It has been known that the rectification property of inward rectifier K⁺ channel (Kir) is mainly due to block of outward current by cytoplasmic Mg²⁺ and polyamines. Crystal structure analysis of the cytoplasmic region of Kir3.1 has revealed that positively (R228, R260) and negatively (E224, D259, E299) charged residues are on the wall of the inner vestibule in Kir2.1. In this study, we aimed to approach to the functional significance of these charged residues, and compared the electrophysiological properties of single/double mutants of them. We examined the inward rectification property of the mutants expressed in Xenopus oocytes under two-electrode voltage clamp. We observed that E224Q, D259N and E229Q single point mutations weakened rectification, and that R228Q and R260Q single point mutations on the background of E224Q made the rectification property recovered. We next analyzed susceptibilities of the mutants to blockade by intracellular blockers using the inside-out patch clamp technique. We observed that the decrease in the net negative charge in the cytoplasmic pore reduced the blocking sensitivity to Mg²⁺ and spermine, and the voltage dependency of Mg²⁺ blocking. It also influenced K⁺ permeation; i.e. unexpectedly weakened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these results suggest that the net negative charge in the cytoplasmic pore increases the local concentration of cations such as Mg²⁺, spermine as well as K⁺, and also extend the electrostatic field to the inner vestibule. [Jpn J Physiol 54 Suppl:S132 (2004)]

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An inwardly rectifying K⁺ channel, Kir5.1, is expressed abundantly in brain but its functional role is still largely unknown. Because Kir5.1 is coexpressed with Kir4.1 in retinal Muller cells, in this study we have compared the biochemical and immunological properties of Kir5.1 and Kir4.1 in the brain. We have found that not only Kir4.1 but also Kir5.1 are expressed mainly in brain astrocytes. These two Kir subunits differentially assemble in a region-specific manner: the heteromer of Kir4.1/Kir5.1 was identified in the areas such as neocortex and glomerulus of olfactory bulb, while the homomer of Kir4.1 was in those such as hippocampus and thalamus. The homomeric assembly of Kir5.1 was not identified. The Kir4.1/Kir5.1 and Kir4.1 channels in astoryctes were concentrated on the membrane facing pia and blood vessels as well as at the processes surrounding synapses. Because each multimer exhibits a unique channel property, homomeric Kir4.1- and heteromeric Kir4.1/Kir5.1-channels may play differential roles in the K⁺-buffering action of brain astrocytes. In addition, we found that these Kir channels could associate with a PDZ-domain containing protein, syntrophins, which are components of the dystrophin-accociated protein complex (DAPC). Because DAPC has been suggested to regulate the polarized distribution of Kir4.1, the syntrophins may be involved in targeting the Kir channels to selected membrane-domains in the astrocytes. [Jpn J Physiol 54 Suppl:S132 (2004)]

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Large conductance, voltage- and Ca²⁺-activated K⁺ channel (Maxi-K) is distributed in a variety of epithelial secretory cells, seemingly displaying cell-specific mechanisms of fluid secretion, where an effective Cl⁻ exit takes place through the membrane negativity created by the activation of K⁺ channels. Rat and mouse pancreatic acinar cells are peculiar in this sense because of the lack of Maxi-K channel, which, once activated, can fairly influence the membrane potential due to its large unitary conductance. However, as evidenced here, this is not entirely correct. The expression of Maxi-K channels is controlled in an age-dependent manner. We studied their presence in mouse pancreatic acinar cells, during the animal age from 5- to 84-week after birth, with patch-clamp measurements and RT-PCR detection of mRNA of mSlo, the mouse Maxi-K channel gene. Channel activity on the plasma membrane started to appear around 12 postnatal weeks and the rate of the channel presence steeply increased up to 84 weeks. We could detect mSlo mRNA in pancreas of old (58 and 64 weeks) animals but not of young (7 or 8 weeks). Acetylcholine activated Maxi-K channels most immediately after its administration among the pancreatic Ca²⁺-activated ion channels, implying Maxi-K channels locate very close to the zone triggering the first rise in internal Ca²⁺. These results suggest that the pancreatic secretory machinery is progressively reorganized and the mode of fluid secretion is reshaped with aging. [Jpn J Physiol 54 Suppl:S132 (2004)]

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Removal of external K⁺ ions abolishes not only inward currents but also outward currents through the inwardly rectifying K⁺ channel which determines the resting potential. This suggests that external K⁺ ions are essential for the channel activation. To investigate how extracellular K⁺ ions act the channel, we made single points mutants in which one of negative charged amino acids in the extracellular loops of the murine inwardly rectifying K⁺ channel (Kir2.1) was neutralized. cDNA was transfected into COS-1 cells using the liposome method, and voltage clamp experiments were done after 24-72 h. COS-1 cells transfected with D112N did not show inward rectification under whole-cell recording in the normal Tyrode solution. Cells transfected with tandem tetramers with one wild-type (WT) and three D112N mutant subunits (WT-(D112N)3) also did show inward rectification, while cells transfected with tandem tetramers with two wild-type and two D112N subunits (WT2-(D112N)2) showed inward rectification. Channels from WT2-(D112N)2 had the single-channel conductance similar to that of wild-type channels. It is suggested that two negative charges at the D112 site may be required for K⁺ ions to bind to and activate the channel. [Jpn J Physiol 54 Suppl:S133 (2004)]

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G-protein-gated potassium (K_G) channel,which is directly activated by G-protein βγ subunits,and is responsible for cholinergic regulation of heart beats.Cardiac K_G channel is known to exhibit a characteristic apparentvoltage-dependent behavior, named "relaxation", which has been shown toreflect the voltage-dependent control of receptor mediated cyclic reactionof G-protein (G-protein-cycle) by regulators of G-protein signaling (RGS)proteins. Based on these results, we have succeeded in establishing aphysiological G-protein-cycle model.We use the Monod-Wyman-Changeux allosteric model for the interaction between theK_G channel and the heterotrimeric G-protein βγ subunit.The parameters of the allosteric model are estimated by the experimental results(Corey and Clapham, 2001) of the population distribution of the number of bindingG_βγ with the GIRK4 homotetramer. The derived parameter set ofthe allosteric model is consistent with the dose-dependence of the channelactivity. The model of receptor-G-protein interaction is based on the modelpresented by Thomsen et al (1988). The rate constants of each reactions in theG-protein-cycle are decided to reproduce simultaneously both thedose-dependence of the steady-state channel activation and the time courseof the open probability. The physiological relevance of the relaxation behaviorin heart beat regulation can be evaluated by incorporating this model intoaction potential simulation, Luo-Rudy model. [Jpn J Physiol 54 Suppl:S133 (2004)]

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Application of adenosine (Ade) induces K⁺-current response in the follicular cells surrounding a Xenopus oocyte under voltage clamp. This Ade-induced K⁺ current response is produced by activation of Gs, subsequent adenylate cyclase, cAMP-dependent protein kinase, and by opening of the ATP-sensitive K channels. Application of 20 nM insulin initially augmented the Ade-induced response, and subsequently depressed. The depressing effect of insulin on the K⁺ current response was irreversible during the washout for more than 3 hours with normal perfusuate. Prior application of 30 nM Lavendustin A (Lav.A), an inhibitor of protein tyrosine kinase, markedly blocked the depressing effect of insulin on the Ade-induced response. These results suggested that the depressing effect of insulin on the Ade-induced K⁺ current response might be regulated by activation of protein tyrosine kinase through the stimulation of insulin receptor. [Jpn J Physiol 54 Suppl:S133 (2004)]

