Activation and Deactivation Kinetics of α2A- and α2C-Adrenergic Receptor-activated G Protein-activated Inwardly Rectifying K+ Channel Currents

Abstract

Although G protein-coupled receptor-mediated signaling is one of the best studied biological events, little is known about the kinetics of these processes in intact cells. Experiments with neurons from ␣ 2A -adrenergic receptor knockout mice suggested that the ␣ 2A -receptor subtype inhibits neurotransmitter release with higher speed and at higher action potential frequencies than the ␣ 2C -adrenergic receptor. Here we investigated whether these functional differences between presynaptic ␣ 2 -adrenergic receptor subtypes are the result of distinct signal transduction kinetics of these two receptors and their coupling to G proteins. ␣ 2A -and ␣ 2C -receptors were stably expressed in HEK293 cells at moderate (ϳ2 pmol/mg) or high (17-24 pmol/mg) levels. Activation of G protein-activated inwardly rectifying K ؉ (GIRK) channels was similar in extent and kinetics for ␣ 2A -and ␣ 2C -receptors at both expression levels. However, the two receptors differed significantly in their deactivation kinetics after removal of the agonist norepinephrine. ␣ 2C -Receptor-activated GIRK currents returned much more slowly to base line than did ␣ 2A -stimulated currents. This observation correlated with a higher affinity of norepinephrine at the murine ␣ 2C -than at the ␣ 2A -receptor subtype and may explain why ␣ 2C -adrenergic receptors are especially suited to control sympathetic neurotransmission at low action potential frequencies in contrast to the ␣ 2A -receptor subtype. G protein-coupled receptors (GPCRs)1 transfer a large diversity of extracellular signals into the cell interior, including light, neurotransmitters, and hormones. Although GPCRs represent some of the best studied signaling molecules, relatively little information exists about the kinetics of signal transduction by these receptors (except for rhodopsin) in intact cells. However, more detailed knowledge about the kinetic properties of GPCR signal transduction would be of particular interest to determine the physiological significance of closely related receptor subtypes, which can be activated by the same endogenous agonist but differ in their biological function.Functional data on ␣ 2 -adrenergic receptor subtypes suggest that they differ in their signaling kinetics. Interestingly, several physiological differences were identified between presynaptic ␣ 2A -and ␣ 2C -receptor subtypes (1). In mouse atria, the ␣ 2A -subtype inhibited norepinephrine release at high stimulation frequencies whereas the ␣ 2C -receptor operated at lower levels of sympathetic nerve activity (1). Moreover, inhibition of norepinephrine release mediated by the ␣ 2A -subtype occurred much faster than inhibition by the ␣ 2C -receptor. These findings indicate that two presynaptic receptors in the inhibitory feedback loop of transmitter release may differentially regulate synaptic transmission. Several explanations may account for these functional differences. ␣ 2 -Adrenergic receptor subtypes have been shown to differ in their signal transduction, agonistdependent internalization and receptor...

Agonist unbinding from receptor dictates the nature of deactivation kinetics of G protein-gated K + channels

“…It is noticeable that the ligands we use in these studies are largely synthetic agonists developed for enhanced affinity to their cognate receptor. However, other investigators have noticed significantly different deactivation rates between the ␣ 2A and ␣ 2C receptors with physiological agonists and postulated that agonist unbinding may be limiting with the latter (47). In this regard our deactivation kinetics for noradrenaline at the ␣ 2A receptor, in the clonal isolate used here, are slower than those of others (33,47).…”

“…However, other investigators have noticed significantly different deactivation rates between the ␣ 2A and ␣ 2C receptors with physiological agonists and postulated that agonist unbinding may be limiting with the latter (47). In this regard our deactivation kinetics for noradrenaline at the ␣ 2A receptor, in the clonal isolate used here, are slower than those of others (33,47). Data (unpublished observations) indicate that it is possible to modulate the deactivation rate with RGS overexpression, and thus slow kinetics per se do not imply that agonist unbinding is rate-limiting.…”

Inhibition of insulin secretion via distinct signaling pathways in alpha2-adrenoceptor knockout mice

“…glucose-depolarized islet cells, suggesting that the receptors are not coupled to ion channels or are not expressed in b cells. However, both, a 2A -and a 2C -AR, when stably expressed in HEK293 cells, have been found to activate G protein-activated inwardly rectifying K þ channels although with different activation and deactivation kinetics (35). These results indicate that the a 2A -AR is the main receptor in b cells and mediates hyperpolarization, inhibition of adenylyl cyclase and inhibition of secretion.…”

Formation of a Ternary Complex among NHERF1, β-Arrestin, and Parathyroid Hormone Receptor

“…To generate a PTHR-CFP construct, enhanced CFP preceded by a 6-amino acid linker sequence was fused to the human HA-PTHR by PCR. Human M3 muscarinic acetylcholine receptor was purchased from UMR cDNA Resource Center, and murine ␣ 2A -adrenergic receptor has been described previously (29). To generate pcDNA3.1(ϩ)-sEYFP-NHERF1-HA, sEYFP with KpnI and XhoI restriction sites at 5Ј and 3Ј was amplified via PCR using pcDNA3.1-sEYFP vector as template.…”