Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein

Abstract

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca(2+) act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT(-/-)) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.

Figure 1

(A) Properties of FRET-MT. Shown is FRET-MT cDNA construct of human type IIa metallothionein (hMTIIAa), flanked by enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP). (B) Scheme of the above construct (FRET-MT) showing possible unfolding, metal release and consequent changes in FRET induced by EDTA/NaCl. (C–E) Fluorescent emission spectra of lysates as isolated (control) and after addition of 1 mM EDTA/150 mM NaCl (decrease in 535 nm intensity and increase in 480 nm intensity after 8-h incubation) (C); 50 μM Cu¹⁺ (immediate decrease in 480 nm intensity and increase in 535 nm intensity) (D); and 100 μM of NO (immediate decrease in 535 nm intensity and increase in 480 nm intensity) (E).

Figure 2

Regulation of FRET-MT function in endothelial cells. Emission intensity ratio (535 nm/480 nm) changes over time after addition of 10 μM carbachol (indicated by arrow) (A); 20 μM bradykinin (indicated by arrow) (B); or 1 μM of the calcium ionophore Br-A23187 (indicated by arrow) (C). (D) Histogram of percent emission ratio changes showing control (time, 2,000 s), carbachol, bradykinin, and Br-A23187 with relative error bars. (E) Pseudo-color representation of a SPAEC before and after addition of Br-A23187.

Figure 3

eNOS-generated NO regulates FRET-MT. Shown are emission ratio changes (535 nm/480 nm) in response to S-nitrosylglutathione (100 μM, 100 μM DTT was present throughout the experiment) (A) [Inset shows individual 535-nm fluorescence intensity (left axis) and 480-nm fluorescence intensity (right axis)]; ≈1 mM NO (B); 1 μM BrA23187 in the presence of 15 μM W-7 (C); and 1 μM BrA23187 in the presence of 1 mM l-NAME using arginine-depleted cells (D). (E) Histogram of percent emission changes with relative error bars (see text for details).

Figure 4

Lack of myogenic reactivity in mesenteric arteries from MT^−/− mice can be restored by inhibition of nitric oxide synthase. The percent myogenic tone (difference between passive diameter and reactive diameter) of mesenteric arteries from MT^+/+ (closed circles) and MT^−/− (open circles) mice (n = 5, with relative error bars) was measured with stepwise increases in luminal pressure of 20 mmHg in the presence of l-NAME (A), the absence of l-NAME (B), and after adding l-arginine after the pretreatment with l-NAME (D).