FIGURE 2.

Inhibition of CaMKII specifically in caveolae abolished phosphorylation of the β2a subunit of the L-type calcium channel. (A) Schematic diagram of a fusion protein comprised of green fluorescent protein (GFP) tagged with caveolae-binding domain (CBD) and autocamtide-2-related inhibitory peptide (AIP), a CaMKII-specific inhibitory molecule. (B) Phosphorylation of the β2a subunit of the L-type calcium channel at threonine 498, a CaMKII phosphorylation site, in NRCMs expressing the β2a subunit or β-galactosidase (β-gal) as a control by adenoviral gene transfer. The additional adenoviral expression of a fusion protein, either CBD-GFP or CBD-GFP-AIP, was induced, and phosphorylation or expression levels were assessed by immunoblot analysis using the indicated antibodies. Phosphorylation of the overexpressed β2a subunit was substantially attenuated by CBD-GFP-AIP expression, indicating that phosphorylation of this protein occurs exclusively in caveolae. (C) Schema of the proposed mechanism based on immunoblot analysis. Phosphorylation of the β2a subunit by CaMKII induces a Ca²⁺ influx, which in turn elicits CaMKII activation to develop a positive feedback loop between the two molecules in the caveolae microdomain of NRCMs. Expression of CBD-GFP-AIP, which binds to caveolin-3, inhibits caveolae-specific CaMKII activation to terminate the positive feedback loop and abolish phosphorylation of the β2a subunit. MOI: multiplicity of infection. These figures were prepared with minor modifications from Tonegawa et al. (2017).