Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15675号 / 工博第3333号 / 新制||工||1503(附属図書館) / 28212 / 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 跡見 晴幸, 教授 濵地 格 / 学位規則第4条第1項該当

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18594号 / 工博第3955号 / 新制||工||1608(附属図書館) / 31494 / 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 梅田 眞郷, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15408号 / 工博第3287号 / 新制||工||1495(附属図書館) / 27886 / 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 跡見 晴幸 / 学位規則第4条第1項該当

Brain function implies complex information processing in neuronal circuits, critically dependent on the molecular machinery that enables signal transmission across synaptic contacts between neurons. The types of ion channels and receptors in the neuronal membranes vary with neuron types and brain regions and determine whether neuronal responses will be excitatory or inhibitory and often allow for functional synaptic plasticity which is thought to be the basis for much of the adaptability of the nervous system and for our ability to learn and store memories. The present thesis is a study of synaptic transmission in the medial preoptic nucleus (MPN), a regulatory center for several homeostatic functions but with most clearly established roles in reproductive behaviour. The latter behaviour typically shows several distinct phases with dramatically varying neuronal impulse activity and is also subject to experience-dependent modifications. It seems likely that the synapses in the MPN contribute to the behaviour by means of activity-dependent functional plasticity. Synaptic transmission in the MPN, however, has not been extensively studied and is not well understood. The present work was initiated to clarify the synaptic properties in the MPN. The aim was to achieve a better understanding of the functional properties of the MPN, but also to obtain information on the functional roles of ion channel types for neurotransmission and its plastic properties in general. The studies were carried out using a brain slice preparation from rat as well as acutely isolated neurons with adhering nerve terminals. Presynaptic nerve fibres were stimulated electrically or, in a few cases, by raised external K+ concentration, and postsynaptic responses were recorded by tight-seal perforated-patch techniques, often combined with voltage-clamp control of the post-synaptic membrane potential. Glutamate receptors of α-amino-3-hydroxy-5-methyl-4-izoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) types were identified as mediating the main excitatory synaptic signals and γ-aminobutyric acid (GABA)A receptors as mediating the main inhibitory signals. Both types of signals were suppressed by serotonin. The efficacy of AMPA-receptor-mediated transmission displayed several types of short-term plasticity, including paired-pulse potentiation and paired-pulse depression, depending on the stimulus rate and pattern. The observed plasticity was attributed to mainly presynaptic mechanisms. To clarify some of the presynaptic factors controlling synaptic efficacy, the role of presynaptic L-type Ca2+ channels, usually assumed not to directly control transmitter release, was investigated. The analysis showed that (i) L-type channels are present in GABA-containing presynaptic terminals on MPN neurons, (ii) that these channels provide a means for differential control of spontaneous and impulse-evoked GABA release and (iii) that this differential control is prominent during short-term synaptic plasticity. A model where Ca2+ influx through L-type channels may lead to reduced GABA release via effects on Ca2+-activated K+ channels, membrane potential and other Ca2+-channel types explains the observed findings. In addition, massive Ca2+ influx through L-type channels during high-frequency stimulation may contribute to increased GABA release during post-tetanic potentiation. In conclusion, the findings obtained in the present study indicate that complex neurotransmission mechanisms and different forms of synaptic plasticity contribute to the specific functional properties of the MPN.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are able to change their firing rate and pattern in response to small changes in external osmolality due to the involvement of osmosensitive ion channels. The firing rate and pattern determine the release of vasopressin (VP), a primary hormone regulating osmolality by controlling water excretion from the kidney. Both VP- and oxytocin (OT)-MNCs display irregular and infrequent fire when plasma osmolality is near normal, and they progressively increase the frequency of firing to fast continuous firing with increases in osmolality. VP-MNCs also respond to osmotic stimulation by adopting a phasic pattern of firing, which maximizes neuropeptide secretion. Sustained dehydration also causes structural and functional adaptations in MNCs.<p> Voltage-dependent Ca2+ channels play many important roles not only in the regulation of cell excitability but also in intracellular signal transduction, and L-type Ca2+ channel-mediated Ca2+ signals initiate intracellular signal transduction events that activate long-lasting changes in brain function and behavior. Our electrophysiological and immunocytochemical studies demonstrate that 16-24 h of water deprivation causes a significant increase in the amplitude of L-type Ca2+ current (from 55.5 ± 6.2 to 99.1 ± 10.0 pA) but not in other types of Ca2+ current. This increase occurred in both VP- and OT-MNCs. Such an increase in L-type Ca2+ current may contribute to modulation of firing rate and pattern, regulation of vasopressin release, structural adaptation in MNCs during sustained dehydration.