Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase |
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Authors: | Wenjuan Duan Juefei Zhou Wei Li Teng Zhou Qianqian Chen Fuyu Yang Taotao Wei |
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Institution: | 1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 2. Research Department of Bacterial Vaccine, Chengdu Institute of Biological Products Co. Ltd, Chengdu 610023, China; 3. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; 4. University of Chinese Academy of Sciences, Beijing 100049, China |
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Abstract: | The activation and deactivation of Ca2+- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved F?rster resonance energy transfer (FRET), we determined the occurrence of Ca2+-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca2+ concentrations (Ca2+]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal Ca2+]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation. |
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Keywords: | plasma membrane calcium ATPase neuronal nitric oxide synthase calcium nitric oxide lipid raft F?rster resonance energy transfer |
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