[en] We studied gamma-aminobutyric acid (GABA)-mediated currents in short-term cultured dorsal unpaired median (DUM) neurons of cockroach Periplaneta americana using the whole cell patch-clamp technique in symmetrical chloride solutions. All DUM neurons voltage-clamped at -50 mV displayed inward currents (I(GABA)) when 10(-4) M of GABA was applied by pneumatic pressure-ejection pulses. The semi-logarithmic curve of I(GABA) amplitude versus the ejection time yielded a Hill coefficient of 4.0. I(GABA) was chloride (Cl-) because the reversal potential given by the current-voltage (I-V) curve varied according to the value predicted by the Nernst equation for Cl- dependence. In addition, I(GABA) was almost completely blocked by bath application of the chloride channel blockers picrotoxin (PTX) or 3,3-bis(trifluoromethyl)bicyclo-[2,2,1]heptane-2,2-diacarbonitrile (BIDN). The I-V curve for I(GABA) displayed a unexpected biphasic aspect and was best fitted by two linear regressions giving two slope conductances of 35.6 +/- 2.1 and 80.9 +/- 4.1 nS for potentials ranging from 0 to -30 and -30 to -70 mV, respectively. At -50 mV, the current amplitude was decreased by cadmium chloride (CdCl2, 10(-3) M) and calcium-free solution. The semi-logarithmic curve for CdCl2-resistant I(GABA) gave a Hill coefficient of 2.4. Hyperpolarizing voltage step from -50 to -80 mV was known to increase calcium influx through calcium-resting channels. According to this protocol, a significant increase of I(GABA) amplitude was observed. However, this effect was never obtained when the same protocol was applied on cell body pretreated with CdCl2. When the calmodulin blocker N-(6-aminohexyl)-5-chloro-1-naphtalene-sulfonamide or the calcium-calmodulin-dependent protein kinase blocker 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) was added in the pipette solution, I(GABA) amplitude was decreased. Pressure ejection application of the cis-4-aminocrotonic acid (CACA) on DUM neuron cell body held at -50 mV, evoked a Cl- inward current which was insensitive to CdCl2. The Hill plot yielded a Hill coefficient of 2.3, and the I-V curve was always linear in the negative potential range with a slope conductance of 32.4 +/- 1.1 nS. These results, similar to those obtained with GABA in the presence of CdCl2 and KN-62, indicated that CACA activated one subtype of GABA receptor. Our study demonstrated that at least two distinct subtypes of Cl--dependent GABA receptors were expressed in DUM neurons, one of which is regulated by an intracellular Ca2+-dependent mechanism via a calcium-dependent protein kinase. The consequences of the modulatory action of Ca2+ in GABA receptors function and their sensitivity to insecticide are discussed.
Aguayo L.G., Espinosa F., Kunos G., Satin L.S. (1998) Effects of intracellular calcium on GABAA receptors in mouse cortical neurons. Pflügers Arch 435:382-387.
Akaike N. (1990) GABA-gated Cl- currents and their regulation by intracellular free Ca2+. Chloride Channels and Carriers in Nerves, Muscle, and Glial Cells , edited by Alvarez-Leefmans FJ and Russel JM. New York: Plenum; 261-272.
Alix P., Grolleau F., Hue B. (2000) Modulation by intracellular calcium of a GABA receptor-mediated chloride current via a calcium/calmodulin-dependent protein kinase in insect neurosecretory cells. Eur J Neurosci 12:43.
Anthony N.M., Harrison J.B., Sattelle D.B. (1993) GABA receptor molecules of insects. Comp Mol Neurobiol 63:172-209.
Aydar E., Beadle D.J. (1999) The pharmacological profile of GABA receptors on cultured insect neurones. J Insect Physiol 45:213-219.
Bloomquist J.R. (1996) Ion channels as targets for insecticides. Annu Rev Entomol 41:163-190.
Bornhorst J.A., Falke J.J. (2000) Attractant regulation of the aspartate receptor-kinase complex: Limited cooperative interactions between receptors and effects of the receptor modification state. Biochemistry 39:9486-9493.
Browning M.D., Bureau M., Dudek E.M., Olsen R.W. (1990) Protein kinase C and cAMP-dependent protein kinase phosphorylate the β subunit of the purified γ-aminobutyric acid A receptor. Proc Natl Acad Sci USA 87:1315-1318.