<p> The mechanisms underlying the transition of the electrical behaviour are not completely understood. Ion channels, especially osmosensitive ion channels, play key roles in the modulation of MNC firing. A voltage-gated, 4-AP- and TEA-insensitive slowly activating outward current displayed a significant increase in about 66% of MNCs when the osmolality of the external solution was acutely increased from 295 to 325 mosmol kg-1. The responding cells showed an increase in net outward current from 12.3 ± 1.3 pA/pF to 21.4 ± 1.8 pA/pF. The reversal potential of this current was near the equilibrium for K+ and shifted with changes of K+ concentrations in external solution, suggesting that this current is a K+-selective current. The KCNQ/M current selective blockers linopirdine (150 µM) and XE991 (5 µM) suppressed this current. The IC50 of XE991 blockade was 3.9 ìM. The KCNQ/M channel openers retigabine (10 µM) and flupirtine (10 µM) significantly increased the current and shifted its activation curve toward more negative potentials. E4031, a specific blocker of ERG K+ channels, did not significantly block this current. The results from immunocytochemistry suggest that MNCs express KCNQ2, KCNQ3, KCNQ4, and KCNQ5, but not KCNQ1. These data suggest that this osmosensitive current could be a KCNQ/M current. Studies using single unit extracellular recording in hypothalamic explants showed that 10 µM XE991 increased MNC firing rate and that 20 µM retigabine decreased firing rate or caused a cessation of firing. These data suggest that a KCNQ/M current contributes to the regulation of MNC firing. KCNQ/M channels play key roles in regulating neuronal excitability in many types of central neurons. Slow activation of this current during firing might suppress activity by hyperpolarizing the cells and thus contribute to a transition between fast continuous and burst firing.<p> Our studies will be beneficial to understand the mechanisms that control VP and OT in response to acute changes in osmolality and also the mechanisms underlying MNC adaptation during sustained dehydration.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are able to change their firing rate and pattern in response to small changes in external osmolality due to the involvement of osmosensitive ion channels. The firing rate and pattern determine the release of vasopressin (VP), a primary hormone regulating osmolality by controlling water excretion from the kidney. Both VP- and oxytocin (OT)-MNCs display irregular and infrequent fire when plasma osmolality is near normal, and they progressively increase the frequency of firing to fast continuous firing with increases in osmolality. VP-MNCs also respond to osmotic stimulation by adopting a phasic pattern of firing, which maximizes neuropeptide secretion. Sustained dehydration also causes structural and functional adaptations in MNCs.<p> Voltage-dependent Ca2+ channels play many important roles not only in the regulation of cell excitability but also in intracellular signal transduction, and L-type Ca2+ channel-mediated Ca2+ signals initiate intracellular signal transduction events that activate long-lasting changes in brain function and behavior. Our electrophysiological and immunocytochemical studies demonstrate that 16-24 h of water deprivation causes a significant increase in the amplitude of L-type Ca2+ current (from 55.5 ± 6.2 to 99.1 ± 10.0 pA) but not in other types of Ca2+ current. This increase occurred in both VP- and OT-MNCs. Such an increase in L-type Ca2+ current may contribute to modulation of firing rate and pattern, regulation of vasopressin release, structural adaptation in MNCs during sustained dehydration.<p> The mechanisms underlying the transition of the electrical behaviour are not completely understood. Ion channels, especially osmosensitive ion channels, play key roles in the modulation of MNC firing. A voltage-gated, 4-AP- and TEA-insensitive slowly activating outward current displayed a significant increase in about 66% of MNCs when the osmolality of the external solution was acutely increased from 295 to 325 mosmol kg-1. The responding cells showed an increase in net outward current from 12.3 ± 1.3 pA/pF to 21.4 ± 1.8 pA/pF. The reversal potential of this current was near the equilibrium for K+ and shifted with changes of K+ concentrations in external solution, suggesting that this current is a K+-selective current. The KCNQ/M current selective blockers linopirdine (150 µM) and XE991 (5 µM) suppressed this current. The IC50 of XE991 blockade was 3.9 ìM. The KCNQ/M channel openers retigabine (10 µM) and flupirtine (10 µM) significantly increased the current and shifted its activation curve toward more negative potentials. E4031, a specific blocker of ERG K+ channels, did not significantly block this current. The results from immunocytochemistry suggest that MNCs express KCNQ2, KCNQ3, KCNQ4, and KCNQ5, but not KCNQ1. These data suggest that this osmosensitive current could be a KCNQ/M current. Studies using single unit extracellular recording in hypothalamic explants showed that 10 µM XE991 increased MNC firing rate and that 20 µM retigabine decreased firing rate or caused a cessation of firing. These data suggest that a KCNQ/M current contributes to the regulation of MNC firing. KCNQ/M channels play key roles in regulating neuronal excitability in many types of central neurons. Slow activation of this current during firing might suppress activity by hyperpolarizing the cells and thus contribute to a transition between fast continuous and burst firing.<p> Our studies will be beneficial to understand the mechanisms that control VP and OT in response to acute changes in osmolality and also the mechanisms underlying MNC adaptation during sustained dehydration.

