Shingo Kimura

Regulation of dopamine-induced response by recycling of the receptor associated with capacitance change through caveolae- and SNARE-dependent process

in DKO mice. On the other hand, the reduction of GluA4 in -7-KO granular layer reflected its loss at mossy fiber-granule cell synapses, whereas the reduction of GluA1 and GluA4 in -7-KO molecular layer was caused, at least partly, by their loss in Bergmann glia. These findings suggest -2 and -7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and -7 alone promotes glial expression.

Quisqualic acid produces a novel K+-current response in identified neurons of aplysia ganglion

Extracellular application of either quisqualic acid (QA) or Phe-Met-Arg-Phe-NH1 (FMRFamide) to the identified neurons of Aplysia ganglion elicits a slow K’-current response under voltage clamp. The QA-induced K’-current response was markedly depressed in the presence of CNQX, an antagonist for non-NMDA receptors. On the other hand, application of kainate and AMPA, agonists for non-NMDA receptors, did not induce any significant response in the same neurons. QA-induced Kf-current response was not depressed at all by intracellular injection of guanosine S--O-(2-thiodiphosphate) (GDP@), while the FMRFamide-induced response was completely blocked by GDP-OS in the same cell. However. both the QAand FMRFamide-induced K’-current responses decreased markedly when the temperature was lowered to 15 “C from 22 “C. These results indicate that the QA-induced Kt-current response does not conform in its pharmacological characteristics to any of the known glutamate-induced responses in vertebrate.

Regulatory roles of calmodulin-dependent protein kinase and phosphatase on the receptor-induced K+-current responses

009 EGUIJ,TORY ROLES OF CALMODULIN-DEPENDENT PROTEIN KINASE AND PHOSPHATASE ON THE RECEPTOR-INDUCED K+-CURRENT RESPONSES SATOSHI KAWASAKI’, SHINGO KIMURA’, REIKO FUJITA’, KOICHIRO TAKASHInlA’, MITSUHIKO MATSUMOT02, KAZUHIKO SASAKI’ ‘Dept. of Physiology, School of Medicine, Iwate Medical Univ., Morioka 020-8505, ‘Lab. 01’ Physics, School of Allied Medical Sciences, Hirosaki Univ., Hirosaki 036-8203 Application of dopamine, acetylcholine , or Phe-Met-Arg-Phe-NH, to identified neurons of Aplysia ganglia induces identical K’-current responses which are produced by activation of specific G-proteins of the Gi family. These K’-current responses were depressed by intracellular injection of either KN-9.3, an inhibitor for Ca”-calmodulin dependent protein kinase, W-7, a calmodulin inhibitor, or U-73 122, a phospholipase C inhibitor, irrespective of their receptor tj pes. In contrast, intracellular injection of cypermethrin, an inhibitor of calcineurin, significantly augmented the K’-current responses. Howe\ier, all the above inhibitors did not affect at all the K’-current response elicited by direct activation of G-protein via intracellular apphcation of GTPyS. These results suggest that both Ca”-calmodulin dependent protein kinase and calcineurin are physiologically activated by stimulation of these receptors, regulating the opening process of the receptor-operated Kkhannels, and that the acting site of these enzymes might be localized somewhere between the receptors and G-protein.

221 Inversion of an inward current induced by dopamine after the application of GTP-analog observed in the neurons of Aplysia

Kohji Sate’ , Kazuko Sakata2, Shoichi Shimada2, Masaya Tohyama2 We constructed a cell line which stably expresses glycine transporter 1 (GlyTl) proteins. The cell line showed significant 142C-giycine uptake and could keep steep glycine concentration gradient between intracellular and extracellular space (in>out). Using this cell line, we investigated glycine release mediated by this transporter. The 142C-Glycine release was enhanced by extracellular glycine and sarcosine, a selective inhibitor of the transporter, in a dose-dependent manner. In addition, the replacement of extracellular Na+ with Lis’ or extracellular Clwith acetatemarkedly increased the release. Furthermore, we investigated the effects of extracellular Ca2+ and K+. The removal of these ions also showed enhancement of the release. These results suggest that glycine transporter 1 protein, which might be involved in the NMDA receptor neurotransmission, can release glycine into the extracellular space in the vicinity of synpases, and that the release might be influenced by the extrace

251 Uncoupling between receptor and G-protein by protein kinase A & C in the receptor-induced K+-current responses

KAZUHIKO SASAKI’, SATOSHI KAWASAKI’, SHINGO KIMURA’, REIKO FUJITAl , KOICHIRO TAKASHIMA’, MITSUHIKO MATSUMOT02, MAKOTO SATO’ Application of either acetylcholine, histamine, dopamine, or Phe-Met-Arg-Phe-NH2 induces a G-protein mediated K+-current response in the ganglion cells of Aplysia under voltage clamp. Application of CAMP, an activator of protein kinase A (PKA), or phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), markedly depressed the transmitter-induced responses. Intracellular injection of the catalytic subunit of either PKA or PKC mimicked the effects of CAMP or PDBu. Furthermore, application of okadaic acid, an inhibitor of phosphatase 1 & 2A, facilitated the effects of both CAMP and PDBu. On the other hand, Kf-channel opening of the same cell induced by guanosinethiotriphosphate (GTPgammaS) was not depressed by either CAMP or PDBu. These results suggested that the acting sites of both PKA and PKC might be somewhere between the receptor and G-protein, and that the phosphorylation of these sites would impair the functional coupling between these proteins.

S2-1 G-proteins regulating ionic channel activities: Modulatory effects of various protein kinases on the receptor induced K+-current responses

A decade ago, we published in Nature a hypothesis that K’-channel opening produced by various receptor stimulation is triggered by activation of PTX-sensitive Gproteins regardless of the transmitter or hormone receptors. This time, we identified the neurons of Aplysia ganglion, each of which responded with K+-channel opening to DA, ACh, HA, or FMRF-amide. Intracellular injections of cAMP or catalytic subunit of protein ffinase A depressed all of the receptor-induced c-current responses. Extracellular application of phorbol dibutyrate or intracellular injection of the catalytic subunit of protein Kinase C also depressed all of the receptor-induced p-current responses. Intracellular injection of GTPgS induces irreversible opening of the K+-channel by directly activating PTX-sensitive G-proteins. This c-current response was not depressed by either kinase. Intracellular application of various protein phosphatase inhibitors augmented or depressed depending on the preceding protein phosphorylation by different protein kinases. These results suggested that the receptor-induced K+-current responses are regulated by both intrinsic protein kinase and phosphatase activities.