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Variant Specific Cleavage of the Na + ‐Ca 2+ Exchanger NCX3 During Excitotoxicity
Author(s) -
Michel Lauriane,
Hoenderop Joost,
Bindels René
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.lb620
Subject(s) - calpain , calpastatin , cleavage (geology) , microbiology and biotechnology , chemistry , exon , biology , biochemistry , gene , enzyme , paleontology , fracture (geology)
Recent studies highlighted the crucial role of the Na + ‐Ca 2+ exchanger isoform 3, NCX3, during stress exposure, allowing for a significant Ca 2+ entry in neuronal cells. Consequently, the hyperfunctionality of NCX3 triggered via its cleavage by the calpain protease, is considered to play a neuroprotective effect in numerous pathologies such as Alzheimer's disease and ischemia.NCX3 family contains three variants hypothesized to carry diverse capacity of exchange. We aimed in dissecting the molecular mechanisms responsible for NCX3 cleavage during excitotoxicity.The capacity of exchange of each variant expressed in HEK293T cells was measured by recording single cell [Ca 2+ ] i in response to Na + removal. Additionally, the influence of calpain on NCX3 was investigated using overexpression of the endogeneous inhibitor, Calpastatin, and silencing of the calpain family. We observed that the variants containing the exon B, NCX3‐B and NCX3‐BC, are predominantly expressed in the brain. Measurements of NCX3 revealed a significant decrease in the capacity of exchange of NCX3‐AC and NCX3‐BC during overexpression of Calpastatin. A similar effect was observed by silencing calpain‐1. Site‐directed mutagenesis was performed on a hypothesized cleavage site for calpain located in the exon C of NCX3. The mutation of L 600 and L 601 , at the cleavage site, caused a loss of hyperfunctionality, as observed during the silencing of calpain‐1. Calpain activity triggers an increase in the capacity of exchange of NCX3. However only the variants containing the exon C, NCX3‐AC and NCX3‐BC, are sensitive to calpain cleavage. The predominant expression of NCX3‐BC observed in the brain together with the identification of the cleavage site in NCX3 allows for potential drug design targeting these specific residues.