Interaction of G Protein β Subunit with Inward Rectifier K⁺Channel Kir3

Abstract

G protein ␤␥ subunits bind and activate G protein-coupled inward rectifier K ϩ (GIRK) channels. This protein-protein interaction is crucial for slow hyperpolarizations of cardiac myocytes and neurons. The crystal structure of G␤ shows a sevenbladed propeller with four ␤ strands in each blade. The G␤/G␣ interacting surface contains sites for activating GIRK channels. Furthermore, our recent investigation using chimeras between G␤ 1 and yeast ␤ (STE4) suggested that the outer strands of blades 1 and 2 of G␤1 could be an interaction area between G␤1 and GIRK. In this study, we made point mutations on suspected residues on these outer strands and investigated their ability to activate GIRK1/GIRK2 channels. Mutations at Thr-86, Thr-87, and Gly-131, all located on the loops between ␤-strands, substantially reduced GIRK channel activation, suggesting that these residues are G␤/GIRK interaction sites. These mutations did not affect the expression of G␤1 or its ability to stimulate PLC␤2. These residues are surface-accessible and located outside G␤/G␣ interaction sites. These results suggest that the residues on the outer surface of blades 1 and 2 are involved in the interaction of G␤␥ with GIRK channels. Our study suggests a mechanism by which different effectors use different blades to achieve divergence of signaling. We also observed that substitution of alanine for Trp-332 of G␤1 impaired the functional interaction of G␤1 with GIRK, in agreement with the data on native neuronal GIRK channels. Trp-332 plays a critical role in the interaction of G␤1 with G␣ as well as all effectors so far tested.

A Computational Model Predicts That Gβγ Acts at a Cleft Between Channel Subunits to Activate GIRK1 Channels

“…Previously published mutagenesis data supports the importance of both of these relative front and back regions. Studies indicate that Trp 99 (36), as well as residues Trp 334 , Thr 86 , Thr 87 , Gly 130 (45), and the nearby Thr 128 (46) all play a role in regulation of GIRK channels by Gβγ. Because the largest cluster model has a relatively low interface RMSD compared to the final model, it may be speculated to represent an alternate binding mode or represent an “encounter complex” resulting from favorable microcollisions prior to conformational descent down the final steep energy well (47, 48).…”

The Roles of Gβγ and Gα in Gating and Regulation of GIRK Channels

“…Because crystal structure captures a certain static conformation (pose) of the channel-Gβγ complex, it is plausible that additional poses of GIRK and Gβγ, with partly distinct sets of contact points, exist. Indeed, several mutagenesis-based studies indicate that additional amino acid residues in Gβ may participate in regulating GIRK basal or evoked activities (Albsoul-Younes et al, 2001;Mirshahi, Robillard, Zhang, Hebert, & Logothetis, 2002;Zhao et al, 2003). For a detailed discussion on structural and functional aspects of Gβγ interaction with the channel, refer Chapters 1 and 4.…”

ArabidopsisN-MYC DOWNREGULATED-LIKE1, a Positive Regulator of Auxin Transport in a G Protein–Mediated Pathway

“…While mammals have five Gb subunits and 12 Gg subunits, Arabidopsis has a single gene encoding Gb (AGB1) and at least two genes encoding Gg subunits (AGG1 and AGG2). Well-known Gbg effectors in animals are phosducin, potassium channels, phospholipases, adenyl cyclases, mitogen-activated protein kinases, and phosphoinositol-3-kinase (Crespo et al, 1994;Tsukada et al, 1994;Xu et al, 1995;Akgoz et al, 2002;Zhao et al, 2003;Kino et al, 2005;Rebois et al, 2006;Chen et al, 2008).…”