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

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.

Molecular Determinants for G Protein βγ Modulation of Ionotropic Glycine Receptors

“…Biochemical and functional data studies determined multiple binding and modulatory sites in voltage-gated calcium channels and GIRK channels, indicating a high degree of complexity (19,20,23,35,36). Despite this, the presence of basic residues inside the critical regions for binding and modulation appears to be a common feature.…”

“…However, for GIRK channels no basic residues have been found to be important for G␤␥ regulation, indicating vast types of molecular determinants in the heterodimer effects (35,36).…”

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

“…Two-electrode Voltage Clamp Recording and AnalysisWhole-cell currents were measured by conventional twomicroelectrode voltage clamp with a GeneClamp 500 amplifier (Axon Instruments, Union City, CA), as reported previously (24,33). A high potassium solution was used to superfuse oocytes (96 mM KCl, 1 mM NaCl, 1 mM MgCl 2 , 5 mM KOH/ HEPES (pH 7.4)).…”

“…cRNAs were transcribed in vitro using the "Message Machine" kit (Ambion, Austin, TX). Oocytes were isolated and microinjected as described previously (24,33). Expression of channel proteins in Xenopus oocytes was accomplished by injection of the desired amount of cRNA.…”

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

“…, and Gly 336 of GIRK1 (3)(4)(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).…”