NCX3-B with its NCX3-AC equivalent Lys(580) recapitulated the functional properties of NCX3-AC regarding Ca(2+) sensitivity, Lys(580) presumably acting through a structure stabilization of the Ca(2+) binding site. The higher Ca(2+) uptake capacity of NCX3-AC compared with NCX3-B is in line with the necessity to restore Ca(2+) levels in the sarcoplasmic reticulum during prolonged exercise. The latter result, consistent with the high expression in the slow-twitch muscle, suggests that this variant may contribute to the Ca(2+) handling beyond that of extruding Ca(2+)

Differential regulation of the Na+-Ca2+ exchanger 3 (NCX3) by protein kinase PKC and PKA.

Contains fulltext : 174540.pdf (publisher's version ) (Closed access)Isoform 3 of the Na+-Ca2+ exchanger (NCX3) participates in the Ca2+ fluxes across the plasma membrane. Among the NCX family, NCX3 carries out a peculiar role due to its specific functions in skeletal muscle and the immune system and to its neuroprotective effect under stress exposure. In this context, proper understanding of the regulation of NCX3 is primordial to consider its potential use as a drug target. In this study, we demonstrated the regulation of NCX3 by protein kinase A (PKA) and C (PKC). Disparity in regulation has been previously reported among the splice variants of NCX3 therefore the activity of Ca2+ uptake and extrusion of the two murine variants was measured using fura-2-based Ca2+ imaging and revealed that both variants are similarly regulated. PKC stimulation diminished the Ca2+ uptake performed by NCX3 in the reverse mode, triggered by a rise in [Ca2+]i or [Na+]i, whereas an opposite response was observed upon PKA stimulation, with a significant increase of the Ca2+ uptake after a rise in [Ca2+]i. The latter stimulation affected similarly the efflux capacity of NCX3 whereas Ca2+ extrusion capacity remained unaffected under activation of PKC. Next, using site-directed mutagenesis, the sensitivity of NCX3 to PKC was abolished by singly mutating its predicted phosphorylation sites T529 or S695. The sensitivity to PKC might be due to the influence of T529 phosphorylation on the Ca2+-binding domain 1. Additionally, we showed that stimulation of NCX3 by PKA occurred through residue S524. This effect may well participate in the fight-or-flight response in skeletal muscle and the long-term potentiation in hippocampus.1 juli 201

Function and regulation of the Na+-Ca2+ exchanger NCX3 splice variants in brain and skeletal muscle

Isoform 3 of the Na(+)-Ca(2+) exchanger (NCX3) is crucial for maintaining intracellular calcium ([Ca(2+)]i) homeostasis in excitable tissues. In this sense NCX3 plays a key role in neuronal excitotoxicity and Ca(2+) extrusion during skeletal muscle relaxation. Alternative splicing generates two variants (NCX3-AC and NCX3-B). Here, we demonstrated that NCX3 variants display a tissue-specific distribution in mice, with NCX3-B as mostly expressed in brain and NCX-AC as predominant in skeletal muscle. Using Fura-2-based Ca(2+) imaging, we measured the capacity and regulation of the two variants during Ca(2+) extrusion and uptake in different conditions. Functional studies revealed that, although both variants are activated by intracellular sodium ([Na(+)]i), NCX3-AC has a higher [Na(+)]i sensitivity, as Ca(2+) influx is observed in the presence of extracellular Na(+). This effect could be partially mimicked for NCX3-B by mutating several glutamate residues in its cytoplasmic loop. In addition, NCX3-AC displayed a higher capacity of both Ca(2+) extrusion and uptake compared with NCX3-B, together with an increased sensitivity to intracellular Ca(2+). Strikingly, substitution of Glu(580) in NCX3-B with its NCX3-AC equivalent Lys(580) recapitulated the functional properties of NCX3-AC regarding Ca(2+) sensitivity, Lys(580) presumably acting through a structure stabilization of the Ca(2+) binding site. The higher Ca(2+) uptake capacity of NCX3-AC compared with NCX3-B is in line with the necessity to restore Ca(2+) levels in the sarcoplasmic reticulum during prolonged exercise. The latter result, consistent with the high expression in the slow-twitch muscle, suggests that this variant may contribute to the Ca(2+) handling beyond that of extruding Ca(2+)

Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H2O2), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H2O2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H2O2 Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H2O2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H2O2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies.Virginie Montiel, Ramona Bella, Lauriane Y. M. Michel, Hrag Esfahani, Delphine De Mulder ... et al