Gβγ-activated Inwardly Rectifying K+(GIRK) Channel Activation Kinetics via Gαiand Gαo-coupled Receptors Are Determined by Gα-specific Interdomain Interactions That Affect GDP Release Rates

Qingli Zhang, Alec Dickson, Craig A. Doupnik
2004 Journal of Biological Chemistry  
G␤␥-activated inwardly rectifying K ؉ (GIRK) channels have distinct gating properties when activated by receptors coupled specifically to G␣ o versus G␣ i subunit isoforms, with G␣ o -coupled currents having ϳ3-fold faster agonist-evoked activation kinetics. To identify the molecular determinants in G␣ subunits mediating these kinetic differences, chimeras were constructed using pertussis toxin (PTX)-insensitive G␣ oA and G␣ i2 mutant subunits (G␣ oA(C351G) and G␣ i2(C352G) ) and examined in
more » ... and examined in PTX-treated Xenopus oocytes expressing muscarinic m2 receptors and Kir3.1/3.2a channels. These experiments revealed that the ␣-helical N-terminal region (amino acids 1-161) and the switch regions of G␣ i2 (amino acids 162-262) both partially contribute to slowing the GIRK activation time course when compared with the G␣ oA(C351G) -coupled response. When present together, they fully reproduce G␣ i2(C352G) -coupled GIRK kinetics. The G␣ i2 C-terminal region (amino acids 263-355) had no significant effect on GIRK kinetics. Complementary responses were observed with chimeras substituting the G␣ o switch regions into the G␣ i2(C352G) subunit, which partially accelerated the GIRK activation rate. The G␣ oA /G␣ i2 chimera results led us to examine an interaction between the ␣-helical domain and the Ras-like domain previously implicated in mediating a 4-fold slower in vitro basal GDP release rate in G␣ i1 compared with G␣ o . Mutations disrupting the interdomain contact in G␣ i2(C352G) at either the ␣D-␣E loop (R145A) or the switch III loop (L233Q/A236H/E240T/ M241T), significantly accelerated the GIRK activation kinetics consistent with the G␣ i2 interdomain interface regulating receptor-catalyzed GDP release rates in vivo. We propose that differences in G␣ i versus G␣ o -coupled GIRK activation kinetics are due to intrinsic differences in receptor-catalyzed GDP release that rate-limit G␤␥ production and is attributed to heterogeneity in G␣ i and G␣ o interdomain contacts. Cardiac and neuronal G␤␥-gated inwardly rectifying K ϩ channels (GIRKs) 1 are activated by G protein-coupled recep-
doi:10.1074/jbc.m403359200 pmid:15123672 fatcat:uskh6fubhbfldc3izshst5p46u