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

yes / Ion channels represent a large and growing family of target proteins regulated by gasotransmitters such as nitric oxide, carbon monoxide and, as described more recently, hydrogen sulfide. Indeed, many of the biological actions of these gases can be accounted for by their ability to modulate ion channel activity. Here, we report recent evidence that H2S is a modulator of low voltage-activated T-type Ca2+ channels, and discriminates between the different subtypes of T-type Ca2+ channel in that it selectively modulates Cav3.2, whilst Cav3.1 and Cav3.3 are unaffected. At high concentrations, H2S augments Cav3.2 currents, an observation which has led to the suggestion that H2S exerts its pro-nociceptive effects via this channel, since Cav3.2 plays a central role in sensory nerve excitability. However, at more physiological concentrations, H2S is seen to inhibit Cav3.2. This inhibitory action requires the presence of the redox-sensitive, extracellular region of the channel which is responsible for tonic metal ion binding and which particularly distinguishes this channel isoform from Cav3.1 and 3.3. Further studies indicate that H2S may act in a novel manner to alter channel activity by potentiating the zinc sensitivity/affinity of this binding site. This review discusses the different reports of H2S modulation of T-type Ca2+ channels, and how such varying effects may impact on nociception given the role of this channel in sensory activity. This subject remains controversial, and future studies are required before the impact of T-type Ca2+ channel modulation by H2S might be exploited as a novel approach to pain management. / This work was supported by grants from the British Heart Foundation, the Medical Research Council, and the Hebei Medical University

No / T-type Ca2+ channels are a distinct family of low voltage-activated Ca2+ channels which serve many roles in different tissues. Several studies have implicated them, for example, in the adaptive responses to chronic hypoxia in the cardiovascular and endocrine systems. Hydrogen sulfide (H2S) was more recently discovered as an important signalling molecule involved in many functions, including O2 sensing. Since ion channels are emerging as an important family of target proteins for modulation by H2S, and both T-type Ca2+ channels and H2S are involved in cellular responses to hypoxia, we have investigated whether recombinant and native T-type Ca2+ channels are a target for modulation by H2S. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS, selectively inhibits Cav3.2 T-type Ca2+ channels heterologously expressed in HEK293 cells, whilst Cav3.1 and Cav3.3 channels were unaffected. Sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn2+ to this channel. Chelation of Zn2+ using TPEN prevented channel inhibition by H2S. H2S also selectively inhibited native T-type channels (primarily Cav3.2) in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca2+ channel Cav3.2. Results have important implications for the proposed pro-nociceptive effects of this gasotransmitter. Implications for the control of cellular responses to hypoxia await further study.

No / T-type Ca2+ channels regulate proliferation in a number of tissue types, including vascular smooth muscle and various cancers. In such tissues, up-regulation of the inducible enzyme heme oxygenase-1 (HO-1) is often observed, and hypoxia is a key factor in its induction. HO-1 degrades heme to generate carbon monoxide (CO) along with Fe2+ and biliverdin. Since CO is increasingly recognized as a regulator of ion channels (Peers et al. 2015), we have explored the possibility that it may regulate proliferation via modulation of T-type Ca2+ channels. Whole-cell patch-clamp recordings revealed that CO (applied as the dissolved gas or via CORM donors) inhibited all 3 isoforms of T-type Ca2+ channels (Cav3.1-3.3) when expressed in HEK293 cells with similar IC50 values, and induction of HO-1 expression also suppressed T-type currents (Boycott et al. 2013). CO/HO-1 induction also suppressed the elevated basal [Ca2+ ]i in cells expressing these channels and reduced their proliferative rate to levels seen in non-transfected control cells (Duckles et al. 2015). Proliferation of vascular smooth muscle cells (both A7r5 and human saphenous vein cells) was also suppressed either by T-type Ca2+ channel inhibitors (mibefradil and NNC 55-0396), HO-1 induction or application of CO. Effects of these blockers and CO were non additive. Although L-type Ca2+ channels were also sensitive to CO (Scragg et al. 2008), they did not influence proliferation. Our data suggest that HO-1 acts to control proliferation via CO modulation of T-type Ca2+ channels.