Positive and Negative Coupling of the Metabotropic Glutamate Receptors to a G Protein–activated K 1 Channel, GIRK, in Xenopus Oocytes

Metabotropic glutamate receptors (mGluRs) control intracellular signaling cascades through activation of G proteins. The inwardly rectifying K 1 channel, GIRK, is activated by the bg subunits of G i proteins and is widely expressed in the brain. We investigated whether an interaction between mGluRs and GIRK is possible, using Xenopus oocytes expressing mGluRs and a cardiac/brain subunit of GIRK, GIRK1, with or without another brain subunit, GIRK2. mGluRs known to inhibit adenylyl cyclase (types 2, 3, 4, 6, and 7) activated the GIRK channel. The strongest response was observed with mGluR2; it was inhibited by pertussis toxin (PTX). This is consistent with the activation of GIRK by G i /G o -coupled receptors. In contrast, mGluR1a and mGluR5 receptors known to activate phospholipase C, presumably via G proteins of the G q class, inhibited the channel’s activity. The inhibition was preceded by an initial weak activation, which was more prominent at higher levels of mGluR1a expression. The inhibition of GIRK activity by mGluR1a was suppressed by a broad-specificity protein kinase inhibitor, staurosporine, and by a specific protein kinase C (PKC) inhibitor, bis-indolylmaleimide, but not by PTX, Ca 2 1 chelation, or calphostin C. Thus, mGluR1a inhibits the GIRK channel primarily via a pathway involving activation of a PTX-insensitive G protein and, eventually, of a subtype of PKC, possibly PKCm . In contrast, the initial activation of GIRK1 caused by mGluR1a was suppressed by PTX but not by the protein kinase inhibitors. Thus, this activation probably results from a promiscuous coupling of mGluR1a to a G i /G o protein. The observed modulations may be involved in the mGluRs’ effects on neuronal excitability in the brain. Inhibition of GIRK by phospholipase C–activating mGluRs bears upon the problem of specificity of G protein (GIRK interaction) helping to explain why receptors coupled to G q are inefficient in activating GIRK. key words: G proteins • ion channel • modulation • phosphorylation • protein kinase C i n t r o d u c t i o n Glutamate is a major excitatory neurotransmitter in the brain. It acts on receptors of two major types: ( a ) ionotropic glutamate receptors, which are ligand-gated ion channels and include the AMPA/kainate and NMDA receptors, and ( b ) metabotropic glutamate receptors (mGluRs) 1 that activate G proteins (for reviews see Hollman and Heinemann, 1994; Nakanishi, 1994; Pin and Duvoisin, 1995). mGluRs participate in synaptic plasticity phenomena, playing a role both in long-term potentiation (LTP) and long-term depression (LTD), as well as in neurotoxicity and neuroprotection (for reviews see Bliss and Collingridge, 1993; Bear and Malenka, 1994; Nakanishi, 1994; Pin and Duvoisin, 1995). mGluRs form a family of eight genes cloned to-date, mGluR1– mGluR8 (Masu et al., 1991; Houamed et al., 1991; Tanabe et al., 1992; Abe et al., 1992; Tanabe et al., 1993; Nakajima et al., 1993; Okamato et al., 1994; Saugstad et al., 1994; Duvoisin et al., 1995) that encode proteins with structural similarity but low sequence homology to members of the seven-transmembrane domain receptors superfamily; thus, mGluRs may belong to a novel superfamily of G protein–coupled receptors (Nakanishi, 1994; Gomeza et al., 1996). mGluRs are subdivided into groups according to their signal transduction mechanisms, pharmacological properties, and levels of sequence homology between the genes. Group I includes mGluR1 (the longest splice variant is called mGluR1a) and mGluR5, group II includes mGluR2 and mGluR3, and group III includes the rest (Nakanishi, 1994; Pin and Duvoisin, 1995). Group I mGluRs activate phospholipase C (PLC) and are thus believed to couple to the G q class of G proteins. When expressed in Xenopus oocytes, these receptors activate a large endogenous Ca 2 1 -dependent chloride current, a fact that enabled molecular cloning by functional expression of the first mGluR, mGluR1 (Masu et al., 1991; Houamed et al., 1991). Group II and group III receptors inhibit adenylyl cyclase (AC) activity, suggesting that they couple to G proteins of the G i /G o class (Gilman, 1987). Address correspondence to Dr. Nathan Dascal, Department of Physiology and Pharmacology, Tel Aviv University, Sackler School of Medicine, Ramat Aviv 69978, Israel. Fax: 972-3-6409113; E-mail: dascaln@