Local membrane charge regulates β(2) adrenergic receptor coupling to G(i3)

The β(2) adrenergic receptor (β(2)AR) signals through both G(s) and G(i) in cardiac myocytes, and the G(i) pathway counteracts the G(s) pathway. However, G(i) coupling is much less efficient than G(s) coupling in most cell-based and biochemical assays, making it difficult to study β(2)AR−G(i) interactions. Here we investigate the role of phospholipid composition on G(s) and G(i) coupling. While negatively charged phospholipids are known to enhance agonist affinity and stabilize an active state of the β(2)AR, we find that they impair coupling to G(i3) and facilitate coupling to G(s). Positively charged Ca(2+) and Mg(2+), known to interact with the negative charge on phospholipids, facilitates G(i3) coupling. Mutational analysis suggests that Ca(2+) coordinates an interaction between phospholipid and the negatively charged EDGE motif on the amino terminal helix of G(i3). Taken together, our observations suggest that local membrane charge modulates the interaction between β(2)AR and competing G protein subtypes.