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Lucifer Yellow (LY) has been used to mark cells electrophysiologically investigated or to visualize coupled cells with gap junctions. We, however, showed that LY radicals produced by the illumination of LY with the moderate intensity light inhibited the inactivation of voltage-gated Na⁺ channels by increasing the mean open time of the channel (J. Physiol., 550.1;159-167, 2003). In the present study, we investigated the effect of illuminated LY (2 mg/ml) on voltage-gated K⁺ channels and on ligand-gated channels of cultured hippocampal neurons under whole-cell clamp conditions. The light was 4850 lx at source, a moderate strength for microscopy, and band-passed (400-440 nm). The width of action potentials increased with increasing exposure time, and was 3.9 times longer than the control after 5 min exposure. After 4 min exposure, the magnitude of delayed rectifier K⁺ currents was 1.4 times larger than the control at +65.3 mV, and that of inward rectifier K⁺ currents was 1.6 times larger at –134.7 mV. The pretreatment of LY-injected neurons with 1 mM dithiothreitol for 10 min inhibited the elongation of action potential width and the enhancement of both K⁺ current in magnitude. In contrast, exposed LY hardly changed the magnitude of NMDA- and GABA-induced currents. These results suggest that LY radicals modify a part or parts of voltage-gated channel proteins that regulate the channel open times. [Jpn J Physiol 54 Suppl:S133 (2004)]

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In the mesencephalic trigeminal sensory neurons (MTNs), both the Na⁺-K⁺ pump current (I-p) and the h-current (I-h) participate in generating the resting potential, while these two currents have been thought to be produced independently.
We show here functional coupling between Na⁺-K⁺ pumps and h-channels in the rat MTNs. Under whole-cell voltage-clamp condition, activation of I-h in response to hyperpolarizing command pulse leads to the generation of two types of ouabain-sensitive I-p with temporal profiles similar to those of instantaneous and slow components of I-h, reflecting Na⁺-transients in a restricted cellular space (microdomain). Moreover, the I-p activated by instantaneous I-h can facilitate the subsequent activation of slow I-h. Such counteractive and cooperative interactions were also disclosed by replacing extracellular Na⁺ with Li⁺. These observations indicate that the interactions are mediated by Na⁺ ions and reflected in Na⁺-transient in presumed microdomain. Consistent with these findings, h-channels and Na⁺-K⁺ pumps were colocalized in plasma membrane of MTNs covered by numerous spines. Therefore, the bidirectional interactions between Na⁺-K⁺ pumps and h-channels are likely to be mediated by a novel mechanism of Na⁺-microdomain, presumably created by the morphological specialization of spines in primary sensory neurons of MTN. The presence of Na⁺-microdomain was further investigated by measuring Na⁺ transient just beneath the plasma membrane and at the center of soma, using sodium-binding benzofuran isophthalate (SBFI). [Jpn J Physiol 54 Suppl:S134 (2004)]

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Persistent sodium current plays important roles in neuronal excitabilities. Nav1.6, a widely expressed sodium channel isoform in CNS and PNS, is known as one of the major contributors of persistent currents in cell soma. On the other hand, Nav1.6 is localized at the axon initial segment (AIS) and node of Ranvier and thereby responsible for fast saltatory conduction of action potential along myelinated fibers, where, by classical measurements, persistent component is not so prominent. One possibility is that some factors in node and AIS may inhibit persistent current which could be a default property of Nav1.6 channel. We show that ankyrin G, which is required for clustering of Nav1.6 channels in node and AIS, significantly reduces persistent current of Nav1.6 channel in tsA201 cells and Xenopus oocyte. This inhibition was not observed in coexpresssion with another ankyrin isoform, ankyrin B. By testing chimeras between ankyrin G and B, it was shown that the membrane-binding domain in ankyrin G gives such specificity. The suppression of persistent current by ankyrin G seems to be mediated by direct binding of Nav1.6 to ankyrin G, since deletion of ankyrin-binding consensus sequence of Nav1.6 reduced the modification of inactivation by ankyrin G. These results raise an intriguing possibility that ankyrin-dependent gating of sodium channel might account in part for functional diversities or pathophysiological abnormalities of brain Nav channels. [Jpn J Physiol 54 Suppl:S134 (2004)]

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Disturbance of renal Na⁺ reabsorption develops hypertension in Dahl salt-sensitive (DS) rat. We previously reported that high sodium diet decreased the plasma aldosterone level, evertheless high sodium diet elevated mRNA expression of epithelial Na⁺ channel (ENaC) and blood pressure in the kidney of DS rat, suggesting that ENaC expression is induced independent of aldosterone. Therefore, we studied whether aldosterone regulates ENaC expression in DS rat. In addition to ENaC expression, we assessed the effect of aldosterone on serum and glucocorticoid-regulated kinase 1 (sgk1) contributing to renal Na⁺ reabsorption. Aldosterone (1.5 mg/kg B.W.) was subcutaneously injected into adrenalectomized DS and Dahl salt-resistant (DR) rats kept with normal diet and water supplemented with 9 g/l NaCl for 2 weeks after adrenalectomy. RNA was extracted from the kidney 6 h after the injection. In DR rats, aldosterone injection increased expression of alpha-ENaC and sgk1. On the other hand, in DS rats, aldosterone injection elevated expression of alpha-ENaC similar to DR rats, but not sgk1. These observations suggest that the sgk1-mediated alpha-ENaC-inducing action of aldosterone normally observed in salt-resistant rats is disordered in salt-sensitive hypertensive rats, and that an sgk1-independent alpha-ENaC-inducing mechanism would cause hypertension in a salt-sensitive aldosterone-independent manner in DS rats. Supported by JSPS 15659052 (YM), 15590189 (NN), 15790120 (HM). [Jpn J Physiol 54 Suppl:S134 (2004)]

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The amiloride-sensitive epithelial Na⁺ channel (ENaC) controls Na⁺ transport into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and denoted as α, β, γ, and δ. ENaCδ can associate with β and γ subunits and generate a constitutive current two orders magnitude larger than that of homomeric ENaCδ. However, the distribution pattern of ENaCδ is not consistent with that of β and γ subunits. ENaCδ is expressed mainly in the brain in contrast to β and γ subunits which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaCδ and investigated the detailed tissue distribution in humans. When hENaCδ was expressed in Xenopus oocytes and CHO cells, a reduction of extracellular pH activated this channel and the acid-induced current was abolished by amiloride. The most striking finding was that the desensitization of the acid-evoked current was much slower, dissociating from the kinetics of acid-sensing ion channels (ASICs) in degenerin/ENaC family. RNA dot-blot analyses showed that ENaCδ mRNA was widely distributed throughout the brain, and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaCδ was expressed in the cerebellum and the hippocampus. In conclusion, hENaCδ activity is regulated by protons, indicating that it may contribute to the pH-sensation or/and pH-regulation in human brain. [Jpn J Physiol 54 Suppl:S134 (2004)]

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Both Tetrodotoxin-sensitive (TTX-S) and resistant (TTX-R) voltage-gated Na⁺ channel are expressed in human neuroblastoma NB-1 cells , but the gene encoding the TTX-R Na⁺ channel is not identified. In this study, we have cloned α subunit of TTX-R Na⁺ channel from NB-1 cells by reverse transcriptase-polymerase chain reaction (RT-PCR) and designated it as hNbR1. The longest open reading frame of NbH1 encodes 2016 amino acid residues. The presence of a cysteine residue (c373) in the pore region domain I suggests that hNbR1 is resistant to TTX. Sequence comparisons indicated that hNbR1 is highly homologous to human cardiac SCN5A (99.2% nucleotide homology) except the region between domain I S3 and S4. At least one alternative splicing variant, which is characterized by a deletion (54 amino acids) the intracellular loop between domains II and III, is found. These results suggest that SCN5A is more widely distributed than previously thought and contributes to TTX-R Na⁺ current in neural tissues. [Jpn J Physiol 54 Suppl:S135 (2004)]

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Ciguatoxins (CTXs) are receptor site-5 neurotoxins that open voltage-dependent Na channels, but their physiological effects are poorly understood. We studied channel modification induced by the ciguatoxin congener CTX3C (1 μmol/l) in three different Na channel isoforms (rNa_v1.2, rNa_v 1.4, and rNa_v1.5) transduced into HEK cells. In all three isoforms, the toxin shifted the activation potential for the Na current (V_1/2 of the activation curve) in the negative direction, resulting in a 30-mV hyperpolarizing shift of threshold potential. The toxin also shortened time-to-peak current from 0.62 to 0.52 ms in rNa_v1.2. CTX3C shifted the inactivation potential (V_1/2 of the inactivation curve) of all isoforms in the negative direction by 15 to 18 mV. CTX3C suppressed I_Na amplitude at -20 mV to a similar extent in all three isoforms (i.e., 80-85% of the control value). Recovery from slow inactivation induced by a prolonged (500 ms) depolarizing prepulse to 0 mV was dramatically delayed: recovery time constants were increased from 38 ± 8 to 588 ± 151 ms (n=5), 53 ± 6 to 338 ± 85 ms (n=4), and 23 ± 3 ms to 232 ± 117 ms (n = 3) in rNa_v1.2, rNa_v1.4, and rNa_v1.5, respectively. Thus, CTX3C exhibited a multimodal action on sodium channels, with simultaneous stimulatory and inhibitory aspects presumably related to the large molecular size and lipophilicity of this membrane-spanning toxin. [Jpn J Physiol 54 Suppl:S135 (2004)]

