Abstract 13159: SCN5A and Nedd4.2 S-Nitrosylation by nNOS Decreases SCN5A Ubiquitinization: A Mechanism for Increased Peak and Late Sodium Current
Introduction: The voltage-gated sodium channel (SCN5A) and its channel interacting proteins (ChIPs) constitute a macromolecular complex that regulates the function, expression, and turnover of the sodium channel protein. Nitric oxide (NO) from neural nitric oxide synthase (nNOS) activity causes S-nitrosylation of SCN5A and increased late sodium current (INa), and excessive late INa underlies the molecular phenotype of LQT syndrome types 3, 9 and 12. We previously showed that in the macromolecular complex of SCN5A-nNOS-syntrophin α1 (SNTA), sarcolemmal Ca2+ ATPase (PMCA4b) and caveolin 3 (Cav3) are negative regulators of nNOS activity. Nedd4.2 is an enzyme from the ubiquitin ligase 3 family that regulates SCN5A ubiquitination and consequent internalization and degradation. S-nitrosylation of parkin, a protein of the ligase 3 family, reduces ubiquitin ligase activity. We hypotehsized that increased nNOS activity causes Nedd4.2 nitrosylation resulting in decreased SCN5A internalization and increased peak INa.
Methods: We co-expressed the nNOS-SCN5A-SNTA macromolecular complex with Nedd4.2 alone, or Nedd4.2 plus either PMCA4b or Cav3 in HEK-293 cells. INa was recorded using the voltage clamp method with standard bath and pipette solutions. Protein nitrosylation was detected using the biotin switch assay (BSA) followed by Western blots.
Results: In HEK-293 cells expressing the macromolecular complex SCN5A-nNOS-SNTA and an empty vector (control), peak INa was 343±85 pA/pF; coexpression of Nedd4.2 reduced peak INa from control levels to 152±65, and coexpression of Nedd4.2 in tandem with PMCA4b or Cav3 further reduced peak INa from control levels to 20±15 or 43±17 pA/pF, respectively (n= 6-10 cells per group). In the latter two sets of cells, incubation with the NO donor SNAP (200 µM for 2 hours at 37 °C) restored peak INa to near control levels (~300 pA/pF, p<0.05, n= 3-6). BSA revealed that Nedd4.2 was S-nitrosylated under these conditions.
Conclusions: These results identify Nedd4.2 S-nitrosylation as a novel mechanism of sodium channel regulation, and recognize that abnormal S-nitrosylation of interacting/regulatory proteins like Nedd4.2 may contribute to pathophysiological processes that can potentially cause arrhythmia syndromes.
- © 2011 by American Heart Association, Inc.