The mechanism of action of notexin in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture by using whole-cell patch clamp recording. Exposure of the culture to notexin dose-dependently enhanced the frequency of spontaneous synaptic currents (SSCs). Either buffering of intracellular Ca2+ rise by BAPTA or replacing culture medium with Ca2+-free Ringer¡¦s solution effectively hampered notexin effect, suggesting Ca2+ influx is requisite for this facilitation. Pretreatment of the cultures with a TRP channel inhibitor SKF96365, instead of voltage-dependent L-type Ca2+ channel blockers nifedipine or verapamil, significantly abolished the SSC facilitating effect of notexin. Furthermore, knockdown the expression of TRPC channels by TRPC-specific morpholino abruptly abolished notexin effect, suggesting that TRPC channel is the major entranceway of extracellular Ca2+. The notexin-enhanced SSC frequency was also obviously reduced under intracellular Ca2+ store depletion by pretreatment of the cultures with pharmacological Ca2+-ATPase inhibitor thapsigargin or CPA. Bath application of membrane-permeable inositol 1,4,5-triphosphate (IP3) inhibitor, XeC, effectively occluded the facilitation of SSC frequency elicited by notexin. Furthermore, notexin-induced SSC frequency facilitation was blocked in the presence of phospholipase C (PLC) inhibitor, U73122. Taken collectively, these results suggest that notexin elicits a Ca2+ release from the IP3-sensitive intracellular Ca2+ store which resulted in further store depletion-operated Ca2+ influx through membrane TRPC channels of the presynaptic nerve terminal. This is done via PLC signaling cascade, leading to an enhancement of spontaneous transmitter release. Notexin-induced synaptic facilitation is potentially reduced while structural modification with phenylglyoxal. In addition, bath application of PLA2 inhibitor either aristolochic acid or glycyhirrzin effectively abolished notexin effect, suggesting the PLA2 activity is involved in notexin-induced SSC frequency facilitation. Previously it has been suggested local accumulation of PLA2-induced lipid metabolites myristoyl lysophosphatidylcholine (mLPC) and oleic acid (OA) promotes the fusion of the hemifused synaptic vesicles with plasma membrane, hence facilitating the neurotransmitter release in cultured cerebella granular neurons and thus resulted in bulges formation along the neurite. Our real-time morphometric analysis and synaptic activity assays showed that bulges formation along the neurite appeared significantly earlier and was induced at lower notexin concentrations than synaptic activity facilitation. Bath application of either mLPC, OA alone or their mixtures failed to mimic the notexin-induced facilitation in spontaneous transmitter release. Attenuation of PLA2 activity by chemical modification (notexin-80) resulted in correlated decrease of notexin-induced synaptic facilitation but not the degenerative morphological sign. Moreover, PLP-notexin, a site-specific modification of notexin with full intact PLA2 activity, shows significant loss the ability of notexin-induced neurite degeneration. Overall, results from our studies suggest the morphologic changes and synaptic facilitating effect induced by notexin are resulted from different cellular mechanisms.

The transient receptor potential (TRP) channel superfamily is a non-selective Ca2+-permeable cation channels involved in sensory physiology. Here we show that 2-aminoethoxydiphenyl borate (2-APB), a compound commonly used as TRP channel inhibitor, dose-dependently induce a significant facilitation on the frequency of spontaneous neurotransmitter release at developing Xenopus neuromuscular junction through, surprisingly, TRP channel activation. Bath application of universal TRP channel inhibitors either SKF96365, flufenamic acid or RuR cease the 2-APB-induced synaptic facilitation. Exclusion of Ca2+ from culture medium or bath application of the pharmacological Ca2+ channel inhibitor cadmium, membrane-permeable Ca2+ chelator BAPTA-AM, effectively hampered the facilitation of neurotransmitter release induced by 2-APB, suggesting Ca2+ influx is requisite for 2-APB-induced synaptic facilitation. Blockade of the voltage-dependent Ca2+ channel with either nifedipine, verapamil or £s-CTX failed to abolish the SSC facilitating effect of 2-APB. Electrophysiological recording of 2-APB induced single channel currents by using cell-attached patch-clamp technique reveals 2-APB evoked a robust single channel activity recorded at different pipette voltages. Furthermore, the 2-APB-evoked single-channel events are significantly abolished in the presence of SKF96365. Either pretreatment of the cultures with inhibitor of phospholipase C (U73122) or tyrosine kinase (Genistein) abolishes 2-APB induced potentiation of synaptic transmission. The structure of PMA is analogous to diacylglycerol (DAG), which abolishes 2-APB induced synaptic facilitation. 2-APB no longer elicited any changes in SSC frequency when serum is eliminated from culture medium. Overall, results from our current study provide evidences that 2-APB induces the opening of TRP channels and Ca2+ influx which resulting in facilitation of spontaneous neurotransmitter release at developing Xenopus neuromuscular synapse. Serum may activate tyrosine kinase to turn on PI3K and phospholipase C. Then phospholipase C cleavage PIP2 to IP3 and diacylglycerol, and diacylglycerol induced TRP channel opening. 2-APB potentiates and sensitizes the TRP channel, increasing Ca2+ inffux. Elevated [Ca2+]i resulted in enhancement of neurotransmitter release from presynaptic nerve terminal.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21047号 / 薬科博第90号 / 新制||薬科||10(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 中山 和久, 教授 金子 周司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

