Functional Reconstitution of a GIRK1-Chimera and its Regulation by the βγ Subunits of G Proteins

The molecular details of ion channel regulation by G proteins remain unknown. A first step in this direction is to define the characteristics of interacting proteins of known structure in isolation and in complex form. The three-dimensional structure of a GIRK1-chimera determined by single particle electron microscopy at 25A is consistent with the crystal structure (Nishida et al., 2007, EMBO J 26:4005-15). We have functionally reconstituted this GIRK1-chimera into a 1:1 ratio of phosphatidylethanolamine to phosphatidylserine planar lipid bilayers. The GIRK1-chimera produces a conductance of approximately 23 pS that shows Mg2+-dependent inwardly rectifying K+ currents and an absolute requirement on the presence of phosphatidylinositol-4,5-bisphosphate for activation. These currents are blocked by PIP2 antibody and poly-lysine applied from the cis but not the trans side. Moreover, the channel shows a high affinity for diC8-PIP2 (EC50 ∼ 7.5 μM). GIRK1-chimera channel currents are blocked by Ba2+ and the GIRK peptide blocker tertiapin when applied from the trans but not the cis side. Interestingly, Gβγ applied from the cis side inhibits GIRK1-chimera currents and shifts phosphoinositide sensitivity by decreasing the apparent affinity to PIP2. This is in contrast to the Gβγ effects on full-length GIRK1∗ channels assayed in Xenopus oocytes or planar lipid bilayers.