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Grayanotoxin (GTX) exerts selective effects on voltage dependent sodium channels by eliminating fast inactivation and causing a hyperpolarizing shift in voltage dependence of channel activation. We have detected several sites that affect GTX action in all S6 transmembrane segments of four domains, and succeeded in distinguishing specific roles in the two sites (Na_v1.4-Tyr-1586 and Na_v1.4-Phe-1579) in D4S6 by measuring binding and unbinding constants of GTX (k_on and k_off) in mutant sodium channels. The former regulates the accessibility of the toxin to the receptor and the latter behaves as a receptor. Systematic substitutions of Na_v1.4 at Phe-1579 increased both k_on and k_off resulting in no prominent effect on dissociation constant (K_d). Such substitutions of Phe-1579 with several amino acids indicated that the size of molecule was the most critical factor to affect GTX binding. Systematic substitutions of Tyr-1586 with amino acids having hydrophobic or aromatic side chains decreased k_off causing drastic increase in the affinity of the toxin. In this study we applied similar analysis on the sites in D1. Na_v1.4-Leu-437 in D1S6 had a similar property for Tyr-1586, indicating this site also behaves as a receptor site. These results indicate that a receptor for GTX may be composed of multiple sites broadly distributed in different domains. [Jpn J Physiol 54 Suppl:S135 (2004)]

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PURPOSE To examine the properties of a newly identified voltage-gated sodium channel of bovine ciliary muscle cells.
METHODS Bovine ciliary myocytes freshly dispersed by collagenase treatment were used. Whole-cell membrane potential was held constant at -50 mV and ramp pulses of 200 ms duration either ascending or descending between -120 mV and 80 mV at a rate of 1 V/s were applied. The bath was perfused with a HEPES-Krebs solution (pH 7.4, 30°C), and borosilicate-glass pipette electrodes were filled with 100 mM-Cs⁺ solution containing 70 nM-Ca²⁺ and 200 μM-GTP (pH 7.0).
RESULTS Ascending ramp-pulse evoked an inward current having a peak of 40 to 200 pA at -10 to 0 mV, which was not observed when descending ramp pulse protocol was used. The current was abolished by tetrodotoxin (1 μM), whereas it was not affected by carbachol (2 μM) or verapamil (1 μM). A similar current was observed in response to the ascending ramp pulse when all extracellular metal cations were isosmotically replaced with either Li⁺ or Na⁺. No currents were evoked when Cs⁺, Mg²⁺, Sr²⁺ or Ba²⁺ was used as the substitute ion. A small but clearly discernible inward current was observed when Ca²⁺ was the substitute.
DISCUSSION The bovine ciliary muscle possesses a voltage-dependent sodium channel similar to the ones widely distributed in neurons or skeletal muscle. This channel, which is measurably permeable to Ca²⁺, may serve as a pathway for Ca²⁺ entry from the extracellular space in the early phase of the contraction. [Jpn J Physiol 54 Suppl:S135 (2004)]

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Microglia participate in both neuroprotective and neuropathological functions. Voltage-gated proton (H⁺) channels, highly expressed in microglia, sense subtle pH disturbances and secrete a massive amount of H⁺. We hypothesize that the H⁺ channel plays a crucial role in H⁺-signaling in the CNS. Here the H⁺ channel activity in different types of cell acidosis was examined in rat microglia under actions of intrinsic pH buffers employing perforated whole-cell recordings. When washing pre-loaded NH₄Cl imposed acute cell acidosis, the H⁺ channel was activated immediately upon a drop in intracellular pH. The equilibrium potential for H⁺ (Er) shifted to more negative voltages, but the IV curve was identical. The activation was transient at voltages higher than the channel threshold, and sustained at voltages lower than the threshold, indicating that relief of the cell acidosis terminated the channel activation. When prolonged cell acidosis was induced by Na-lactate (pH 6.8), activation of the H⁺ channels was maintained accompanying by Er shift to negative voltages and enhancement of the IV curve. It is suggested that cell acidosis by washing NH₄Cl activates the H⁺ channel due to the inherent pH-dependency, but during lactoacidosis, pH-independent mechanisms also contribute to the activation. The H⁺ channel, as a real-time pH monitor, is a useful tool in investigating H⁺-signaling. [Jpn J Physiol 54 Suppl:S136 (2004)]

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The human cone cyclic nucleotide-gated (CNG) channel comprises hCNGA3 α-subunit and hCNGB3 β-subunit. Mutations in both hCNGA3 and hCNGB3 have been linked to rod monochromacy. In the present study we examined the functional consequences of the rod monochromacy-associated T565M and E593K mutations in hCNGA3, which reside in the cyclic nucleotide-binding domains (CNBD) of the α-subunit. Macroscopic patch current was recorded from HEK293T cells expressing the recombinant CNG channels using the inside-out patch-clamp methods. The apparent affinity for cGMP (160 μM) in hCNGA3 homomeric channels containing the T565M mutation was markedly lower than that (9.0 μM) in wild-type hCNGA3 channels. On the other hand, hCNGA3 channels containing the E593K mutation exhibited a significantly higher apparent affinity for cGMP (3.0 μM) compared with wild-type hCNGA3 channels. When the hCNGA3 was associated with the hCNGB3, the apparent affinity for cGMP was significantly reduced (15.0 μM). However, association of the hCNGA3 containing the T565M or E593K mutation with the hCNGB3 did not appreciably affect the apparent affinity for cGMP. The present results thus suggest that the rod monochromacy-associated mutations (T565M and E593K) in the hCNGA3 lead to a marked alteration in the sensitivity for cGMP in human cone CNG channels and thereby contribute at least partly to the pathogenesis of rod monochromacy. [Jpn J Physiol 54 Suppl:S136 (2004)]

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The four subtypes (HCN1-4) of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in mammals show distinct activation and deactivation kinetics and cAMP sensitivities, which may lead to their distinct physiological roles. We previously elucidated that S1 and S1-S2 loop, and S6-cyclic nucleotide binding domain (CNBD) loop are important for activation kinetics by hyperpolarizing voltage steps. However, those regions affected deactivation kinetics much less than activation kinetics. Therefore, we investigated the structural basis for deactivation with the whole-cell patch clamp technique by using HCN1, HCN4, and chimeric channels in a mammalian expression system (COS-7). The deactivation time constant of HCN4 was about 7-fold longer than that of HCN1 when compared at 0 mV. In chimeras between HCN1 and HCN4, the replacement of the regions from the N-terminus to S3 and from S6 to CNBD affected the deactivation kinetics in a graded manner. The substitution of the region from S3 to S6 between HCN1 and HCN4, which includes the putative voltage sensor S4 and the pore region, did not affect the deactivation at all and affected the activation kinetics little. Furthermore, we investigated the effect of cAMP on deactivation kinetics. We especially focused on the single point mutation in S2, which was already reported to alter the effect of cAMP on activation kinetics. These results show that HCN channels modified not the region from S3 to S6 but the regions from N-terminus to S3 and from S6 to CNBD to create distinct subtypes in evolution. [Jpn J Physiol 54 Suppl:S136 (2004)]

