Differential regulation of the Na+-Ca2+ exchanger 3 (NCX3) by protein kinase PKC and PKA. (July 2017) - Michel, Lauriane Y.M.
Graphical abstract: Highlights: Na + -Ca 2+ exchanger isoform 3 (NCX3) is involved in the control of Ca 2+ homeostasis in many cell types. Ca 2+ uptake by NCX3 is increased or diminished during PKA or PKC stimulation, respectively. PKA and PKC phosphorylate NCX3 to modulate its activity. PKA-dependent activation of NCX3 participates in fight-or-flight response in skeletal muscles. Abstract: Isoform 3 of the Na + -Ca 2+ exchanger (NCX3) participates in the Ca 2+ 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 Ca 2+ uptake and extrusion of the two murine variants was measured using fura-2-based Ca 2+ imaging and revealed that both variants are similarly regulated. PKC stimulation diminished the Ca 2+ uptake performed by NCX3 in the reverse mode, triggered by a rise in [Ca 2+ ]i or [Na + ]i, whereas an opposite response was observed upon PKA stimulation, with a significant increase of the Ca 2+ uptake after a rise in [Ca 2+ ]i . The latter stimulation affected similarly the efflux capacity of NCX3 whereas Ca 2+ 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 Ca 2+ -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.