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Biomedical subjects

Y Kurachi

Publications and source records attributed to Y Kurachi.

At least 55 records · Page 3Linked to original sources
PubMed1998

The expression of late infantile neuronal ceroid lipofuscinosis (CLN2) gene product in human brains.

We raised polyclonal antibodies against a gene product responsible for late infantile neuronal ceroid lipofuscinosis (CLN2). By Western blotting, all three antisera recognized the CLN2 protein at approximately 49 kDa in human brain homogenates. Immunohistochemistry using the antisera demonstrated the granular labelling in the cytoplasm of cerebral neurons and glial cells. The immunoreactivity on Western blots was absent from the brain of a patient with CLN2. Our results suggest the usefulness of these antibodies for the diagnosis of CLN2, which currently requires demonstration of characteristic ultrastructure by electron microscopy.

Adolescent↗
PubMed1998

Intracellular nucleotide-mediated gating of SUR/Kir6.0 complex potassium channels expressed in a mammalian cell line and its modification by pinacidil.

1. We have examined the properties of intracellular nucleotide-mediated gating of K+ channel constructs composed of the sulphonylurea receptor 2B and the inwardly rectifying K+ channel subunits Kir6.1 and Kir6.2 (SUR2B/Kir6.1 and SUR2B/Kir6.2 complex K+ channels) heterologously expressed in human embryonic kidney (HEK) 293T cells. In the cell-attached form, both types of K+ channel were activated by pinacidil. 2. In inside-out (IO) patches, the SUR2B/Kir6.2 channels opened spontaneously and were inhibited by intracellular ATP (ATPi). Pinacidil attenuated the ATPi-mediated channel inhibition in a concentration-dependent manner. In contrast, the SUR2B/Kir6.1 channels required intracellular nucleoside di- or tri-, but not mono-, phosphates for opening. The potency of adenine, guanine or uracil nucleotides to activate SUR2B/Kir6.1 channels was enhanced by pinacidil. 3. In the presence of pinacidil, adenine and guanine, but not uracil, nucleotides exhibited bell-shaped concentration-dependent activating effects on SUR2B/Kir6.1 channels. This was due to channel inhibition caused by adenine and guanine nucleotides, which was unaffected by pinacidil. 4. From power density spectrum analysis of SUR2B/Kir6.1 currents, channel activation could be described by the product of two gates, a nucleotide-independent fast channel gate and a nucleotide-dependent slow gate, which controlled the number of functional channels. Pinacidil specifically increased the potency of nucleotide action on the slow gate. 5. We conclude that Kir6.0 subunits play a crucial role in the nucleotide-mediated gating of SUR/Kir6.0 complex K+ channels and may determine the molecular mode of pinacidil action.

Adenosine Diphosphate↗
PubMed1998

Cloning and functional expression of a novel cardiac two-pore background K+ channel (cTBAK-1).

We have cloned from a mouse heart cDNA library a novel K+ channel subunit that has two pore-forming domains and four transmembrane regions. Its amino acid sequence shares 25% identity with mouse TWIK-1, 22% with mouse TREK-1, and 33% with a putative K+ channel of Caenorhabditis elegans (C40C9). Strikingly abundant mRNA for this clone was expressed in the heart. The mRNA was also detected in kidney, brain, skin, testis, lung, skeletal muscle, small intestine, and stomach but not in liver, thymus, or spleen. Reverse transcription-polymerase chain reaction analyses of single cells showed that the mRNA of the clone was expressed in both atrial and ventricular myocytes per se. Xenopus oocytes injected with the cRNA of the clone expressed a Ba2+-sensitive K+-selective current with an almost linear steady-state current-voltage relationship. In cell-attached patches, the expressed channel exhibited short-lasting openings with a mean open time of approximately 2 milliseconds and a unitary conductance of approximately 16 pS (150 mmol/L [K+]o). The K+ current was insensitive to intracellular Na+ (50 mmol/L), Ca2+ (0.1 mmol/L), H+ (pH 6.4), and arachidonic acid (10 micromol/L) in inside-out patches. Thus, the current flowing through the channel may contribute to the cardiac cellular electrical activity as a linear background K+ conductance. Therefore, we designated the clone cTBAK (cardiac two-pore background K+ channel).

Amino Acid Sequence↗
PubMed1998

A novel junction-like membrane complex in the optic nerve astrocyte of the Japanese macaque with a possible relation to a potassium ion channel.