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[PURPOSE] The human cone photoreceptor CNG channel is thought to comprise hCNGA3- and hCNGB3-subunits. The purpose was to examine functional role of hCNGB3 in modulation of human cone CNG channels and to characterize functional consequences of rod monochromacy-associated mutations in hCNGB3 (S435F and D633G). [METHODS] Macroscopic patch currents were recorded from HEK293 cells expressing homomeric and heteromeric channels using inside-out patch-clamp technique. [RESULTS] Both hCNGA3 homomeric and hCNGA3/hCNGB3 heteromeric channels were activated by cGMP, with K_1/2 of 11.1±1.0 and 26.2±1.9 μM, respectively. The hCNGA3 channels appeared to be more sensitive to inhibition by extracellular Ca²⁺ compared with hCNGA3/hCNGB3 channels. Coexpression of either of rod monochromacy-associated mutants of hCNGB3 with hCNGA3 significantly reduced K_1/2 value for cGMP but little affected the sensitivity to extracellular Ca²⁺, compared with wild-type heteromeric channels. The selectivity of homomeric and heteromeric channels for monovalent cations was largely similar. Immunoprecipitation experiments showed association of hCNGA3-subunit with both of wild-type and mutant hCNGB3-subunits. [CONCLUSIONS] The rod monochromacy-associated S435F and D633G mutations in hCNGB3 evokes a significant increase in the apparent affinity for cGMP, which should alter cone function and thereby contribute at least partly to pathogenesis of the disease. [Jpn J Physiol 54 Suppl:S136 (2004)]

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We reported a novel feedback regulation of pyramidal cell firing in the rat visual cortex, in which spike-induced Ca²⁺ influx triggers Ca²⁺ release from IP₃ receptors (IP₃Rs) sensitized by a prior increase in IP₃ (IP₃-assisted CICR), thereby activating SK channels in an enhanced fashion (Yamamoto et al., 2002; Yamada et al., 2004). Activation of group I metabotropic glutamate receptors (mGluRs) was shown to suffice to provide the requisite IP₃. Homer/Vesl proteins, which bind both IP₃Rs and mGluRs, may well be involved in this feedback. To probe this possible Homer/Vesl involvement, we have studied whether an activity-dependently inducible member of Homers/Vesls, Homer1a/Vesl1S, affects pyramidal cell excitability. Injection of Homer1a/Vesl1S protein through whole-cell pipettes elicited hyperpolarization by 5-10 mV. This hyperpolarization was prevented by co-applying either the mGluR antagonist MPEP, PLC inhibitor U-73122, IP₃R blocker heparin, Ca²⁺ indicator/chelator fura-2, or BK class Ca²⁺-activated K⁺ channel antagonist charybdotoxin, but not by the SK channel blocker dequalinium. These findings suggest that activation of mGluR by Homer1a/Vesl1S produces IP₃, then causes inositol-induced Ca²⁺ release and a consequent BK channel opening, and finally leads to hyperpolarization. To our surprise, neither was this membrane stabilization triggered by spike firing nor achieved by SK channel opening, therefore depending unlikely on IP₃-assisted CICR. [Jpn J Physiol 54 Suppl:S137 (2004)]

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In the ciliary muscle, muscarinic stimulation evokes an inward current which probably results from opening of non-selective cation channels (NSCCs). They may possibly serve as routes of Ca²⁺ influx required for sustained contraction of this smooth muscle that lacks voltage-gated Ca²⁺ channels. We examined here the properties of the NSCCs by current-noise analysis in the porcine ciliary muscle.
METHODS Whole-cell membrane currents were recorded by voltage clamp in porcine ciliary myocytes freshly dispersed with collagenase. The bath was perfused with a physiological saline buffered with 10 mM-HEPES. Pipettes were filled with a K⁺-free solution containing 100 mM Cs aspartate, 5 mM-BAPTA ([Ca²⁺]=70 nM) and 200 μM-GTP (pH 7.0). Experiments were carried out at 30°C. For RT-PCR, specific primers for TRPC homologues were designed on the basis of cDNA sequences reported for several mammalian species.
RESULTS & DISCUSSION Under voltage clamp at -50 mV, CCh (2 μM) evoked inward currents accompanied by a marked increase in noise. The plot of variance against mean of the current exhibited two linear components, indicating the existence of two types of NSCCs with unitary conductances of 35 pS and 95 fS. FFT analysis of the noise gave power spectra to which a double-Lorentzian function was fitted with corner frequencies of 14 ± 3 and 82 ± 5 Hz (n=35). RT-PCR identified high level of mRNA of several TRPC homologues which have been shown to constitute NSCCs modulated by G-protein-linked mechanisms. [Jpn J Physiol 54 Suppl:S137 (2004)]

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The area postrema (AP) has been considered to play an important role in controlling the antonomic functions. We found that ACh evoked an inward current at a holding potential of -70mV in acutely dissociated AP neurons of rat, using the whole cell patch clamp method. The ACh-induced current was inhibited by nicotinic antagonists, but not by atropine, and the current was also induced by nicotinic agonists. The current-voltage relationship showed a marked inward rectification. The ACh-induced current was reduced in the absence of external Na⁺, but was still substantial at 10mM Ca²⁺, suggesting some permeability to Ca²⁺ of receptor channel. The ACh response was significantly inhibited by serotonin, but not by noradrenaline. Serotonin did not affect the ATP-induced current. Our results indicate the presence of nicotinic receptors on AP neurons, and suggest that serotonergic nerve modifies the nicotinic transmission. [Jpn J Physiol 54 Suppl:S137 (2004)]

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In guinea-pig intestinal smooth muscle, M2 muscarinic receptors activate nonselective cationic current (I_cat). Nonetheless, M3 receptors which link to the G_q/11 protein/phospholipase C (PLC) system have also been suggested to play a crucial role in I_cat activation via Ca²⁺-independent mechanisms. Therefore, we examined using whole-cell patch clamp techniques whether PLC is involved in I_cat activation in single smooth muscle cells. 1) The PLC inhibitor U73122 (0.1-1 μM) concentration-dependently inhibited I_cat evoked by ascending application of carbachol (CCh; 1-300 μM), and completely blocked at 1 μM. It also inhibited I_cat evoked by intracellularly-applied GTPγS (400 μM). 2) The inactive analogue U73343 (0.1-1 μM) did not significantly affect CCh- or GTPγS-evoked I_cat. 3) Antibodies against the α subunit of G_q/11 proteins blocked only an oscillatory component of CCh-evoked I_cat such as that linked to G_q/11 protein-regulated PLC activity. 4) OAG (10-100 μM), an analogue of diacylgrycerol (DAG), evoked no or only small current by itself and remained CCh-evoked I_cat unchanged. These results indicate that PLC plays a crucial role in I_cat activation, and also suggest that the PLC's role is neither regulated by G_q/11 proteins nor mediated by DAG. [Jpn J Physiol 54 Suppl:S137 (2004)]

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KCNQ channels are known as "M-channels", which are suppressed by stimulating co-expressed muscarinic acetylcholine receptors. PKC-dependent pathway has been proposed as a possible mechanism of receptor-induced inhibition of M-current (Hoshi et al. 2003). We investigated the effects of a PKC activator phorbol 12-myristate -13-acetate (PMA) on homomeric KCNQ channels heterologously expressed in Xenopus oocytes. Upon application of 100 nM PMA to KCNQ2 channel, the conductance-voltage (G-V) relationship plot was shifted by 18 mV. A Similar but a smaller shift of G-V plot (12 mV) could be also induced by stimulation of co-expressed M1 receptor. PMA further shifted the G-V plot after M1-receptor stimulation, while M1-receptor stimulation failed to shift the G-V plot further after PMA application. These results indicate that the shift of G-V plot is caused exclusively via PKC-dependent pathway. We also found that the G-V plot of KCNQ1 channel was not clearly shifted either by PMA or M1-receptor stimulation. We constructed KCNQ2-based chimeras to identify the region responsible for the PKC-dependent shift of G-V plot. KCNQ2 with a part of the c-terminus cytoplasmic region (V521~G590) replaced with the corresponding region of KCNQ1 showed slight shift of G-V plot by PMA. This region may be responsible for the shift of G-V plot induced by PKC-dependent pathway. [Jpn J Physiol 54 Suppl:S138 (2004)]