Buckingham S.D., Hosie A.M., Roush R.L., Sattelle D.B. (1994) Actions of agonists and convulsant antagonists on a Drosophila melanogaster GABA receptor (Rdl) homo-oligomer expressed in Xenopus oocytes. Neurosci Lett 181:137-140.
Buckingham S.D., Hue B., Sattelle D.B. (1994) Actions of bicuculline on cell body and neuropilar membranes of identified insect neurons. J Exp Biol 186:235-244.
Chen L., Wang H., Vicini S., Olsen W. (2000) The γ-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics. Proc Natl Acad Sci USA 97:11557-11562.
Chen Q.X., Stelzer A., Kay A.R., Wong R.K. (1990) GABAA receptor function is regulated by phosphorylation in acutely dissociated guinea pig hippocampal neurons. J Physiol (Lond) 420:207-221.
Churn S.B., DeLorenzo R.J. (1998) Modulation of GABAergic receptor binding by activation of calcium- and calmodulin-dependent kinase II membrane phosphorylation. Brain Res 809:68-76.
David J.A., Pitman R.M. (1996) Muscarinic receptor activation modulates ligand-gated ion channels in an insect motoneuron via changes in intracellular calcium. Proc R Soc Lond B Biol Sci 263:469-474.
Di Angelotonio S., Nistri A. (2001) Calibration of agonist concentrations applied by pressure pulse or via rapid solution exchanger. Neurosci Res 110:155-161.
Dubreil V., Sinakevitch I.G., Hue B., Geffard M. (1994) Neuritic GABAergic synapses in insect neurosecretory cells. Neurosci Res 19:235-240.
Eldefrawi A.T., Eldefrawi M.E. (1987) Receptors for γ-aminobutyric acid and voltage-dependent chloride channels as targets for drugs and toxicants. FASEB J 1:262-271.
Feigenspan A., Bormann J. (1994) Modulation of GABAC receptors in rat retinal bipolar cells by protein kinase C. J Physiol (Lond) 481:325-330.
Ffrench-Constant R.H., Rocheleau T.A., Steichen J.C., Chalmer A.E. (1993) A point mutation in a Drosophila GABA receptor confers insecticide resistance. Nature 363:449-451.
Filippova N., Dudley R., Weiss D.S. (1999) Evidence for phosphorylation-dependent internalization of recombinant human pl GABAC receptors. J Physiol (Lond) 518:385-399.
Goodman C.S., Spitzer N.C. (1980) Embryonic development of neurotransmitter receptors in grasshoppers. Receptors for Neurotransmitters Hormones and Pheromones in Insects , edited by Sattelle DB, Hall, LM, and Hildebrand. Amsterdam: Elsevier/North-Holland; 195-208.
Grolleau F., Lapied B. (1996) Two distinct low-voltage-activated Ca2+ currents contribute to the pacemaker mechanism in cockroach dorsal unpaired median neurons. J Nearophysiol 76:963-976.
Grolleau F., Lapied B. (2000) Dorsal unpaired median neurons in the insect central nervous system: Toward a better understanding of the ionic mechanisms underlying spontaneous electrical activity. J Exp Biol 203:1633-1648.
Grolleau F., Sattelle D.B. (2000) Single channel analysis of the blocking actions of BIDN and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a Drosophila cell line. Br J Pharmacol 130:1833-1842.
Hainzl D., Cole L.M., Casida J.E. (1998) Mechanisms for selective toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl photoproduct. Chem Res Toxicol 11:1529-1535.
Hamill O.P., Marty A., Neher E., Sakmann B., Sigworth F.J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch Arch 391:85-100.
Han M.H., Yang X.L. (1999) Zn2+ differentially modulates kinetics of GABAC vs GABAA receptors in carp retinal bipolar cells. Neuroreport 10:2593-2597.
Harvey R.J., Schmitt B., Hermans Borgmeyer I., Gundelfinger E.D., Betz H., Darlison M.G. (1994) Sequence of a Drosophila ligand-gated ion-channel polypeptide with an unusual amino-terminal extracellular domain. J Neurochem 62:2480-2483.
Heine M., Wicher D. (1998) Ca2+ resting current and Ca2+-induced Ca2+ release in insect neurosecretory neurons. Neuroreport 9:3309-3314.
