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 Gβ1γ2 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 Gβ1γ2 dimers with the immobilized Kir3.4ct domain were temporally resolved as a change in refractive index detected by surface plasmon resonance. Specific binding of Gβ1γ dimers to Kir3.4ct was characterized by a dissociation rate (kd) 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 Gβ1γ2 to Kir3.4ct. The estimated equilibrium dissociation binding constant (KD) was ∼800 nM. These studies demonstrate that Gβγ 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 Gβ1γ2 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