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

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 ␤␥ subunits (G␤␥) upon stimulation of G protein-coupled receptors, such as M2 acetylcholine receptor. The binding of G␤␥ 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 G␤␥ 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 G␤␥, by ITC and NMR. The ITC analyses indicated that four G␤␥ molecules bind to a tetramer of the CP region of GIRK with a dissociation constant of 250 M. The NMR analyses revealed that the G␤␥ 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.G protein-activated inwardly rectifying potassium channel (GIRK) 3 is a member of the inwardly rectifying potassium channel (Kir) family, which regulates heart rate and neuronal excitability (1, 2). The Kir proteins function as tetramers, consisting of a transmembrane (TM) region and a cytoplasmic pore (CP) region. The helix bundle at the cytoplasmic side of the TM region is assumed to be a K ϩ -ion gate. The opening and closing in the gate (gating) of Kirs are regulated by a variety of cytoplasmic factors. The gating of GIRK is triggered by the binding of its CP region with the heterotrimeric G-protein ␤␥ subunits (G␤␥), which are released from the pertussis toxin-sensitive G protein ␣ subunit (G␣ i/o ), subsequent to the stimulation of a G protein-coupled receptor, such as M2 acetylcholine receptor.Extensive mutational analyses to identify the GIRK residues that are critical for the G␤␥-induced activation revealed several critical residues such as His 57 , Leu 262 , Leu 333, and Gly 336 of GIRK1 (3-5). However, when these residues were mapped on the recently reported crystal structures of Kirs, they did not form a cluster on the protein surface (6 -9). Therefore, no clear consensus has been obtained regarding the region of GIRK that is essential for G␤␥ binding and/or GIRK activation. One of the reasons might be a structural alteration introduced by the mutagenesis (10), which could change the gating property of the channel. Although the various crystal structures have provided little information about the conformational change involved in the gating of the channel, FRET analyses have clearly demonstrated the conformational rearrangements in the CP region of GIRK upon G␤␥ binding (11). However, the resolution of the structural information obtained by the FRET analyses is low, primarily due to the large size of the fluorescent probe proteins.To reveal the structural mechanism by which G␤␥ binding activates GIRK, we have investigated the direct interacti...

Structural Basis for Modulation of Gating Property of G Protein-gated Inwardly Rectifying Potassium Ion Channel (GIRK) by i/o-family G Protein α Subunit (Gαi/o)

“…The anchoring site is relevant to precoupling and priming for GIRK when the site accommodates G␣ i/o (GDP)␤␥ and to the binding of G␣ i/o (GTP) when G␤␥ shifts to the activation site, which was revealed by our NMR analyses as the border of the two neighboring subunits of the GIRK tetramer (12). In this (39) is shown in a surface representation.…”

“…Thus, the K d value for the binding of G␣ i3 and GIRK CP-L was estimated as 1 mM. Although this K d value is quite large as a value for a proteinprotein interaction, it would fall in the nanomolar to micromolar range on the cell membrane, considering the reduced dimensionality effects (12,47,48). The K d value on the order of 1 mM is 4 times of the K d value of 0.25 mM that we previously reported for the GIRK CP -G␤␥ interaction (12), which is consistent with the report that the affinity of the cytoplasmic region of GIRK1 for G␤␥ was 4 -5-fold stronger than that for G␣ i3 (15).…”

“…First, the binding model of G␤␥-GIRK was built by using HADDOCK with parameters listed in supplemental Table 2. The G␤␥ binding residues on GIRK CP-L identified in our previous study (12) and the residues on G␤␥ reported to be important for GIRK binding (54,55) were defined as the active residues in the software. Then the G␣-GIRK complex model shown in Fig.…”

“…GIRK (12,33). Briefly, G␣ i3 was purified to homogeneity by chromatography on a HIS-Select Nickel Affinity Gel (Sigma) column, His-tag cleavage by Pre-Scission TM Protease (GE Healthcare), and removal of the cleaved His tags and the PreScission TM Protease (GE Healthcare) by HIS-Select Nickel Affinity Gel (Sigma).…”

“…The TCS experiments were performed as described with minor modifications (12,36). In the TCS experiments observing G␣ i3 , a solution containing uniformly 2 H, 15 N-labeled G␣ i3 (0.3 mM) and unlabeled GIRK CP-L (0.25 mM as a tetramer) was prepared in buffer (10 mM HEPES-NaOH (pH 6.5), 50 mM KCl, 5 mM DTT, 1 mM DSS, 0.8 mM GTP␥S, 20% 1 H 2 O, 80% 2 H 2 O).…”