Local membrane charge regulates β₂ adrenergic receptor coupling to G_i3

The β₂ adrenergic receptor (β₂AR) 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 β₂AR-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 β₂AR, we find that they impair coupling to G_i3 and facilitate coupling to G_s. Positively charged Ca²⁺ and Mg²⁺, known to interact with the negative charge on phospholipids, facilitates G_i3 coupling. Mutational analysis suggests that Ca²⁺ 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 β₂AR and competing G protein subtypes.