Specificity of Gβγ signaling to Kir3 channels depends on the helical domain of pertussis toxin-sensitive Gα subunits

Acetylcholine signaling through muscarinic type 2 receptors activates atrial G protein-gated inwardly rectifying K⁺ (Kir3) channels via the βγ subunits of G proteins (Gβγ). Different combinations of recombinant Gβγ subunits have been shown to activate Kir3 channels in a similar manner. In native systems, however, only Gβγ subunits associated with the pertussis toxin-sensitive Gα_i/o subunits signal to K⁺ channels. Additionally, in vitro binding experiments supported the notion that the C terminus of Kir3 channels interacts preferentially with Gα_i over Gα_q. In this study we confirmed in two heterologous expression systems a preference of Gα_i over Gα_q in the activation of K ⁺ currents. To identify determinants of Gγβ signaling specificity, we first exchanged domains of Gα_i and Gα_q subunits responsible for receptor coupling selectivity and swapped their receptor coupling partners. Our results established that the G proteins, regardless of the receptor type to which they coupled, conferred specificity to Kir3 activation. We next tested signaling through chimeras between the Gα_i and Gα_q subunits in which the N terminus, the helical, or the GTPase domains of the Gα subunits were exchanged. Our results revealed that the helical domain of Gα_i (residues 63-175) in the background of Gα_q could support Kir3 activation, whereas the reverse chimera could not. Moreover, the helical domain of the Gα_i subunit conferred "Gα_i- like" binding of the Kir3 C terminus to the Gα_q subunits that contained it. These results implicate the helical domain of Gα_i proteins as a critical determinant of Gγβ signaling specificity.