CLONING AND EXPRESSION OF A COMPLEMENTARY-DNA ENCODING A MOLLUSCAN OCTOPAMINE RECEPTOR THAT COUPLES TO CHLORIDE CHANNELS IN HEK293 CELLS

A cDNA encoding a G-protein coupled receptor was cloned from the centr al nervous system of the pond snail Lymnaea stagnalis. The predicted a mino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an oc topamine receptor (Lym oa(1)) that we recently cloned from Lymnaea. Af ter stable expression of the cDNA in HEK293 cells, we found that [H-3] rauwolscine binds with high affinity to the receptor (K-D = 6.2 . 10(- 4) M). Octopamine appears to be the most potent naturally occurring ag onist to displace the [H-3]rauwolscine binding (K-i = 3.0 . 10(-7) m). Therefore, the receptor is considered to be an octopamine receptor an d is consequently designated Lym oa(2). The novel receptor shares litt le pharmacological resemblance with Lym oa(1), indicating that the two receptors represent different octopamine receptor subfamilies. Octopa minergic stimulation of Lym oa(2) does not induce changes in intracell ular concentrations of cAMP or inositol phosphates. However, electroph ysiological 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 n ot require the activation of either protein kinase A or C, it can be b locked by inhibition of protein phosphorylation.