Referenced in: none

Automatically associated channels: Kir2.4 , Kir3.1 , Kir3.4 , Slo1

Title: Time resolved kinetics of direct G beta 1 gamma 2 interactions with the carboxyl terminus of Kir3.4 inward rectifier K+ channel subunits.

Authors: C A Doupnik, C W Dessauer, V Z Slepak, A G Gilman, N Davidson, H A Lester

Journal, date & volume: Neuropharmacology, 1996 , 35, 923-31

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/8938723

Abstract
The direct interaction of recombinant G beta 1 gamma 2 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 beta 1 gamma 2 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 beta 1 gamma 2 dimers to Kir3.4ct was characterized by a dissociation rate (kd) of approximately 0.003 s-1. Association kinetics were dominated by a concentration-independent component (time constant approximately 50 s) which complicates models of binding and may indicate conformational changes during binding of G beta 1 gamma 2 to Kir3.4ct. The estimated equilibrium dissociation binding constant (Kd) was approximately 800 nM. These studies demonstrate that G beta gamma 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 beta 1 gamma 2 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.