G protein Gating of a Kir3.1-Prokaryotic Kir Channel Chimera Functionally Reconstituted in Planar Lipid Bilayers

Abstract

Functional reconstitution of the purified chimeric Kir3.1 channel constituting the cytosolic domain of mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 was achieved in lipid bilayers by addition of PIP2 from the intracellular side. Chimera had the typical traits of an inwardly rectifying potassium channel (Kir); PIP2- and Mg2+-dependence. Additionally, the chimera exhibited typical sidedness of a Kir3 channel: channel activity could be blocked by external (Trans) Tertiapin Q and by internal (Cis) poly Lysine and PIP2 antibody. Chimeric channels could also be stimulated by internal application of ethanol. Either of the G-protein subunits Gα-GDP or Gβγ alone or together and in either order of application inhibited PIP2-activated channel currents. In contrast, addition of GTPγS, following inhibition by both Gα-GDP and Gβγ, caused channel stimulation. Alternatively, addition of GTPγS following inhibition by Gα-GDP had no effect but further addition of Gβγ caused channel stimulation. Thus, gating of the chimeric channel required both activated forms of α, (α-GTPγS) and βγ subunits of G-proteins. This result is reminiscent of the requirement of both active Gαs and Gβγ subunits for activation of Gβγ-sensitive isoforms of adenylyl cyclase. Mammalian Kir3 channels expressed in native or heterologous systems do not exhibit a requirement for activated G-protein subunits and this interesting difference ought to addressed in future. A 3D reconstruction of the chimera by single particle electron microscopy indicated a structure consistent with the crystal structure. Our results confirm that the chimera is a reasonable structural and functional model for regulation of effectors by G protein subunits. Moreover our ability to reconstitute modulation of channel currents by G protein subunits in planar lipid bilayers offers a unique opportunity to dissect precise roles for each component of the signaling complex.