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.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
