Neuronal nitric oxide synthase inhibits store‐operated Ca2+ entry in cardiomyocytes via S‐nitrosylation of stromal interaction molecule‐1

Abstract

Store‐operated Ca2+ entry (SOCE) mediated by stromal interacting molecule‐1 (STIM1) and Orai1 represents a major route of Ca2+ entry in mammalian cells. The luminal domain of STIM1 is critical to Ca2+ sensing, STIM1 oligomerization and SOCE activation. Notably, the luminal domain contains two cysteine sites (Cys49 and Cys56). It is not known if neuronal nitric oxide synthase (nNOS), which is associated to the sarcoplasmic reticulum (SR) membrane, regulates STIM1 oligomerization and SOCE activity in cardiomyocytes via S‐nitrosylation of these cysteine sites. Here, we show that nNOS deficiency or treatment with a nNOS inhibitor L‐VNIO significantly increased SOCE in cardiomyocytes. Further, the Ca2+ release‐activated Ca2+ channel current (ICRAC) was significantly enhanced in cardiomyocytes treated with L‐VNIO and in nNOS−/− cardiomyocytes. Consistently, STIM1 S‐nitrosylation was significantly decreased in nNOS−/− hearts. Treatment of HEK293 cells co‐expressing YFP‐Orai1 and mCherry‐STIM1 with the NO donor S‐nitrosoglutathione (GSNO) inhibited STIM1 puncta formation and ICRAC. Remarkably, no functional inhibition was observed in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Mechanistically, we found that NO donors caused Cys49 and Cys56‐specific structural changes associated with reduced protein backbone mobility, significantly increased the thermal stability and suppressed Ca2+‐depletion‐dependent oligomerization of the luminal Ca2+‐sensing region of STIM1. Collectively, our data reveal that nNOS inhibits SOCE in cardiomyocytes through NO‐mediated STIM1 S‐nitrosylation which suppresses oligomerization via enhanced luminal domain stability and rigidity.Support or Funding InformationCanadian Institutes of Health Research