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Physiological and Pharmacological Characteristics of Quisqualic Acid-Induced K؉-Current Response in the Ganglion Cells of Aplysia

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Physiological and Pharmacological Characteristics of Quisqualic Acid-Induced K؉-Current Response in the Ganglion Cells of Aplysia Japanese Journal of Physiology, 51, 511–521, 2001 Physiological and Pharmacological Characteristics of Quisqualic Acid-Induced K1-Current Response in the Ganglion Cells of Aplysia Shingo KIMURA, Satoshi KAWASAKI, Koichiro TAKASHIMA, and Kazuhiko SASAKI Department of Physiology and Advanced Medical Science Research Center, School of Medicine, Iwate Medical University, Morioka, 020–8505 Japan Abstract: The extracellular application of ei- the application of either kainate or AMPA, ago- ther quisqualic acid (QA) or Phe-Met-Arg-Phe- nists for non-NMDA receptors, produced no type NH2 (FMRFamide) induces an outward current in of response in the same neurons. The QA-in- identified neurons of Aplysia ganglion under volt- duced K1-current response was not depressed age clamp. The time course of the QA-induced at all by an intracellular injection of either gua- response is significantly slower than that induced nosine 59-O-(2-thiodiphosphate) (GDP-bS) or by FMRFamide. The reversal potential for both guanosine 59-O-(3-thiotriphosphate) (GTP-gS), responses was 292 mV and was shifted 17 mV but the FMRFamide-induced response was in a positive direction for a twofold increase in markedly blocked by both GDP-bS and GTP-gS the extracellular K1 concentration. The QA-in- in the same cell. Furthermore, the QA- and FMR- duced response was markedly depressed in the Famide-induced K1-current responses were both presence of Ba21, a blocker of inward rectifier decreased markedly when the temperature was K1-channel, whereas TEA, a Ca21-activated K1- lowered to 15°C, from 23°C. These results sug- 1 1 channel (BKCa) blocker, or 4-AP, a transient K gested that the QA-induced K -current response (A)-channel blocker, had no effect on the re- is produced by an activation of a novel type of sponse. The QA-induced K1-current was signifi- QA-receptor and that this response is not pro- cantly suppressed by CNQX and GYKI52466, duced by an activation of the G protein. [Japa- antagonists of non-NMDA receptors. However, nese Journal of Physiology, 51, 511–521, 2001] Key words: quisqualic acid, glutamate receptor, molluscan neuron, potassium channel. Glutamate is a major neurotransmitter for excitatory tic potentials are produced by the activation of synaptic transmission in the central nervous system ionotropic glutamate receptors (iGluRs), which con- (CNS) of the vertebrate and the invertebrate. Besides sist of receptor–cation channel complexes as revealed producing the fast excitatory synaptic potentials in the by cDNA cloning. The iGluRs have been subdivided CNS, the glutamatergic synapse plays an important into the NMDA-, AMPA-, and kainate-receptor sub- role in the formation of synaptic plasticity, such as types by their pharmacological profiles [1]. Glutamate learning and memory. An excessive release of gluta- can also activate metabotropic glutamate receptors mate from the synaptic terminal is thought to be one (mGluRs), which are coupled to G proteins and a sub- cause for apoptosis, neuronal death. Glutamatergic sequent intracellular messenger system, such as phos- synaptic transmission is also important for the under- phatidyl inositol (PI) turnover. Usually an activation standing of various neuronal diseases. of the mGluRs produces slow synaptic potentials and In glutamatergic synapse, fast excitatory postsynap- primarily mediates the modulatory function of the cell Received on February 21, 2001; accepted on June 11, 2001 Correspondence should be addressed to: Shingo Kimura, Department of Physiology and Advanced Medical Science Research Center, School. of Medicine, Iwate Medical University, Uchimaru 19–1, Morioka, 020–8505 Japan. Tel: 181–19–651–5111 ext 3344, Fax: 181–19– 625–3447, E-mail: [email protected] Japanese Journal of Physiology Vol. 51, No. 4, 2001 511 S. KIMURA et al. response. At least eight subtypes of mGluRs in mam- (QA)-receptor that is quite different from those of malian CNS, termed as mGluR1–mGluR8, have been mammalian neurons. Furthermore, this response is identified by cDNA cloning [2]. produced by an increase in K1 permeability without In invertebrate neurons, the application of gluta- activation of the G protein, but it is possibly regulated mate induces an excitatory response similar to the ver- by a certain enzymatic process. tebrate neurons [3, 4]. Furthermore, inhibitory re- sponses are also induced by the stimulation of another METHODS type of glutamate receptor in molluscan neurons (see review by Cleland [5]). For instance, in the neurons of Preparation and perfusion media. The buc- Planorbarius corneus and Aplysia californica, an ap- cal ganglion or abdominal ganglion was isolated from plication of glutamate produces a hyperpolarizing re- Aplysia kurodai and fixed in a perfusing chamber. The sponse because of the opening of the