G_i-Dependent Localization of β₂-Adrenergic Receptor Signaling to L-Type Ca²⁺ Channels

Abstract

A plausible determinant of the specificity of receptor signaling is the cellular compartment over which the signal is broadcast. In rat heart, stimulation of β₁-adrenergic receptor (β₁-AR), coupled to G_s-protein, or β₂-AR, coupled to G_s- and G_i-proteins, both increase L-type Ca²⁺ current, causing enhanced contractile strength. But only β₁-AR stimulation increases the phosphorylation of phospholamban, troponin-I, and C-protein, causing accelerated muscle relaxation and reduced myofilament sensitivity to Ca²⁺. β₂-AR stimulation does not affect any of these intracellular proteins. We hypothesized that β₂-AR signaling might be localized to the cell membrane. Thus we examined the spatial range and characteristics of β₁-AR and β₂-AR signaling on their common effector, L-type Ca²⁺ channels. Using the cell-attached patch-clamp technique, we show that stimulation of β₁-AR or β₂-AR in the patch membrane, by adding agonist into patch pipette, both activated the channels in the patch. But when the agonist was applied to the membrane outside the patch pipette, only β₁-AR stimulation activated the channels. Thus, β₁-AR signaling to the channels is diffusive through cytosol, whereas β₂-AR signaling is localized to the cell membrane. Furthermore, activation of G_i is essential to the localization of β₂-AR signaling because in pertussis toxin-treated cells, β₂-AR signaling becomes diffusive. Our results suggest that the dual coupling of β₂-AR to both G_s- and G_i-proteins leads to a highly localized β₂-AR signaling pathway to modulate sarcolemmal L-type Ca²⁺ channels in rat ventricular myocytes.