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Neurons of male rat major pelvic ganglia (MPG) can be classified into sympathetic and parasympathetic neurons according to the electrophysiological properties; membrane capacitance, expression of T-type Ca²⁺ channels and GABA_A receptors, and the firing patterns during depolarization. Serotonin (5-hydroxytryptamine, 5-HT), a synaptic transmitter, localized immunohistochemically within the pelvic ganglia; but its functional role remains unclear in autonomic transmissions. In the present study, we investigated the function and molecular expression of 5-HT₃ receptor in MPG neurons using electrophysiological, calcium imaging and RT-PCR techniques. Focal application of 5-HT elicited fast inward currents and depolarized membrane potential in parasympathetic neurons showing phasic firing patterns (n=32). MDL7222 (1 μM) and Y25130 (10 μM), selective 5-HT₃ receptor antagonists, completely abolished the 5-HT-induced inward current and depolarization (n=10). In the fluo-3-loaded cells, 5-HT also induced a rapid increase in intracelluar [Ca²⁺]. Conversely, sympathetic neurons expressing GABA_A receptor did not respond to 5-HT application at all (n=22). RT-PCR analysis revealed that MPG neurons contain mRNAs for 5-HT_3A and 5-HT_3B subunits. Taken together, our data suggest that parasympathetic MPG neurons express 5-HT₃ receptors whose activation can mediate fast synaptic transmission. This study was supported by KOSEF Grant (R05-2003-000-11448-0). [Jpn J Physiol 54 Suppl:S138 (2004)]

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Glycine is a major inhibitory neurotransmitter in central or peripheral nervous system, and the response is mediated by several glycine receptors (GlyRs) . Here, we report cloning and analysis of a novel variant of GlyRα1 subunit. The variant, designated as GlyRα1^del, involves truncated cytoplasmic region between transmenbrane domain (TM)3 and TM4, and the truncation is contributed by an alternative start site of exon 9. Flag-tagged GlyRα1^del, transfected and overexpressed in COS-7 cells, revealed an about 44 kDa immunoreactive band by immunoblotting. Immunofluorescence analysis showed that it seemed to localize at cell surface as similarly as GlyRα1. We further transfected HEK293 cells with GlyRα1 or GlyRα1^del, and currents activated by glycine were examined using the whole-cell patch-clamp recording technique. Maximal currents (holding potential at -45 mV) and I-V curves for GlyRα1^del showed no clear difference from that for GlyRα1. We also examined the property on the glycine dose-response curve. In the cells with GlyRα1^del, EC₅₀ and Hill coefficient for the glycine current were 71.5 ± 3.4 μM (mean ± SEM) and 2.8 ± 0.1, respectively. By contrast, those with GlyRα1 were 43.3 ± 7.5 μM and 2.7 ± 0.2, respectively. Further experiments are in progress to evaluate the functional characterization of GlyRα1^del. [Jpn J Physiol 54 Suppl:S138 (2004)]

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Long-term administration of histamine H₂ receptor (H₂R) antagonist (inverse agonist) induces up-regulation of the H₂R, which may be relevant to rebound hypersecretion of gastric acid after withdrawing the treatment. However the underlying mechanisms remains to be elucidated. To clarify the mechanisms of antagonist-induced up-regulation of H₂R, human H₂R-GFP fusion protein (H₂R-GFP) was generated, functionally expressed in HEK293 cells, and used for the experiments. Binding affinity of [³H]tiotidine was not significantly different between H₂R-GFP and wild-type H₂R expressed in HEK293 cells. Visualization of H₂R-GFP revealed that antagonist induced recycling of both the agonist-induced internalized H₂R and the constitutively internalized H₂R, localizing in recycling endosome, within 2 hours. Persistent treatment with antagonist for at least 3 hours induced up-regulation of H₂R-GFP estimated by binding assay, and also induced the increase in GFP fluorescence in plasma membrane under a microscope. During the treatment with cimetidine, an H₂R antagonist, H₂R mRNA was not augmented, and after its pretreatment, the inhibition of protein synthesis with cycloheximide had no effect on maintaining H₂R up-regulation. These findings suggested that, upon antagonist (inverse agonist) exposure, alteration of the intracellular cycling between receptor endocytosis and recycling precedes the H₂R up-regulation, probably with suppressing H₂R degradation. [Jpn J Physiol 54 Suppl:S138 (2004)]

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Locus coeruleus (LC) nucleus and mesencephalic trigeminal nucleus (MesV) are innervated by orexin-like immunoreactive axons, and neurons in these nuclei express orexin receptors (OXRs), OX₁R or OX₂R, respectively (Greco, M.A. et al., 2001). We analyzed effects of ORX-A/B on dissociated LC or MesV neurons by using whole-cell patch clamp techniques. In current-clamp mode about 50% of both LC and MesV neurons were depolarized by application of 10⁻⁷ M ORX-A (3-10 mV), while ORX-B (10⁻⁷ M) depolarized about 40% LC or MesV neurons (3-5 mV). LC neurons normally showed a rapid increase in a firing frequency (from 0.2 Hz to 1.0 Hz) and returned to control levels soon after removal of ORX-A/B. These effects were maintained in the presence of TTX, CNQX, AP5 or bicucullin. It indicates direct effect of ORX-A/B on these neurons. MesV neurons rarely fired spontaneously, even either in a current-applied depolarizing condition, but showed the membrane depolarization and the firing of action potentials in an orexin application. In voltage clamp analysis, we demonstrated that ORX-A/B enhanced a non-selective cationic conductance (NSCC) and inhibited conductance of the sustained K⁺ current (I_K). The activation of an NSCC and inhibition of I_K mediate the depolarization and broadening of the action potentials caused by ORX-A/B in both LC and MesV neurons. [Jpn J Physiol 54 Suppl:S139 (2004)]

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AMPA receptors (AMPARs) mediate fast synaptic transmission at the calyx of Held. At the postnatal day 7 (P7) EPSCs are characterized with slow decay time and strong paired-pulse depression (PPD), but the decay time becomes faster and the magnitude of PPD decreases as animals mature. Using fast glutamate application to outside-out patches excised from postsynaptic MNTB neurons, we examined whether the desensitization and deactivation of postsynaptic AMPARs might contribute to these developmental changes. Throughout development (P6-21) the decay time constant of miniature (m) EPSCs was similar to that of AMPAR deactivation. Cyclothiazide (CTZ) almost abolished AMPAR desensitization, and prolonged AMPAR deactivation. CTZ also prolonged mEPSC decay time, with the magnitude of prolongation being inexplicable simply by that of AMPAR deactivation, particularly at the early postnatal period. CTZ also abolished PPD of glutamate-induced AMPAR currents, and reduced the magnitude of synaptic PPD at P7, but not after P14. In the single cell RT-PCR analysis the abundance of GluR4 flop subunit showed a positive correlation with the mEPSC decay time at P7, but not after P14. We conclude that postsynaptic AMPAR desensitization contributes to the decay time of EPSCs and synaptic depression at the early period, but its contributions become less as animals mature. [Jpn J Physiol 54 Suppl:S139 (2004)]

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The ionotropic glutamate receptor mediate fast neuronal transmission in vertebrate central nerve system. Although several models have been proposed, the subunit stoichiometry and symmetry of ionotropic glutamate receptors (iGluRs) remain unclear. The recent theory that iGluR consists of two dimers in a tetramer formation raised the possibility that the iGluR have twofold symmetric structure, in contrast to fourfold symmetric structure of potassium channels. We have developed a new approach to investigate the conformation of functional channels. The N-terminal extracellular LIVBP domain of GluR1 subtype of iGluR was replaced by leucine zipper peptides designed to form stable symmetric dimers, trimers, tetramers or pentamers. Although the LIVBP domain has been proposed to form initial dimers, channel function was only restored by the tetramer-forming peptide. Furthermore, engineered metal bridge method, which measures the distance between cysteine residues, indicated that the channel domain adopts fourfold symmetric tetramer in functional GluR1 channels. Therefore, we propose that GluR1 assembles as a fourfold symmetric tetramer, and the LIVBP domain plays an important role in achieving this conformation. [Jpn J Physiol 54 Suppl:S139 (2004)]