Cl2-channel [6, connective tissues covering the caudal surface of the 7]. Furthermore, Bolshakov et al. [8], Katz and Levi- buccal ganglion or the dorsal surface of the abdominal tan [9], Kehoe [10], and Watanabe et al. [11] have re- ganglion were carefully removed under a dissecting ported other types of hyperpolarizing responses pro- microscope to expose the cells to the perfusing duced by an increase in K1 conductance after receptor medium. Neurons of the buccal ganglia responded to stimulation in the neurons of Planorbarius corneus, either QA or FMRFamide, but not to GABA, whereas Aplysia californica, and Euhadra peliomphala. neurons of the abdominal ganglia responded to GABA We previously hypothesized that most of the recep- and FMRFamide, but not to QA. We therefore used tor-induced K1-current responses are produced by an the buccal ganglia in most experiments, except the activation of the common G protein, Gi or Go, regard- temperature dependence of the GABA-induced re- less of their receptor types, based on experimental evi- sponse. The artificial Aplysia serum solution con- dence [12]. Actually, recent studies on invertebrates tained (in mM) NaCl, 587; MgCl2, 52; KCl, 11.5; 1 neurons have demonstrated that a slow K -current re- CaCl2, 14; and Tris, 50.1 (pH 7.4) [15]. The cells were sponse induced by glutamate is mediated by pertussis continuously perfused at a constant flow rate of toxin (PTX)-sensitive G protein activation [8]. Studies 5 ml/min and at a temperature of 2362°C. The effec- of some vertebrate neurons have also shown that glu- tive perfusing volume of the chamber was 0.2 ml. tamate evokes a hyperpolarizing response as a result Electrical measurements. Two microelec- of the activation of PTX-sensitive G protein. For ex- trodes filled with 1.8 M potassium citrate with a resis- ample, Holmes et al. have shown that the activation of tance of 5 to 7 MV were inserted into a single cell. Ei- 21 1 Ca -activated K channels (BKCa channel) through a ther the current- or voltage-clamp method was used to group II mGluR is linked to a PTX-sensitive G pro- evaluate the response of the neurons to the bath-ap- tein in basolateral amygdala neurons of rat [13]. plied agonists. The membrane potential was usually Knoflach and Kemp have also reported that a group II held at 265 mV, which was close to the resting poten- mGluR-induced hyperpolarizing response produced tial of the cells studied. To evaluate the change in by an opening of the inward rectifying K1 channel is membrane slope conductance, repetitive 1 s, 5 to coupled to G protein in rat cerebellum neuron, though 10 nA current pulses or 3 to 6 mV voltage pulses at the type of G protein involved in this response is not 0.2 Hz were applied across the cell membrane in the yet identified [14]. current- or voltage-clamp methods, respectively. On the other hand, Kehoe [10] and Watanabe et al. Drug application. Drugs used were L-glutamic [11] demonstrated in molluscan neurons that gluta- acid (Kanto Chemical); quisqualic acid (QA, Sigma); 1 mate produces an outward K -current response, Phe-Met-Arg-Phe-NH2 (FMRFamide, Peptide Insti- which does not appear to be mediated by G protein. tute); g-aminobutyric acid (GABA, Wako); N-methyl- However, pharmacological properties and the mecha- D-aspartic acid (NMDA, RBI); (6)-a-amino-3-hy- nism involved in ionic channel gating in this type of droxy-5-methylisoxazole-4-propionic acid (AMPA, glutamate receptor response have not been studied in RBI); 2-carboxy-4-(1-methyl-ethenyl)-3-pyrrolidin- detail. acetic acid (kainate, RBI); trans-(6)-1-amino- In this paper, we studied physiological and pharma- (1S,3R)-cyclopentanedicarboxylic acid (transACPD, cological characteristics of the slow hyperpolarizing RBI); trans-2,4-azetidine-dicarboxylic acid (tADA, response to stimulation of the glutamate receptor in RBI); L(1)-2-amino-4-phosphonobutyric acid (L- the identified neurons of the buccal ganglion of AP4, RBI); (6)-2-amino-5-phosphonopentanoic acid Aplysia. We report that this type of glutamate receptor (AP-5, RBI); 6-cyano-7-nitroquinoxaline-2,3-dione: belongs to a novel subtype of quisqualic acid HBC complex (CNQX, RBI); 1-(4-aminophenyl)-4- 512 Japanese Journal of Physiology Vol. 51, No. 4, 2001 Quisqualic Acid–Induced K1-Current methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine RESULTS hydrochloride (GYKI52466, RBI); (6)-a-amino- 4-carboxy-a-methyl-benzeneacetic acid ((6)MCPG, Characteristics of quisqualic acid–induced RBI); R(1)-2-amino-3-phosphonopropanoic acid 3- response phosphono-L-alanine (L(1)-AP-3, RBI); guano- In current-clamp experiments, the application of ei- sine 59-O-(2-thiodiphosphate) (GDP-bS, Boehringer ther quisqualic acid (QA) or Phe-Met-Arg-Phe-NH2 Mannheim); guanosine 59-O-(3-thiotriphosphate) (FMRFamide) induces a slow hyperpolarizing re- (GTP-gS, Boehringer Mannheim); ethyleneglycol-bis- sponse associated with a decrease in membrane resis- (b-aminoethylether)N,N,N9,N9-tetraacetic acid (EGTA, tance in both BR6 and BL6 cells [16, 17] of the buc- Dojin); tetraethylammonium (TEA, Kanto Chemical); cal ganglion of Aplysia (Fig.
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