NMR Analyses of the Gβγ Binding and Conformational Rearrangements of the Cytoplasmic Pore of G Protein-activated Inwardly Rectifying Potassium Channel 1 (GIRK1)

Mariko Yokogawa,Masanori Osawa,Koh Takeuchi,Yoko Mase,Ichio Shimada

doi:10.1074/jbc.m110.160754

Abstract

G protein-activated inwardly rectifying potassium channel (GIRK) plays crucial roles in regulating heart rate and neuronal excitability in eukaryotic cells. GIRK is activated by the direct binding of heterotrimeric G protein βγ subunits (Gβγ) upon stimulation of G protein-coupled receptors, such as M2 acetylcholine receptor. The binding of Gβγ to the cytoplasmic pore (CP) region of GIRK causes structural rearrangements, which are assumed to open the transmembrane ion gate. However, the crucial residues involved in the Gβγ binding and the structural mechanism of GIRK gating have not been fully elucidated. Here, we have characterized the interaction between the CP region of GIRK and Gβγ, by ITC and NMR. The ITC analyses indicated that four Gβγ molecules bind to a tetramer of the CP region of GIRK with a dissociation constant of 250 μM. The NMR analyses revealed that the Gβγ binding site spans two neighboring subunits of the GIRK tetramer, which causes conformational rearrangements between subunits. A possible binding mode and mechanism of GIRK gating are proposed.

Highlights

Closing in the gate of Kirs are regulated by a variety of cytoplasmic factors
The validity of this construct was confirmed by its crystal structures [7, 8], which were essentially identical to the structures of the cytoplasmic pore (CP) region in the full-length Kir channels, such as KirBac1.1 [14] and a chimeric channel of KirBac1.3 and GIRK1 [6]
Because the 0.25 eq of GIRKCP tetramer corresponds to the concentration of the GIRKCP monomer, the isothermal titration calorimetry (ITC) result suggests that four G␤␥ molecules bind to one GIRKCP tetramer, where the Kd value for the binding of GIRKCP monomer to G␤␥ is 250 ␮M

Summary

EXPERIMENTAL PROCEDURES

Expression and Purification of the Cytoplasmic Regions of Mouse GIRK1—The N- and C-terminal cytoplasmic regions of mouse GIRK1 (residues 41– 63 and 190 –371) were fused into a single polypeptide, which is hereafter referred to as JANUARY 21, 2011 VOLUME 286 NUMBER 3. The uniformly 2H,15N-labeled GIRKCP samples for NMR analyses were prepared by growing the E. coli host in M9 minimal medium, containing 15NH4Cl, [U-2H]glucose, and [2H/15N]Celtone௡ base powder in 99% 2H2O. The uniformly 2H,15N-labeled GIRKCP for the transferred cross-saturation (TCS) experiments was prepared without the denaturing and refolding procedure, to leave the amide hydrogen atoms as 2H in the core of the protein. The supernatant was purified using HIS-Select௡ Nickel Affinity Gel (Sigma), followed by further purification using His-Trap HP (GE Healthcare). A solution containing the uniformly 2H,15N-labeled GIRKCP (300 ␮M as a tetramer) and the nonlabeled G␤␥ (200 ␮M) was prepared in buffer (10 mM HEPESNaOH (pH 7.5), 50 mM KCl, 2 mM DTT, 1 mM sodium 2,2dimethyl-2-silapentane-5-sulfonate, 20% 1H2O, 80% 2H2O). The selective saturation for the aliphatic protons of G␤␥ was performed with a 15-ms IBURP2 pulse centered at 1.0 ppm

The saturation duration and the relaxation delay were set at

RESULTS

Viewed from the inside of the pore

DISCUSSION