Requirement for an Activated G Protein α (Gα) Subunit for Gβγ Activation of a Purified Mammalian GIRk1 Channel Reconstituted in Planar Lipid Bilayers

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We previously reported functional reconstitution of a GIRK1-chimera into a planar lipid bilayer (Leal-Pinto et al., 2010, JBC 285:39790). This GIRK1-chimera (GIRK1: K41-W82, F181-L371; KirBac1.3: F45-A127) produced a conductance of ∼23 pS that showed K+ currents with Mg2+-dependent inward rectification, an absolute requirement on the presence of phosphatidylinositol-4,5-bisphosphate for activation with a relatively high apparent affinity for diC8-PIP2 (EC50 ∼ 7.5 μM). GIRK1-chimera currents could be blocked by external Ba2+. Interestingly, Gβγ stimulation of activity required activated Galpha (i.e. Galpha-GTPgammaS) subunits.To compare the behavior of the GIRK1 chimera to that of its mammalian counterpart (GIRK1delta∗_K41-L371, including the previously missing N83-M180), we purified hGIRK1delta∗ in Pichia pastoris, and functionally reconstituted it in lipid bilayers. hGIRK1delta∗ showed at least a 4-fold lower affinity to diC8-PIP2 and a smaller single-channel conductance (∼15 pS) than the GIRK1 chimera, consistent with the full-length channel (GIRK1∗ M1-T501) characteristics expressed in cell systems. Both channels displayed similar Mg2+-dependent inward rectification and block by external Ba2+. Interestingly, hGIRK1delta∗ displayed a similar requirement for Gβγ-stimulated activity on activated Galpha (i.e. Galpha-GTPgammaS) subunits, as did the GIRK1 chimera. These results are in contrast to the response of purified GIRK2 in a fluorescence liposome flux assay, which did not require activated Galpha for Gβγ stimulation of activity (Whorton and MacKinnon, 2013, Nature 498:190-7). Our results suggest that the GIRK1 and GIRK2 channel subunits may possess distinct requirements for activation by G protein subunits.