<p>From insects to mammals, the ability to constantly sense environmental stimuli is essential for the survival of most living organisms. Most animals have nocifensive behaviors towards extreme temperatures, mechanical stimuli or irritant chemicals that are considered to be noxious. Nociception is defined as the neural encoding and processing of noxious stimuli. This process starts from the activation of pain detecting peripheral sensory neurons (nociceptors) that can detect noxious mechanical, thermal or chemical stimuli. On the other hand, animals also have the ability to discriminate innocuous temperatures and to direct their locomotions to their favorable environmental temperatures and this behavior is called thermotaxis. </p><p>In this study, I used <italic>Drosophila melanogaster</italic>as a genetic model organism to study the molecular and cellular basis of nociception and thermotaxis. <italic>Drosophila</italic> larvae exhibit a stereotyped defensive behavior in response to nociceptive stimuli (termed nocifensive escape locomotion behavior, NEL). Using this behavior as a readout, we manipulated the neuronal activities of periphery sensory Type II multidendritic neurons and have identified a specific class of neurons, class IV multidendritic neurons, to function as nociceptors in <italic>Drosophila</italic> larvae. </p><p>After identifying the nociceptors, I next investigated several ion channels that are critical molecular components for larval nociception. The Degenerin Epithelial Sodium Channel (DEG/ENaC) protein called pickpocket (ppk) is required specifically for larval mechanical nociception but not for thermal nociception. Being specifically expressed in class IV multidendritic neurons (the nociceptors), pickpocket is likely to function as a first detector of mechanical stimuli and upstream of general neuronal action potential propagation. In addition, I have found that the <italic>Drosophila</italic> orthologue of mammalian TRPA1 gene, <italic>TrpA1</italic>, is required for both mechanical and thermal nociception in <italic>Drosophila</italic> larvae. I have cloned a new isoform of dTRPA1 and have found it to be specifically expressed in class IV md neurons. Unlike the known dTRPA1 isoform that is warmth activated, this new isoform is not directly activated by temperatures between 15-42 °C. Instead, it may function downstream of sensory transduction step in the nociceptors. </p><p>Interestingly, <italic>dTrpA1</italic> mutants are also defective in their thermotaxis behavior within innocuous temperature ranges. In addition to the previously reported defects in avoiding warm temperatures, I have found these flies also failed to avoid cool temperatures between 16-19.5 °C. This defect is likely to be mediated by temperature sensing neurons in the antennae. I have detected antennal expression using a GAL4 reporter of dTrpA1. Significantly, these neurons exhibit elevated calcium levels in response to cooling. dTrpA1 mutants have a premature decay of the cooling response at temperatures below 22 °C during a cooling process. I have also identified another population of cells in the antennae that can respond to temperature changes. These neurons express the olfactory co-receptor Or83b and are known to be olfactory neurons. Calcium oscillations triggered by cooling were detected in these neurons and they were terminated by warming. Severe behavioral defects in avoiding cool temperatures were found in animals lacking <italic>Or83b</italic>. Our results suggest that there are multiple pathways regulating cooling sensation in the fly antennae.</p><p>Taken together, I have shown that <italic>Drosophila</italic> serves as a great model system to study nociception and thermosensation at molecular, cellular and behavioral levels.</p> / Dissertation

