Gbetagamma-activated inwardly rectifying K(+) (GIRK) channel activation kinetics via Galphai and Galphao-coupled receptors are determined by Galpha-specific interdomain interactions that affect GDP release rates.
Gbetagamma-activated inwardly rectifying K(+) (GIRK) channels have distinct gating properties when activated by receptors coupled specifically to Galpha(o) versus Galpha(i) subunit isoforms, with Galpha(o)-coupled currents having approximately 3-fold faster agonist-evoked activation kinetics. To identify the molecular determinants in Galpha subunits mediating these kinetic differences, chimeras were constructed using pertussis toxin (PTX)-insensitive Galpha(oA) and Galpha(i2) mutant subunits (Galpha(oA(C351G)) and Galpha(i2(C352G))) and examined in PTX-treated Xenopus oocytes expressing muscarinic m2 receptors and Kir3.1/3.2a channels. These experiments revealed that the alpha-helical N-terminal region (amino acids 1-161) and the switch regions of Galpha(i2) (amino acids 162-262) both partially contribute to slowing the GIRK activation time course when compared with the Galpha(oA(C351G))-coupled response. When present together, they fully reproduce Galpha(i2(C352G))-coupled GIRK kinetics. The Galpha(i2) C-terminal region (amino acids 263-355) had no significant effect on GIRK kinetics. Complementary responses were observed with chimeras substituting the Galpha(o) switch regions into the Galpha(i2(C352G)) subunit, which partially accelerated the GIRK activation rate. The Galpha(oA)/Galpha(i2) chimera results led us to examine an interaction between the alpha-helical domain and the Ras-like domain previously implicated in mediating a 4-fold slower in vitro basal GDP release rate in Galpha(i1) compared with Galpha(o). Mutations disrupting the interdomain contact in Galpha(i2(C352G)) at either the alphaD-alphaE loop (R145A) or the switch III loop (L233Q/A236H/E240T/M241T), significantly accelerated the GIRK activation kinetics consistent with the Galpha(i2) interdomain interface regulating receptor-catalyzed GDP release rates in vivo. We propose that differences in Galpha(i) versus Galpha(o)-coupled GIRK activation kinetics are due to intrinsic differences in receptor-catalyzed GDP release that rate-limit Gbetagamma production and is attributed to heterogeneity in Galpha(i) and Galpha(o) interdomain contacts.
Zhang
Qingli
Q
Department of Physiology & Biophysics, University of South Florida College of Medicine, Tampa, Florida 33612, USA.
Dickson
Alec
A
Doupnik
Craig A
CA
D000595:Amino Acid Sequence; D000818:Animals; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D037342:Pertussis Toxin / Q000494:pharmacology; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014982:Xenopus laevis
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044387:GTP-Binding Protein beta Subunits; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D020033:Protein Isoforms; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go