Henderson J.E., Soderlund D.M., Knipple D.C. (1993) Characterization of a putative γ-aminobutyric acid (GABA) receptor β subunit gene from Drosophila melanogaster. Biochem Biophys Res Commun 193:474-482.
Hosie A.M., Sattelle D.B. (1996) Allosteric modulation of an expressed homooligomeric GABA-gated chloride channel of Drosophila melanogaster. Br J Pharmacol 117:1229-1237.
Huang R.Q., Dillon C.H. (1998) Maintenance of recombinant type A γ-aminobutyric acid receptor function: Role of protein tyrosine phosphorylation and calcineurin. J Pharmacol Exp Ther 286:243-255.
Hue B. (1991) Functional assay for GABA receptor subtypes of a cockroach giant interneuron. Arch Insect Biochem Physiol 18:147-157.
Hue B. (1998) A picrotoxin-resistant GABA-gated chloride channel receptor subtype in the cockroach central nervous system. Arch Insect Biochem Physiol 37:231-238.
Ikeda T., Zhao X., Nagata K., Kono Y., Shono T., Yeh J.Z., Narahashi T. (2001) Fipronil modulation of γ-aminobutyric acidA receptors in rat dorsal root ganglion neurons. J Pharmacol Exp Ther 296:914-921.
Inoue M., Oomura Y., Yakushiji T., Akaike N. (1986) Intracellular calcium ions decrease the affinity of the GABA receptor. Nature 324:156-158.
Johnston G.A. (1996) GABAc receptors: Relatively simple transmitter-gated ion channels?. Trends Pharmacol Sci 17:319-323.
Kapur J., Haas K.F., MacDonald R.L. (1999) Physiological properties of GABAA receptors from acutely dissociated rat dentate granule cells. J Neurophysiol 81:2464-2471.
Krishek B.J., Xie X., Blackstone C., Huganir R.L., Moss S.J., Smart T.G. (1994) Regulation of GABAA receptor function by protein kinase C phosphorylation. Neuron 12:1081-1095.
Lapied B., Le Corronc H., Hue B. (1989) Ionic species involved in the electrical activity of single adult aminergic neurons isolated from the sixth abdominal ganglion of the cockroach Periplaneta americana. J Exp Biol 144:535-549.
Lapied B., Malecot C.O., Pelhate M. (1990) Sensitive nicotinic and mixed nicotinic-muscarinic receptors in insect neurosecretory cells. Brain Res 533:132-136.
Le Corronc H., Hue B. (1999) A native picrotoxin-resistant GABA-gated chloride channel receptor subtype in cockroach neurons. Pestic Sci 55:1007-1011.
Lees G., Beadle D.J., Neumann R., Benson J.A. (1987) Responses to GABA by isolated insect neuronal somata: Pharmacology and modulation by a benzodiazepine and a barbiturate. Brain Res 401:267-278.
Liu F., Wan Q., Pristupa Z.B., Yu X.M., Wang Y.T., Niznik H.B. (2000) Direct protein-protein coupling enables cross-talk between dopamine D5 and γ-aminobutyric acid A receptors. Nature 403:274-280.
Llano I., Leresche N., Marty A. (1991) Calcium entry increases the sensitivity of cerebellar Purkinje cells to applied GABA and decreases inhibitory synaptic currents. Neuron 6:565-574.
Martina M., Kilic G., Cherubini E. (1994) The effect of intracellular Ca2+ on GABA-activated currents in cerebellar granule cells in culture. J Membr Biol 142:209-216.
McCaman R.E., McKenna D.G., Ono J.K. (1977) A pressure system for intracellular and extracellular ejections of picoliter volumes. Brain Res 136:141-147.
McDonald B.J., Moss S.J. (1997) Conserved phosphorylation of the intracellular domains of GABAA receptor β2 and β3 subunits by cAMP-dependent protein kinase, cGMP-dependent protein kinase protein kinase C and Ca2+/calmodulin type II-dependent protein kinase. Neuropharmacology 36:1377-1385.
Millar N.S., Buckingham S.D., Sattelle D.B. (1994) Stable expression of a functional homo-oligomeric Drosophila GABA receptor in a Drosophila cell line. Proc R Soc Lond B Biol Sci 258:307-314.
Moran O., Dascal N. (1989) Protein kinase C modulates neurotransmitter responses in Xenopus oocytes injected with rat brain RNA. Brain Res Mol Brain Res 5:193-202.