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19753号 / 工博第4208号 / 新制||工||1649(附属図書館) / 32789 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 梅田 眞郷 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21830号 / 農博第2343号 / 新制||農||1068(附属図書館) / 学位論文||H31||N5202(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 澤山 茂樹, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Mucolipidosis type IV (MLIV) is a rare, autosomal recessive, neurodegenerative, lysosomal storage disorder. MLIV is caused by mutations in a gene (MCOLN1) encoding a TRP channel family member known as Mucolipin 1 or TRPML1. TRPML1 is a lysosomal transmembrane protein that appears to be required for normal lysosomal pH regulation, recycling of molecules and membrane reorganisation including lysosomal biogenesis, fusion and exocytosis. The exact function of the channel is unknown but it is permeable to multiple ions including Ca²⁺, Na⁺ and K⁺, possibly also Fe²⁺ and Zn²⁺. How normal TRPML1 function regulates lysosomal processes is not clearly understood. Mutations in the MCOLN1 gene can lead to complete loss of TRPML1 function, partial loss of function or mislocalisation, all of which lead to lysosomal dysfunction, lysosomal lipid storage and ultimately neurodegeneration. The disease processes that lead to neurodegeneration are poorly understood and at present no therapy exists for MLIV. We have discovered that TRPML1 results in regulating lysosomal Ca²⁺ homeostasis that is the opposite of the Ca²⁺ dysregulation associated with Niemann-Pick type C disease (NPC). Our findings indicate that disrupted function of TRPML1 leads to enhanced Ca²⁺ release via the NAADP receptor, recently shown to be the lysosomal two-pore channel TPC2. This indicates that TRPML1 is not the NAADP receptor as suggested by others, indeed NAADP mediated Ca²⁺ release is enhanced with multiple NAADP induced lysosomal Ca²⁺ release events occurring in TRPML1 null cells compared to single releases in normal cells. This phenotype appears to be responsible for the cellular dysfunction associated with MLIV disease cells, enhanced lysosomal fusion, defective endocytosis and potentially even altered lysosomal pH. Several of these phenotypes are normalised by the NAADP receptor specific antagonist Ned-19. These findings illustrate that the NAADP receptor is central to MLIV disease pathology and may be a novel candidate for disease therapy.

Human body detects potentially damaging stimuli by specialized sensory nerve endings in the skin, the nociceptors. Their membranes are equipped with ion channels, molecular sensors, coding the outside stimuli into the trains of action potentials and conducting them to the higher brain centers. The most prominent group of transduction ion channels is the transient receptor potential (TRP) channel family followed by ion channels responsible for generation and conduction of action potentials from the periphery to the brain, the voltage-gated sodium channels (VGSCs). Understanding the mechanisms how particular stimulus is encoded and processed is of particular importance to find therapeutics for various types of pain conditions. We characterized the properties of VGSC subtypes NaV1.9 and NaV1.8 at high temperatures. We showed that NaV1.9 undergo large increase in current with increasing temperatures and significantly contribute to the action potential generation in dorsal root ganglion (DRG) neurons. Ciguatoxins (CTXs) are sodium channels activator toxins causing ciguatera fish poisoning, a disease manifested by sensory and neurological disturbances. We elucidated the mechanism of CTX- induced cold allodynia, a pathological phenomenon where normally innocuous cool temperatures are perceived as pain. We...

TRPA1 is a thermosensitive ion channel from the ankyrin subfamily of Transient Receptor Potential (TRP) receptors. These proteins play essential roles in the transduction of wide variety of environmental and endogenous signals. TRPA1, which is abundantly expressed in primary nociceptive neurons, is an important transducer of various noxious and irritant stimuli and is also involved in the detection of temperature changes. Similarly to other TRP channels, TRPA1 is comprised of four subunits, each with six transmembrane segments (S1-S6), flanked by the cytoplasmic N- and C-terminal ends. In native tissues, TRPA1 is supposed to be regulated by multiple phosphorylation sites that underlie TRPA1 activity under physiological and various pathophysiological conditions. Using mutational approach, we predicted and explored the role of potential phosphorylation sites for protein kinase C in TRPA1 functioning. Our results identify candidate residues, at which phosho-mimicking mutations affected the channel's ability to respond to voltage and chemical stimuli, whereas the phospho-null mutations to alanine or glycine did not affect the channel activation. Particularly, we identify the serine 602 within the N-terminal ankyrin repeat domain 16, the substitution of which to aspartate completely abolished the TRPA1...