Time resolved kinetics of direct G beta 1 gamma 2 interactions with the carboxyl terminus of Kir3.4 inward rectifier K+ channel subunits.
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.
Doupnik
C A
CA
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Dessauer
C W
CW
Slepak
V Z
VZ
Gilman
A G
AG
Davidson
N
N
Lester
H A
HA
D000818:Animals; D000906:Antibodies; D004594:Electrophysiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating; D007700:Kinetics; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D015221:Potassium Channels / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011485:Protein Binding; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
D000906:Antibodies; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase