Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase

The activation and deactivation of Ca^(2+)- 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 Förster resonance energy transfer (FRET), we determined the occurrence of Ca^(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca 2+ concentrations ([Ca^(2+)]_(i)), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca^(2+)]_(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.

Molecular Cloning and Distribution of a Plasma Membrane Calcium ATPase Homolog from the Pearl Oyster Pinctada fucata

Plasma membrane calcium ATPase （PMCA） plays a critical role in transporting Ca^2＋ out of the cytosol across the plasma membrane which is essential both in keeping intracellular Ca^2＋ homeostasis and in biomineralization. In this paper we cloned and localized a gene encoding PMCA from the pearl oyster Pinctada fucata. This PMCA shares similarity with other published PMCAs within the functional domains. Reverse transcription-polymerase chain reaction analysis shows that it is expressed ubiquitously. Furthermore, in situ hybridization reveals that it is expressed in the inner epithelial cells of the outer fold and in the outer epithelial cells of the middle fold, as well as the edge near the shell, which suggests that PMCA may be involved in calcified layer formation. The identification and characterization of oyster PMCA can help to further understand the structural and functional properties of molluscan PMCA, as well as the mechanism of maintaining Ca^2＋ homeostasis and the mechanism of mineralization in pearl oyster.