Time Resolved Kinetics of Direct Gβ1γ2 Interactions with the Carboxyl Terminus of Kir3.4 Inward Rectifier K+ Channel Subunits

Abstract The direct interaction of recombinant G β1γ2 proteins with the carboxyl terminal domain of a G protein-gated inward rectifier K channel subunit, Kir3.4 (GIRK4), was measured in real time using biosensor chip technology. The carboxyl terminus of Kir3.4 (a.a. 186–419) was expressed in bacteria as a glutathione- S -transferase (GST) fusion protein, GST-Kir3.4ct. GST-Kir3.4ct was immobilized to the surface of a biosensor chip by high affinity binding of the GST domain to a covalently attached anti-GST antibody. The association and dissociation rates of G β1γ2 dimers with the immobilized Kir3.4ct domain were temporally resolved as a change in refractive index detected by surface plasmon resonance. Specific binding of G β1γ dimers to Kir3.4ct was characterized by a dissociation rate ( k d ) of ∼0.003 s −1 . Association kinetics were dominated by a concentration-independent component (time constant ∼50 s) which complicates models of binding and may indicate conformational changes during binding of G β1γ2 to Kir3.4ct. The estimated equilibrium dissociation binding constant (K D ) was ∼800 nM. These studies demonstrate that G βγ dimers interact directly with the Kir3.4 channel subunit, and suggest interesting details in the interaction with the major cytosolic carboxyl terminal domain. The slow G β1γ2 dissociation rate measured on the sensor chip is similar in magnitude to a slow component of channel deactivation measured electrophysiologically in Xenopus oocytes expressing Kir3.1/3.4 multimeric channels and a G protein-coupled receptor. Biosensor-based experiments such as those described here will complement electrophysiological studies on the molecular basis of G protein interactions with Kir channels and other ion channel proteins. Copyright © 1996 Published by Elsevier Science Ltd