A novel mutant allele of Ncx1: a single amino acid substitution leads to cardiac dysfunction

Abstract

The biological role and structure-function relationship of the Na + Ca 2+ exchanger NCX1 have been the subject of much investigation. Subtle mutagenesis to study the function of a protein seems only feasible in in vitro systems, but genetic forward screens have the potential to provide in vivo models to study single amino acid substitutions. In a genetic screen in mouse, we have isolated a mutant line carrying a novel mutant allele of the mouse Ncx1 gene. In this allele, a point mutation causes the substitution of a highly conserved asparagine residue (N874) with lysine. Accepted models for NCX1 structure propose that the affected amino acid is located in one of the reentrant membrane loops and experiments in vitro have identified N874 as critical for the ion transport function of NCX1. We found severe circulation defects and defective placentation in homozygous Ncx1 N87K4 mutant embryos, making the phenotype essentially indistinguishable from those of previously described null mutants. By ex vivo analysis, we demonstrated intrinsic functional abnormalities of cardiomyocytes. Western blot analysis and immunohistochemistry demonstrated normal levels and subcellular localization of the altered protein, ruling out the possibility that the abnormalities are a mere consequence of a major disturbance of protein structure. This study confirms and extends studies in vitro indicating the significance of amino acid N874 for the function of the NCX1 protein. It provides an in vivo model for this mutation and demonstrates the potential of forward genetic screens in a mammalian system.

SUMO 2 is essential while SUMO 3 is dispensable for mouse embryonic development

“…Whole‐mount in situ hybridization was conducted as described previously . Briefly, antisense riboprobes were generated by in vitro transcription using the respective cDNA templates and were then labeled with digoxigenin (DIG)‐UTP (Roche).…”

The specific binding of peptide ligands to cardiomyocytes derived from mouse embryonic stem cells

“…Nkx2.5 (NK2 transcription factor related, locus 5) regulates tissue‐specific gene expression and organ differentiation, and plays an important role in the formation of the heart in mice and humans . Ncx1 [Slc8a1 solute carrier family 8 (sodium/calcium exchanger), member 1] is a Na + /Ca 2+ exchanger, and maintains the calcium concentration of the sarcoplasmic reticulum in myocardial cells . cTN1 ( Mus musculus troponin I, cardiac 3) (Tnni3) also has important effects on heart development .…”