Identification of Critical Residues Controlling G Protein-gated Inwardly Rectifying K+ Channel Activity through Interactions with the βγ Subunits of G Proteins

Cheng He,Xixin Yan,Hailin Zhang,Tooraj Mirshahi,Taihao Jin,Aijun Huang,Diomedes E Logothetis

doi:10.1074/jbc.m104851200

Abstract

G protein-sensitive inwardly rectifying potassium (GIRK) channels are activated through direct interactions of their cytoplasmic N- and C-terminal domains with the beta gamma subunits of G proteins. By using a combination of biochemical and electrophysiological approaches, we identified minimal N- and C-terminal G beta gamma -binding domains responsible for stimulation of GIRK4 channel activity. Within these domains one N-terminal residue, His-64, and one C-terminal residue, Leu-268, proved critical for G beta gamma-mediated GIRK4 activity. Moreover, mutations at these GIRK4 sites reduced significantly binding of the channel domains to G beta gamma . The corresponding residues in GIRK1 also showed a critical involvement in G beta gamma sensitivity. In GIRK4/GIRK1 heteromers the GIRK4 His-64 and Leu-268 residues showed greater contributions to G beta zeta sensitivity than did the corresponding GIRK1 His-57 and Leu-262 residues. These results identify functionally important channel interaction sites with the beta gamma subunits of G proteins, critical for channel activity.

Highlights

Activation of atrial potassium (Kϩ) channels underlies the acetylcholine (ACh)1-induced reduction in heart rate during vagal activity [1]
G␤␥ binding of GIRK4 channel fragments was compared with that of the following: (a) the PH domain of the ␤-adrenergic receptor kinase (␤ARK) (e.g. Ref. 24); (b) the corresponding full N- and C-terminal fragments of the IRK1 channel, which is related to GIRK4 but is G␤␥-insensitive (e.g. Ref. 24); and (c) the GST protein alone, which was common to all purified fusion proteins
The binding of the full N-terminal segment G4-(41–92) was consistently weaker than that of the smaller N-terminal segment G4-(41–92). Overall these results suggest that G4(41–92) and G4-(253–348) are, respectively, minimal domains of the GIRK4 channel N and C termini capable of maximal binding to G␤␥

Summary

Introduction

Activation of atrial potassium (Kϩ) channels underlies the acetylcholine (ACh)1-induced reduction in heart rate during vagal activity [1]. This result suggested that distinct channel sites may be involved in basal versus agonist-induced channel activation through the G␤␥ subunits. This result agreed with previous work showing the corresponding GIRK region (e.g. G4(323–348)), and in particular a Leu residue (G4Leu339 or G1Leu333) was critical in G␤␥-mediated agonist-induced activation as well as for maximal binding to G␤␥ (Ref. 24; compare in Fig. 1B G␤␥ binding to GST-G4-(253–322) versus GST-G4-(253–348)).

Results

Conclusion