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Recently, we reported that the expression of Ca²⁺ binding proteins, calbindin-D28k (CB) and parvalbumin correlates with the expression of Ca²⁺ permeable AMPA receptors(AMPA-Rs) in rat forebrain. We analyzed cell surface expression of a Ca²⁺ impermeable subunit, GluR2 and a Ca²⁺ permeable subunit, GluR2Q using an adenoviral-mediated expression system in COS7 cells. Biochemical measurements of cell surface expressed subunits using biotinylation experiments revealed that cell surface expression of GluR2Q is significantly increased with CB coexpression, while that of GluR2 is not. By application of AMPA-R blockers, cell surface expression of GluR2Q is increased in the absence of CB coexpression. This result suggests that the activity of AMPA-Rs formed by GluR2Q subunits suppresses cell surface expression of GluR2Q. To determine whether Ca²⁺ entry via Ca²⁺ permeable AMPA receptor causes the suppression, we used the Ca²⁺ chelater, BAPTA-AM. Application of BAPTA-AM increases cell surface expression of GluR2Q. These results suggest that CB enhances cell surface expression of Ca²⁺ permeable AMPA-Rs by buffering the increased intracellular Ca²⁺ that enters through Ca²⁺ permeable AMPA-Rs. [Jpn J Physiol 54 Suppl:S139 (2004)]

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The rat pheochromocytoma (PC12) cells express the NMDAR1 subunit (NR1) mRNA endogenously, but only a small amount of NR1 protein. Furthermore, expression of functional NMDA receptors (NMDARs) is negligible. We reported previously that the adenoviral-mediated delivery of NMDAR2 (NR2) cDNAs not only induced the expression of functional NMDARs, but also increased the amount of NR1 protein in PC12 cells. Interestingly, the delivery of NR2C was much less prominent than that of NR2B for these effects. The C-terminal cytoplasmic domain of NR2 is known to interact with the scaffolding proteins in the plasma membrane and to play a critical role in the trafficking of NMDARs. To study the role of the C-terminal domain of NR2 for expression of NMDARs, we constructed a chimeric NR2, in which 135 amino acid residues of the C-terminus of NR2C was replaced with 273 residues of the C-terminus of NR2B. The delivery of this chimera cDNA (NR2C₁₁₁₅B) increased NMDAR-mediated currents to 5.8-fold of that of the wild NR2C. It also increased the amount of NR1 protein to 2.8-fold. However, these effects of delivery of the chimera cDNA were still much smaller than those of the wild NR2B cDNA in terms of expression of both functional NMDARs and NR1 protein, indicating that other unknown factors are also involved in the regulation of NMDAR expression. [Jpn J Physiol 54 Suppl:S140 (2004)]

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The dimeric conformation of the G protein-coupled metabotropic glutamate receptor 1α (mGluR1α), which is a key determinant of the downstream signaling from the receptor, is crucial for some forms of synaptic plasticity. Moreover, crystallographic analysis has shown that glutamate and the antagonist (S)-MCPG respectively stabilize the active and resting states of the dimeric conformation of the extracellular domain. In the present study, we investigated the dimeric rearrangement of the cytoplasmic domain caused by ligand binding by analyzing the fluorescence resonance energy transfer (FRET) signal obtained from the plasma membrane, where ligands interact with the mGluR1α, using a total internal reflection field microscopy system. Intermolecular FRET efficiency was altered within 9 s after glutamate application, and the effect was concentration-dependent. (S)-MCPG inhibited the change by shifting the concentration-FRET efficiency relationship rightward. Alterations in FRET efficiency were also elicited by Ca²⁺ and Gd³⁺, which are known to activate mGluR1α. By contrast, intramolecular FRET efficiency was unaffected by glutamate. We therefore conclude that ligand-induced rearrangement of the extracellular dimeric conformation leads to intracellular dimeric rearrangement without altering the respective monomeric conformations. [Jpn J Physiol 54 Suppl:S140 (2004)]

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Using the optical recordings, we studied the role of non-NMDA and NMDA receptors in the hypoxia-induced increases of [Ca2+]i in the superior colliculus (SC) in a fetal rat, which was still connected with the dam by the umbilical cord. The changes of [Ca2+]i in the SC were recorded as fractional changes in the intensity of fluorescent dye (Fura-2) by an optical recording system. Hypoxia was caused by occlusion of the umbilical cord with a small clip. Focal stimulation of the SC was made by a single and train pulse at currents of 0.5-3 mA via a bipolar electrode inserted into the SC. The umbilical cord occlusion caused marked increases in [Ca2+]i. After the pretreatment of CNQX and AP-5, the occlusion-induced increase in [Ca2+]i was remarkably reduced. Picrotoxin enhanced the occlusion-induced increase in [Ca2+]i. Focal SC stimulation induced increases in [Ca2+]i of the SC regions. This response almost completely disappeared during umbilical cord occlusion. At 3 hrs after reperfusion of umbilical blood flow, the SC stimulation-induced increases in [Ca2+]i remained markedly attenuated. These small [Ca2+]i responses were antagonized by CNQX but not by AP-5. These results suggest that hypoxia could alter the function of NMDA receptors in fetal SC neurons, which may be associated with an innate hypoxia tolerance in the fetal brain. [Jpn J Physiol 54 Suppl:S140 (2004)]

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Rat cultured microglia express AMPA (α-amino-hydroxy-5-methyl-isoxazole-4-propionate)-type of glutamate receptors. Here we examined the functional change of glutamate-induced currents before and after activation of microglia with lipopolysaccharide (LPS), using the conventional whole-cell patch clamp technique under the voltage-clamp condition. Glutamate-induced currents at the holding potential of -60 mV were observed in the presence of 100 µM cyclothiazide (CTZ), an inhibitor of desensitization. After treatment of microglia with 10 µg/ml LPS for 30 minutes, the cells showed ameboidal morphology and glutamate-induced currents were significantly inhibited. Immunocytochemistry using specific antibody against GluR2 showed that the subunit was translocated from cytoplasm to cell membrane after activation of microglia with LPS. These results suggest that membrane translocation of GluR2 may contribute to the inhibition of glutamate-induced currents in activated microglia. The results also indicate that translocation of GluR2 is not restricted to neurons but also occurs in microglia. The functional change of microglia through AMPA receptors may contribute to the neuron-microglia interaction under the pathophysiological conditions. [Jpn J Physiol 54 Suppl:S140 (2004)]

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Astrocytes play an essential role in the two-way glial-neuronal communication by forming an elaborate network with neurons. Glutamate is known to be a key mediator for the glia-neuron interaction. Recent studies showed that significant permeability of some anion channels to organic molecules, such as taurine, glutamate and ATP. Thus, there is a possibility that glutamate release from glial cells is mediated by some anion channels. An enzymatic assay showed that release of glutamate from mouse astrocytes in primary culture was small under normal conditions but greatly increased upon a hypotonic challenge or under chemical ischemia. Patch-clamp experiments showed that both maneuvers activated maxi-anion channels in cell-attached patches. This channel was conductive to glutamate with a permeability ratio of P_Glu/P_Cl=0.20±0.01. Sensitivity of glutamate release to NPPB, SITS, arachidonate and Gd³⁺ was very similar to that of glutamate-permeable maxi-anion channel. Neither blocker of gap-junction hemi-channels (carbenoxolone or 1-octanol) nor inhibitor of exocytosis (brefeldin A) affected glutamate release from astrocytes. Thus, our data suggest that the maxi-anion channel serves as a major pathway for glutamate release under hypotonic or ischemic conditions. [Jpn J Physiol 54 Suppl:S141 (2004)]

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Nucleus tractus solitarius (NTS) plays a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. This nucleus receives primary afferent input from a wide variety of peripheral organs and tissues and is essential in integration of autonomic nervous system functions. Glutamate is one of the principal neurotransmitters of the afferents in the NTS. Indeed, it was well established that glutamate receptor exert a role in controlling the excitability of the NTS. Voltage-dependent calcium channels (VDCCs) serve as crucial mediators of membrane excitability and calcium-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The modulation of VDCCs is believed to be an important means of regulating calcium entry and thus has a direct influence on many calcium-dependent processes. This study investigates the modulation of the VDCCs current (ICa) using patch-clamp recording methods. Application of (RS)-3,5-Dihydroxyphenylglycine (DHPG, Group one mGluR agonist) caused both inhibition and facilitation of ICa. In addition, application of L-(+)-2-amino-4-phosphonobutyric acid (AP-4, Group three mGluR agonist) caused facilitation of ICa. Neither (2S, 2'R, 3'R)-2-(2', 3'-dicarboxycyclopropyl)glycine (DCG, Group two mGluR agonist) nor (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, mGluR5 agonist) modulated ICa. [Jpn J Physiol 54 Suppl:S141 (2004)]

