Differences in eNOS activity because of subcellular localization are dictated by phosphorylation state rather than the local calcium environment

Nitric oxide (NO) produced in the endothelium via the enzyme endothelial nitric-oxide synthase (eNOS) is an important vasoactive compound. Wild-type (WT) eNOS is localized to the plasma membrane and perinuclear/Golgi region by virtue of N-terminal myristoylation and palmitoylation. Acylation-deficient mutants (G2AeNOS) remain cytosolic and release less NO in response to Ca ²⁺-elevating agonists; a disparity that we hypothesized was attributed to the greater distance between G2AeNOS and plasma membrane Ca ²⁺ influx channels. The reduced activity of G2AeNOS versus WT was reversed upon disruption of cellular integrity with detergents or sonication. NO production from both constructs relied almost exclusively on the influx of extracellular Ca²⁺, and elevating intracellular Ca²⁺ to saturating levels with 10 μM ionomycin in the presence of 10 mM extracellular Ca²⁺ equalized NO production. To identify the contribution of calcium to the differences in activity between these enzymes, we created Ca ²⁺/CaM-independent eNOS mutants by deleting the two putative autoinhibitory domains of eNOS. There was no difference in NO production between WT and G2A-targeted Ca²⁺-independent eNOS, suggesting that Ca ²⁺ was the factor responsible. When eNOS constructs were fused in-frame to the bioluminescent probe aequorin, membrane-bound probes were exposed to higher [Ca²⁺] in unstimulated cells but upon ionomycin stimulation, the probes experienced equal amounts of Ca²⁺. The WT and G2A enzymes displayed significant differences in the phosphorylation state of Ser⁶¹⁷, Ser⁶³⁵, and Ser¹¹⁷⁹, and mutating all three sites to alanine or restoring phosphorylation with the phosphatase inhibitor calyculin abolished the differences in activity. We therefore conclude that the disparity in NO production between WTeNOS and G2AeNOS is not caused by different localized [Ca²⁺] upon stimulation with ionomycin, but rather differences in phosphorylation state between the two constructs.