RESUMEN
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.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Guanosina Difosfato/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/fisiología , Toxina del Pertussis/farmacología , Canales de Potasio/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Xenopus laevisRESUMEN
'Regulators of G protein Signalling' (RGSs) accelerate the activation and deactivation kinetics of G protein-gated inwardly rectifying K(+) (GIRK) channels. In an apparent paradox, RGSs do not reduce steady-state GIRK current amplitudes as expected from the accelerated rate of deactivation when reconstituted in Xenopus oocytes. We present evidence here that this kinetic anomaly is dependent on the degree of G protein-coupled receptor (GPCR) precoupling, which varies with different Galpha(i/o)-RGS complexes. The gating properties of GIRK channels (Kir3.1/Kir3.2a) activated by muscarinic m2 receptors at varying levels of G protein expression were examined with or without the co-expression of either RGS4 or RGS7 in Xenopus oocytes. Different levels of specific m2 receptor-Galpha coupling were established by uncoupling endogenous pertussis toxin (PTX)-sensitive Galpha(i/o) subunits with PTX, while expressing varying amounts of a single PTX-insensitive subunit (Galpha(i1(C351G)), Galpha(i2(C352G)), Galpha(i3(C351G)), Galpha(oA(C351G)), or Galpha(oB(C351G))). Co-expression of each of the PTX-insensitive Galpha(i/o) subunits rescued acetylcholine (ACh)-elicited GIRK currents (I(K,ACh)) in a concentration-dependent manner, with Galpha(o) isoforms being more effective than Galpha(i) isoforms. Receptor-independent 'basal' GIRK currents (I(K,basal)) were reduced with increasing expression of PTX-insensitive Galpha subunits and were accompanied by a parallel rise in I(K,ACh). These effects together are indicative of increased Gbetagamma scavenging by the expressed Galpha subunit and the subsequent formation of functionally coupled m2 receptor-G protein heterotrimers (Galpha((GDP))betagamma). Co-expression of RGS4 accelerated all the PTX-insensitive Galpha(i/o)-coupled GIRK currents to a similar extent, yet reduced I(K,ACh) amplitudes 60-90 % under conditions of low Galpha(i/o) coupling. Kinetic analysis indicated the RGS4-dependent reduction in steady-state GIRK current was fully explained by the accelerated deactivation rate. Thus kinetic inconsistencies associated with RGS4-accelerated GIRK currents occur at a critical threshold of G protein coupling. In contrast to RGS4, RGS7 selectively accelerated Galpha(o)-coupled GIRK currents. Co-expression of Gbeta5, in addition to enhancing the kinetic effects of RGS7, caused a significant reduction (70-85 %) in steady-state GIRK currents indicating RGS7-Gbeta5 complexes disrupt Galpha(o) coupling. Altogether these results provide further evidence for a GPCR-Galphabetagamma-GIRK signalling complex that is revealed by the modulatory affects of RGS proteins on GIRK channel gating. Our functional experiments demonstrate that the formation of this signalling complex is markedly dependent on the concentration and composition of G protein-RGS complexes.