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Long-term depression of parallel fiber (PF) EPSC in Purkinje cell is considered as cellular basis for motor learning in the cerebellar cortex. This depression of PF-EPSC is caused by reduction in number of AMPA-receptor (AMPA-R) expressed at the postsynaptic membrane of PF-synapse by internalization of AMPA-R, which is suggested to be mediated through endocytosis in clathrin - dynamin dependent manner. Under normal condition, we found that AMPA-R surface expression was regulated by equilibrium between insertion and internalization of AMPA-R through constitutive exo- and endocytosis. For insertion of AMPA-R into PF-synapse membrane, NSF, an ATPase, is possibly involved in constitutive exocytosis since NSF-binding domain is reported in C-terminus of GluR2. To examine whether NSF is involved in constitutive exocytosis of AMPA-R in PF-synapse of cerebellar Purkinje cell, peptide of GluR2 C-terminal region (844-853), which was reported to block binding of NSF and GluR2 C-terminus region, applied through whole-cell patch-pipette to Purkinje cell in rat cerebellar slice. Results showed that this peptide (1mM) enhanced amplitude of PF-EPSC. NSF was suggested to be not involved in GluR2 insertion into PF-synapse through constitutive exocytosis. Since tetanus toxin (TeTX), a blocker of VAMP-mediated exocytosis, suppressed enhancement of PF-EPSC by this peptide, this peptide is suggested to suppress constitutive internalization, rather than constitutive insertion of GluR2 in PF-synapse. Binding of GluR2 and protein essential for internalization of GluR2 may be interfered by this peptide. [Jpn J Physiol 54 Suppl:S141 (2004)]

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The ionotropic glutamate receptor delta2 subunit (GluRdelta2), which is selectively expressed at a parallel fiber-Purkinje neuron (pf-PN) synapse, plays critical roles in the long-term depression and formation of synaptic connections in the cerebellum. Previous study on the mutant mice deficient in GluRdelta2 (delta2 mice) showed that the long-term depression of pf-PN synaptic transmission is impaired, a PN is innervated by multiple climbing fibers and the frequency of climbing fiber input is increased. Here, we examined the GABAergic synaptic inhibition on a PN in the delta2 mice. We found pronounced enhancement of excitation propagation by application of bicuculline (a GABA(A) receptor antagonist) in the molecular layer of cerebellar slice preparation prepared from a delta2 mouse, suggesting the enhanced GABAergic inhibition in the mutant mouse. Whole-cell patch clamp experiments on PNs in slice preparations revealed that amplitudes of miniature IPSCs (mIPSCs) were larger in delta2 mice than in wild type mice. We also found that depolarization induced potentiation (rebound potentiation, RP) was not induced in slices prepared from delta2 mice. These results suggest that enhancement of climbing fiber inputs induces and saturates RP in delta2 mice. [Jpn J Physiol 54 Suppl:S142 (2004)]

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At the GABAergic synapses between an inhibitory interneuron and a Purkinje neuron (PN) in the cerebellum, postsynaptic depolarization results in long-lasting potentiation of the transmission efficacy (rebound potentiation: RP). We previously demonstrated that presynaptic activation coupled with postsynaptic depolarization suppresses the induction of RP through activation of postsynaptic GABA_B receptors. Here we show that the suppressive effect of GABA_BR activation during depolarization of PNs on the RP induction is not transient but sustained for more than 4 days through augmentation of the integrin-mediated intracellular signaling. Five minutes conditioning treatment of cultured cerebellar neurons with the solution containing 50 mM of KCl and baclofen, a selective GABA_BR agonist, suppressed the RP induction for more than 72 hours. This long-lasting suppression of RP (LSRP) was blocked either by inhibition of de novo transcription using actinomycin D or by inhibition of MAPK cascades with either U0126 or PD98059 during the conditioning treatment. Thus, the establishment of LSRP depends on the de novo transcription and MAPK cascades. LSPR was abolished by inhibition of integrins using GRGDSP peptide, a competitive inhibitor of integrin binding to ligand proteins. Function blocking antibody against either integrin α3 or β1 subunits also abolished the LSRP, suggesting that the LSRP is mediated by the integrin α3/β1 heterodimer. We also present data suggesting that src-family of protein tyrosine kinases mediates the LSRP downstream of integrins. [Jpn J Physiol 54 Suppl:S142 (2004)]

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Activity dependent regulation of AMPA receptor (AMPA-R) trafficking is one of the underlying bases of synaptic plasticity in hippocampus. However, detailed characteristics of AMPA-R trafficking has not yet been revealed in cerebellum. We addressed following issues: whether or not reduction mechanisms for AMPA-R activity through constitutive endocytosis share common features with those for long-term depression (LTD) in parallel fiber (PF) synapse in cerebellar Purkinje cell. Amplitude of PF-evoked EPSCs was reduced to 50-60% through constitutive endocytosis of AMPA-R, shown by intracellular application of Tetanus toxin (TeTX). Chelerythrine (10μM), a protein kinase C (PKC) inhibitor, suppressed TeTX-induced reduction in PF-EPSC amplitude. However, BAPTA (20mM) had no effect on this reduction in PF-EPSC amplitude. These results suggest that background activity of PKC is enough for constitutive endocytosis of AMPA-R. Furthermore, constitutive endocytosis is suggested to be independent of intracellular Ca²⁺ concentration. [Jpn J Physiol 54 Suppl:S142 (2004)]

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Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuring spastic paralysis. Although polysialogangliosides act as components of the neurontoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT. We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP. Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje Cell preparation with PIPLC. In addition, treatment of cultured mouse neuronal cells with filipin, which disrupt clustering of GPI-anchored proteins in lipid refts, prevented intraneuronal VAMP cleavage by TeNT. Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for neurotoxin. [Jpn J Physiol 54 Suppl:S142 (2004)]

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Virus-mediated gene transfer to neuronal cells is a powerful technique to study brain functions and to treat congenital and neurodegenerative diseases. However, adenovirus that is conventionally used for gene transfer to neurons works little to transduce cerebellar Purkinje cells. Here we evaluate potential of two other viruses, lentivirus and adeno-associated virus (AAV) to transduce cultured Purkinje cells. Addition of recombinant GFP-expressing lentivirus into the culture medium caused non-specific transduction of the cultured cells including Purkinje cells and glial cells. In contrast, AAV-mediated GFP expression was mostly confined to Purkinje cells. In both viruses, the expression of GFP in Purkinje cells occurred two weeks after infection and lasted as long as the culture was healthy. These data, thus suggest that gene transfer to Purkinje cells using lentivirus or AAV is a promising technique to study the pathophysiology of the cerebellum. [Jpn J Physiol 54 Suppl:S143 (2004)]

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GABA^*(C) receptors have been identified in vertebrate retina and visual neurons. These novel GABA(C) receptors are Cl⁻ pores and have pharmacological properties distinct from those of GABA(A) receptors, namely, resistance to bicuculline and selective activation by cis-4-aminocrotonic acid (CACA). Interestingly, mRNAs of the GABA(C) receptors have also been found in cerebellar Purkinje neurons of the rat (Boue-Grabot et al, 1998: Rozzo et al, 2002). Using brain slices and patch-clamp techniques (J Neurophysiol 90, 3490-3500, 2003), the author has characterized whole-cell membrane currents induced by a specific GABA(C) agonist, CACA, in Purkinje neurons of the rat. CACA (100 microM)-induced currents showed relatively small amplitudes, slow rise times and equilibrium potential similar to those of Cl ions. The dose-response relation of CACA showed no saturation even at 400 microM. These properties suggest that the receptors involved are those of GABA(C) subtype, consistent with previous findings of the mRNAs present in Purkinje neurons. However, since the specificities of the agonists as well as the antagonists to GABA(C) receptors seem to be weaker than previously thought (ex; Wall, 2001), more detailed analyses of GABA(C) receptors in cerebellar neurons are needed. Also, properties of BDNF^**-induced rapid currents , as have recently been reported (Blum et al, 2002), in cerebellar neurons will be presented. (*GABA= γ-aminobutyric acid;　**BDNF=brain-derived neurotrophic factor) [Jpn J Physiol 54 Suppl:S143 (2004)]

