Cloning and Expression of a Complementary DNA Encoding a Molluscan Octopamine Receptor That Couples to Chloride Channels in HEK293 Cells

Abstract

A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis. The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa 1 ) that we recently cloned from Lymnaea. After stable expression of the cDNA in HEK293 cells, we found that [ 3 H]rauwolscine binds with high affinity to the receptor (K D ‫؍‬ 6.2⅐10 ؊9 M). Octopamine appears to be the most potent naturally occurring agonist to displace the [ 3 H]rauwolscine binding (K i ‫؍‬ 3.0⅐10 ؊7 M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa 2 . The novel receptor shares little pharmacological resemblance with Lym oa 1 , indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa 2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl ؊ current in HEK293 cells stably expressing Lym oa 2 . Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation.

Spider Peripheral Mechanosensory Neurons Are Directly Innervated and Modulated by Octopaminergic Efferents

“…These include activation and inhibition of adenylate cyclase, leading to an increase or decrease in cAMP, and activation of an inositol phosphate pathway, leading to increase in intracellular Ca 2ϩ (for review, see Roeder, 1999). Some receptors also activate Cl Ϫ channels (Gerhardt et al, 1997b). Here, we investigated the most common of these pathways, activation of adenylate cyclase leading to increased cAMP concentration.…”

Octopamine promotes rhythmicity but not synchrony in a bilateral pair of bursting motor neurons in the feeding circuit of Aplysia

“…Cloning studies have identified three molluscan G protein-coupled OA receptors (Gerhardt et al, 1997a;Gerhardt et al, 1997b;Chang et al, 2000). These receptors are coupled to distinct second messenger systems and are activated over broadly ranging OA concentrations.…”

“…One OA receptor that is expressed in the Aplysia CNS (designated Ap oa 1 ) had an EC 50 of 30 nmol l -1 for production of cAMP (Chang et al, 2000). By contrast, the activation of second messenger systems by two Lymnaea OA receptors (Lym oa 1 and Lym oa 2 ) required micromolar or higher agonist concentrations (Gerhardt et al, 1997a;Gerhardt et al, 1997b). It is likely, therefore, that the network effects observed at high (mmol l -1 ) OA concentrations and the cellular effects observed with lower (mmol l -1 ) doses in this study both reflect the activation of multiple receptor types and second messenger cascades.…”

Are Caenorhabditis elegans receptors useful targets for drug discovery: Pharmacological comparison of tyramine receptors with high identity from C. elegans (TYRA-2) and Brugia malayi (Bm4)

“…Clearly, TA and OA signaling is complex and is mediated by at least 5 different G-protein coupled receptors expressed at different levels throughout the nervous system [1,6,9,35,36]. Based on studies in insects and C. elegans, TA receptors generally appear to couple to an inhibition of adenyl cyclase activity through Gα o and OA receptors to a stimulation of adenyl cyclase activity, presumably through Gα s , although few studies demonstrating direct G-protein coupling are available [1,22,27,37]. Most importantly, OA receptors have been useful targets for pesticide development and, presumably, TA and OA receptors, might serve a similar role in parasitic nematodes.…”

“…The G-protein coupling of 18 of the 34 receptors is known or strongly suggested through experimentation [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. An unrooted phylogenetic tree of annotated TA/OA receptors using maximal parsimony was compiled using PAUP (Figure 2).…”