Mouginot D., Feltz P., Schlichter R. (1991) Modulation of GABA-gated chloride currents by intracellular Ca2+ in cultured porcine melanotrophs. J Physiol (Lond) 437:109-132.
Moss S.J., Smart T.G., Blackstone C.D., Huganir R.L. (1992) Functional modulation of GABAA receptors by cAMP-dependent protein phosphorylation. Science 257:661-665.
Raymond L.A., Blackstone C.D., Huganir R.L. (1993) Phosphorylation of amino acid neurotransmitter receptors in synaptic plasticity. Trends Neurosci 16:147-153.
Raymond V., Sattelle D.B., Lapied B. (2000) Co-existence in DUM neurones of two GluCl channels that differ in their picrotoxin sensitivity. Neuroreport 11:2695-2701.
Rubin G.M., Yandell M.D., Wortman J.R., Gabor Miklos G.L., Nelson C.R., Hariharan I.K., Fortini M.E., Li P.W., Apweiler R., Fleischmann W., Cherry J.M., Henikoff S., Skupski M.P., Misra S., Ashburner M., Birney E., Boguski M.S., Brody T., Brokstein P., Celniker S.E., Chervitz S.A., Coates D., Cravchik A., Gabrielian A., Galle R.F., Gelbart W.M., George R.A., Goldstein L.S., Gong F., Guan P., Harris N.L., Hay B.A., Hoskins R.A., Li J., Li Z., Hynes R.O., Jones S.J., Kuehl P.M., Lemaitre B., Littleton J.T., Morrison D.K., Mungall C., O'Farrell P.H., Pickeral O.K., Shue C., Vosshall L.B., Zhang J., Zhao Q., Zheng X.H., Zhong F., Zhong W., Gibbs R., Venter J.C., Adams M.D., Lewis S. (2000) Comparative genomics of the eukaryotes. Science 287:2204-2215.
Sattelle D.B., Harrison J.B., Chen H.H., Bai D., Takeda M. (2000) Immunocytochemical localization of putative γ-aminobutyric acid receptor subunits in the head ganglia of Periplaneta americana using an anti-RDL C-terminal antibody. Neurosci Lett 289:197-200.
Sattelle D.B., Lummis S.C., Wong J.F., Rauh J.J. (1991) Pharmacology of insect GABA receptors. Neurochem Res 16:363-374.
Schofield P.R., Darlison M.G., Fujita N., Burt D.R., Stephenson F.A., Rodriguez H., Rhee L.M., Ramachandran J., Reale V., Glencorse T.A., Seeburg P.H., Barnard E.A. (1987) Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature 328:221-227.
Smart T.G. (1997) Regulation of excitatory and inhibitory neurotransmitter-gated ion channels by protein phosphorylation. Curr Opin Neurobiol 7:358-367.
Swope S.L., Moss S.I., Raymond L.A., Huganir R.L. (1999) Regulation of ligand-gated ion channels by protein phosphorylation. Adv Second Messenger Phosphoprotein Res 33:49-78.
Wafford K.A., Sattelle D.B. (1986) Effects of amino acid neurotransmitter candidates on an identified insect motoneuron. Neurosci Lett 63:135-140.
Wang R.A., Cheng G., Kolal M., Randic M. (1995) α-subunit of calcium/calmodulin-dependent protein kinase II enhances γ-aminobutyric acid and inhibitory synaptic responses of rat neurons in vitro. J Neurophysiol 73:2099-2106.
Washio H. (1994) Effects of putative neurotransmitters on dorsal unpaired median neurons of cockroach (Periplaneta americana) thoracic ganglia. J Insect Physiol 40:841-847.
Watson G.B., Salgado V.L. (2001) Maintenance of GABA receptor function of small-diameter cockroach neurons by adenine nucleotides. Insect Biochem Mol Biol 31:207-212.
Wicher D., Walther C., Penzlin H. (1994) Neurohormone D induces ionic current changes in cockroach central neurons. J Comp Neurol 174:507-515.
Yonekura S., Chern Y., Donahue K.A., Feldman L., Vanasse G.J., Sytkowski A.J. (1991) Erythropoietin receptor induced by dimethyl sulfoxide exhibit positive cooperativity associated with an amplified biologic response. Proc Natl Acad Sci USA 88:2535-2539.
Zhang H.-G., Ffrench-Constant R.H., Jackson M.B. (1994) A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms. J Physiol (Lond) 479:65-75.