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To elucidate chronic actions of brain-derived neurotrophic factor (BDNF) on GABAergic synapses, we examined effects of a long-term application (for 10-15 days) of BDNF on synaptic activity of solitary GABAergic neurons cultured on glial microislands. Evoked and miniature inhibitory postsynaptic currents (IPSCs) were recorded in the whole-cell voltage-clamp mode. The application of BDNF at 100 ng/ml enhanced the amplitude of evoked IPSCs and increased the frequency of miniature IPSCs. Such actions of BDNF were blocked by anti-TrkB antibody. In contrast, BDNF did not have a detectable effect on the amplitude of miniature IPSCs and the paired pulse ratio of IPSCs evoked by paired shocks. To evaluate morphological changes, neurons were immunocytochemically stained with anti- microtubule-associated protein 2 antibodies, to visualize somatodendritic region and anti-synapsin I antibodies, to visualize presynaptic sites. A quantitative morphological analysis indicated that BDNF increased the number of dendritic branches and synaptic sites. To visualize functionally active presynaptic sites, neurons were stained with a styryl dye, FM1-43. BDNF increased the number of FM1-43-positive sites that was correlated with the frequency of miniature IPSCs. These results suggest that the chronic treatment with BDNF strengthens GABAergic synaptic transmission. [Jpn J Physiol 54 Suppl:S143 (2004)]

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Brain-derived neurotrophic factor (BDNF) is known to regulate synaptic transmission and plasticity. An acute application of BDNF to visual cortical and hippocampal slices is reported to enhance excitatory synaptic transmission when evaluated by either field potentials or single-cell excitatory postsynaptic currents (EPSCs). As field potentials mostly reflect activities of pyramidal neurons and EPSCs were recorded from pyramidal cell-like neurons, an important question of how BDNF acts acutely on excitatory synaptic transmission to GABAergic neurons has not been answered yet. To address this question, we used visual cortical slices prepared from glutamic acid decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mice. In these slices, GABAergic neurons containing GAD67, GABA synthesizing enzyme, can easily be detected under a fluorescence microscope. So we could record EPSCs evoked by layer IV stimulation from GABAergic neurons in layer II/III of the cortex through whole-cell patch-clamp electrodes. We found that BDNF quickly depressed EPSCs in most GABAergic neurons and this depression lasted after cessation of the BDNF application. Questions of whether two subtypes of GABAergic neurons, parvalbumin-positive and -negative neurons, are affected in the same way, and the depression is pre- or postsynaptic in origin, are under investigation. [Jpn J Physiol 54 Suppl:S143 (2004)]

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Cell-substrate contact formation is crucial for growth cone motility. By monitoring cell-substrate distance by reflection interference contrast microscopy, we found that growth cone maintained stable contacts at its central domain. In contrast, at the peripheral domain, we observed membrane ruffling with transient contact formation. We previously reported that β1 integrin, one of adhesive molecules, is incorporated into the basal membrane of growth cones by exocytosis. However, the relation between integrin incorporation and cell-substrate contact formation is not clear. To elucidate this, we monitored exocytic incorporation of fluorescent-labeled β1 integrin and contact formation in the growth cone. To observe exocytic events, we employed VAMP-pHluorin as a fluorescent probe to monitor intravesicular pH changes during exocytosis. Time lapse imaging showed that β1 integrin was incorporated into a restricted area where the growth cone formed stable contacts. At the peripheral domain, in contrast, exocytic event was not observed. In addition, approx. 50% of incorporated β1 integrins at the central domain went away from their original position. These results suggest that a half of incorporated β1 integrin contributes to form stable contacts at the central domain and that the other half could move to peripheral domain being involved in transient contacts formation. [Jpn J Physiol 54 Suppl:S144 (2004)]

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Adhesive contact formation at growth cones is essential for neurite extension. It has been suggested that incorporation of cell adhesion molecules to the cell membrane is mediated by exocytosis of vesicles that contained molecules for cell adhesion, such as integrin. Rat hippocampal neurons were cultured for 1 to 3 days until they extended neurites with growth cones. Exocytosis of vesicles was monitored with FM4-64, a fluorescent marker for exocytosis, and adhesive contact formation by reflection interference contrast microscopy (RICM). High K⁺ solution (60mM or 120mM) was applied to the growth cone through a glass pipette to induce exocytosis at the growth cone. The number of FM4-64 fluorescence spots increased remarkably during the high K⁺ stimulation. These FM4-64 fluorescence spots disappeared within several seconds, denoting the exocytosis of the FM4-64 containing vesicle. These bright spots were localized at cell adhesion sites that were imaged by RICM. The area of the cell adhesion increased gradually during the stimulation, suggesting close correlation between the vesicle exocytosis and adhesive contact formation at the growth cone. Furthermore immunostaining of integrin and synapsin showed that these molecules are colocalized in the vesicle at the growth cone. These results suggest that vesicles containing synapsin and integrin are transported to growth cones and integrin is incorporated into the basal cell membrane by exocytosis. [Jpn J Physiol 54 Suppl:S144 (2004)]

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Neural activities trigger an increase in local cerebral blood flow (CBF). However, it is unclear how this "coupling" is mediated. Recently, it is proposed that prostanoids and nitric oxide (NO) may serve as vasodilators in the activity-dependent changes in CBF. We investigated this hypothesis with functional brain imaging using flavoprotein autofluorescence. We anesthetized C57BL/6N mice with urethane (1.7 g/kg, i.p) and exposed the skull. The surface of the skull was covered with clear acrylic resin for preventing drying and keeping the transparency of the skull. Cortical images of green autofluorescence (500-550 nm) in blue light (450-490 nm) were recorded through the intact skull at 9 Hz by a cooled CCD camera system attached to an epifluorescence microscope. Neural activities in the primary somatosensory cortex were elicited by vibratory stimulation at 50 Hz for 1 s applied on the contralateral hind paw skin. An activity-dependent increase in the autofluorescence was observed and followed by darkening of the arterial images reflecting vasodilation. Indomethacin (10 mg/kg, i.p) weakly attenuated the arterial darkening without changing neural responses. L-NAME (30 mg/kg, i.p), a nonspecific NOS inhibitor, did not modify neural responses and the arterial darkening by itself. However, co-application of indomethacin and L-NAME almost completely eliminated the arterial darkening, and activity-dependent vasoconstriction was observed instead. These data suggest that prostanoids and NO mediate activity dependent changes in CBF. [Jpn J Physiol 54 Suppl:S144 (2004)]

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We investigated neural plasticity in the rat primary somatosensory cortex after somatosensory frequency discrimination learning with flavoprotein autofluorescence imaging. A water-deprived rat was trained to discriminate the frequency (20 Hz or 40 z) of floor vibration in a Skinner box. Licking a spout only during the floor vibration at either 20 Hz or 40 Hz (S+) was rewarded with water in each rat, while that in response to 40 Hz or 20 Hz stimulation (S-) was not. The rat quickly learned to discriminate between S+ and S- by suppressing behavioral responses to S-. After this learning was achieved in consecutive 3-5 days, the rat was anesthetized with urethane (1.5 g/kg, i.p.), and somatosensory neural activities in response to vibratory skin stimulation at 20 Hz or 40 Hz applied on the contralateral left plantar hindpaw was recorded. Responses were visualized with flavoprotein autofluorescence (500-550 nm) in blue light (450-490 nm) using a cooled CCD camera attached to an epifluorescence binocular microscope. The neural responses to S- were smaller than those to S+ in all of the 8 rats tested. The responses to S- in learned rats were significantly smaller that those in naive rats, while those to S+ were similar to the responses in naive rats. These results suggest that the plastic changes in the somatosensory cortex (suppression of the somatosensory responses to S-) may be a part of essential neural mechanisms underlying the frequency discrimination learning with suppressed behavioral responses to S-. [Jpn J Physiol 54 Suppl:S144 (2004)]

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