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Analysis of the physical interaction between α 1G T‐type calcium channel and endothelial nitric oxide synthase (NOS3) in pulmonary microvascular endothelial cells
Author(s) -
Sellak Hassan,
Chen Hairu,
Alexeyev Mikhail,
Wu Songwei
Publication year - 2016
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.30.1_supplement.980.16
Subject(s) - immunoprecipitation , mutant , nitric oxide synthase , cytoplasm , microbiology and biotechnology , chemistry , cytosol , nitric oxide , biology , biochemistry , gene , enzyme , organic chemistry
We have observed that, in pulmonary microvascular endothelial cells (PMVECs), activation of the site‐specific α 1G T‐type Ca 2+ channel (α 1G ) provides a cytosolic Ca 2+ source necessary for endothelial nitric oxide synthase (NOS3) activation. Furthermore, the interaction between α 1G and NOS3 in PMVECs has been demonstrated by in vitro Förster resonance energy transfer studies, as well as by immunoprecipitation assays. The present work was undertaken to analyze the molecular basis of the α 1G ‐NOS3 interaction. We constructed retroviral expression vectors harboring different deletion mutants of α 1G that encompasses its cytoplasmic domains including N‐terminus (α 1G ‐Nter, 1–80), loop I–II (396–744), loop II–III (966–1,251), loop III–IV (1,517–1,578), and C‐terminus (α 1G ‐Cter, 1,823–2,254). Additionally, Rat NOS3 was cloned in retroviral expression vector and five (5) deletion mutants were generated and similarly cloned, i.e. , NOS3‐F1 (1–730), NOS3‐F2 (1–450), NOS3‐F3 (400–730), NOS3‐F4 (700–1,202), and NOS3‐F5 (850–1,202). PMVECs were retrovirally transduced with full‐length α 1G alone, full‐length α 1G combined with full‐length NOS3, or full‐length α 1G combined with different NOS3 deletion mutants. Immunoprecipitation assays using protein extracts from these transduced cells showed that fragments NOS3‐F1 (1–730) and NOS3‐F5 (850–1,202) interact with full‐length α 1G . In a reverse approach, PMVECs were retrovirally transduced with full‐length NOS3 alone or in combination with each α 1G cytoplasmic domains. Immunoprecipitation and immunoblot assays were performed and results revealed that α 1G loop I–II, loop II–III, and α 1G C‐terminus interact with NOS3. Altogether, these findings demonstrated the existence of constitutive physical interaction between α 1G T‐type Ca 2+ channel and NOS3, and identified the critical domains that mediate α 1G ‐NOS3 interaction. Thus, α 1G ‐NOS3 association may form a signaling complex that governs the inflammatory response of pulmonary microvascular endothelium. Support or Funding Information Supported by HL‐066299 .