The polybasic puzzle: how N-terminal charges modulate Gαi₁ membrane behavior and signaling output

G protein-coupled receptors (GPCRs) mediate essential cellular responses through interactions with heterotrimeric G proteins, particularly the Gαi subfamily. Despite high sequence similarity among Gαi subunits, subtle differences in their N-terminal polybasic regions influence membrane association, heterotrimer stability, and downstream signaling. Here, we investigate the functional significance of two arginine residues (R21 and R32) within the N-terminal helix of Gαi₁. Using site-directed mutagenesis, fluorescence-based techniques including FRAP and FLIM-FRET, cAMP assays, and molecular dynamics simulations, we demonstrate that even charge-conserving arginine-to-lysine substitutions significantly alter membrane dynamics and impair receptor-mediated signaling. Notably, the R21A/R32A double mutant, which completely neutralizes the local electrostatic charge at these positions, disrupts nanoscale membrane organization, destabilizes the Gαi₁β₁γ₂ complex, and abolishes dopamine D₂ receptor-mediated cAMP inhibition. Although these residues do not directly contact Gβγ or adenylyl cyclase, their spatial charge distribution modulates electrostatic interactions with membrane lipids and receptor coupling interfaces. Our findings underscore the critical role of polybasic motif composition in regulating the biophysical and functional properties of peripheral membrane proteins and reveal how minor sequence variations can profoundly impact GPCR-G protein signaling fidelity.