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

Abstract

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 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 receptors (GPCRs) selectively coupled to pertussis toxin (PTX)-sensitive G␣ i/o ␤␥ proteins (1, 2). The time course for GIRK channel activation elicited by application of receptor agonist can be influenced by the multiple intervening steps of the G protein cycle that begin with agonist binding to the GPCR, and end with G␤␥ binding to the GIRK channel subunits that promote gating transitions to the open state. In addition to the G protein activation steps, signal termination with GTP hydrolysis by the G␣ subunit and G␤␥ reassociation also impacts the kinetics and amplitude of agonist-activated GIRK currents. The ternary complex consisting of agonist, GPCR, and G protein influences the time course of agonist-elicited GIRK channel currents (3), supporting the notion that isoform composition of different GPCR-G␣ i/o ␤␥ protein-RGS protein-GIRK channel signaling complexes have different kinetic properties that affect their functional output (4). We recently repor...

RGS4 Regulates Parasympathetic Signaling and Heart Rate Control in the Sinoatrial Node

“…30 Moreover, RGS4 is known to modulate GIRK channel function in heterologous expression systems. 17,31,32 Consistent with an increased level of G␣ i/o signaling, CCh-treated SAN myocytes from RGS4-deficient mice showed increased I KACh as a result of reduced desensitization and altered GIRK gating kinetics. However, because RGS4 functions at the receptor level to inhibit all G␣ i/o -mediated signaling, it is possible that other pathways regulated by parasympathetic stimuli, including adenylyl cyclase activity, phosphodiesterase activity, intracellular cyclic nucleotide levels, protein kinase A activity, HCN, and L-type calcium channels, 2-4 may also be altered in RGS4-null hearts.…”

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

“…In whole-cell recordings in Xenopus oocytes, τ act of I evoked (elicited by acetylcholine (ACh) acting on muscarinic m2 receptors, m2R) was accelerated by coexpression of Gα i3 , whereas Gα i1 was less effective (Ivanina et al, 2004). Since Gα i1 and Gα i3 display similar rates of GDP dissociation (Linder, Ewald, Miller, & Gilman, 1990;Zhang et al, 2004), we hypothesized that the accelerating effect of Gα i3 is related to its GDP-bound form, due to an enhanced formation of GIRK-Gαβγ macromolecular signaling complex (Ivanina et al, 2004). However, later, using fluorescently labeled Gα constructs showing different kinetic properties in coupling to GIRK1/2, we were able to separate between the Gβγ-sequestering function of Gα i3 (as Gα GDP ) and its function as accelerator of GIRK activation.…”

“…Ligand binding is diffusionlimited and also may change in the course of interaction with the GPCR-G protein complex (Ambrosio & Lohse, 2012). Therefore, ligand may become rate-limiting at low doses; activation kinetics reach maximal speed at saturating doses of agonist (Bunemann, Bucheler, Philipp, Lohse, & Hein, 2001;Marcaggi, Mutoh, Dimitrov, Beato, & Kn€ opfel, 2009;Zhang, Dickson, & Doupnik, 2004).…”

RGS proteins: identifying new GAPs in the understanding of blood pressure regulation and cardiovascular function

“…Several papers from Doupnik and co-workers [67][68][69][70][71] have demonstrated a functional link between RGS4 and the regulation of muscarinic M 2 receptormediated activation of GIRK (G-protein-coupled inward rectifying potassium) channels in atrial myocytes ( Figure 2). GIRK channel activation via G αi class proteins coupled to muscarinic M 2 receptors is a key pathway mediating the hyperpolarizing effects of parasympathetic signalling on atrial myocytes [72][73][74].…”