BACKGROUND: A new type of junction-like membrane complex (JMC) was detected between adjacent astrocytes in the optic nerve of Japanese macaque (macaca fuscata). This membrane complex morphologically resembled a cell junction, but a possible role for potassium ion channels could not be denied based on freeze-fracture replica observation. We attempted to determine the chemical nature and function of the novel JMC. METHODS: Using an electron microscope, we observed JMCs in the optic nerve astrocyte. In addition, we observed them using a freeze-fracture replica and immunohistochemistry with connexin 43, a gap junction specific protein. Furthermore, immunolocalization of an inwardly rectifying potassium ion channel, K(AB)-2 (Kir4.1), was studied with a confocal laser-scanning microscope, and an electron microscope using a newly developed pre-embedding method. RESULTS: These JMCs were abundant around the blood vessel in the area just behind the lamina cribrosa. At JMCs the inner leaflet was thicker than the outer leaflet and electron-dense materials were packed in the intercellular space. Freeze-fracture replica observation revealed orthogonal arrays of particles, probably at the place of JMCs, that have been considered a potassium ion channel. No connexin 43 immunoreactivity was detected in JMCs, while K(AB)-2 was mostly localized on either side of the opposing cell membranes of JMC. CONCLUSIONS: These JMCs do not seem to be a simple junction, but relate to a potassium ion channel. The area just behind the lamina cribrosa may be important in terms of conductance of the optic nerve impulse.

Amino Acid Sequence↗
PubMed1998

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells.

The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (KATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a Ki value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATPi) equipotently in the presence and in the absence of intracellular Mg2+ (Mg2+i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATPi only in the presence of Mg2+i. Uridine 5'-diphosphate (UDP) antagonized the ATPi-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATPi-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac KATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the KATP channel.

ATP-Binding Cassette Transporters↗
PubMed1998

Changes in bite force and occlusal contacts in patients treated for mandibular prognathism by orthognathic surgery.

PURPOSE: The purpose of this study was to evaluate changes in bite force and occlusal contacts before and after orthognathic surgery in patients with mandibular prognathism and to compare the findings with those in controls with normal occlusion. PATIENTS AND METHODS: Bite force and occlusal contacts were analyzed in 23 (7 male and 16 female) patients with mandibular prognathism before and after sagittal split ramus osteotomy, and in 20 (10 male and 10 female) controls with normal occlusion. The bite force and occlusal contacts were simultaneously measured by a computerized occlusal analysis system, the T-Scan system, immediately before surgery, and at 6 weeks, 3 months, 6 months, and 1 year postoperatively. RESULTS: Both the bite force and occlusal contacts in the patients were significantly less than those in the controls before surgery. Although both the bite force and occlusal contacts in the patients were improved by the orthognathic surgery, neither approached the level in the controls within 1 year. Bite force was correlated with the number of occlusal contacts in both patient and control groups. CONCLUSION: The postoperative masticatory function does not reach control levels even 1 year after the orthognathic surgery for mandibular prognathism. Therefore, further adjustment of the occlusion should be considered before the end of treatment.

Adult↗
PubMed1998

SUR2 subtype (A and B)-dependent differential activation of the cloned ATP-sensitive K+ channels by pinacidil and nicorandil.

1. The classical ATP sensitive K+ (K(ATP)) channels are composed of a sulphonylurea receptor (SUR) and an inward rectifying K+ channel subunit (BIR/Kir6.2). They are the targets of vasorelaxant agents called K+ channel openers, such as pinacidil and nicorandil. 2. In order to examine the tissue selectivity of pinacidil and nicorandil, in vitro, we compared the effects of these agents on cardiac type (SUR2A/Kir6.2) and vascular smooth muscle type (SUR2B/Kir6.2) of the K(ATP) channels heterologously expressed in HEK293T cells, a human embryonic kidney cell line, by using the patch-clamp method. 3. In the cell-attached recordings (145 mM K+ in the pipette), pinacidil and nicorandil activated a weakly inwardly-rectifying, glibenclamide-sensitive 80 pS K+ channel in both the transfected cells. 4. In the whole-cell configuration, pinacidil showed a similar potency in activating the SUR2B/Kir6.2 and SUR2A/Kir6.2 channels (EC50 of approximately 2 and approximately 10 microM, respectively). On the other hand, nicorandil activated the SUR2B/Kir6.2 channel > 100 times more potently than the SUR2A/Kir6.2 (EC50 of approximately 10 microM and > 500 microM, respectively). 5. Thus, nicorandil, but not pinacidil, preferentially activates the K(ATP) channels containing SUR2B. Because SUR2A and SUR2B are diverse only in 42 amino acids at their C-terminal ends, it is strongly suggested that this short part of SUR2B may play a critical role in the action of nicorandil on the vascular type classical K(ATP) channel.

ATP-Binding Cassette Transporters↗
PubMed1998

Mg2+ and ATP dependence of K(ATP) channel modulator binding to the recombinant sulphonylurea receptor, SUR2B.

1. The binding of modulators of the ATP-sensitive K+ channel (KATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular KATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37 degrees C using the tritiated KATP channel opener, [3H]-P1075. 2. Binding of [3H]-P1075 required the presence of Mg2+ and ATP. MgATP activated binding with EC50 values of 10 and 3 microM at free Mg2+ concentrations of 3 microM and 1 mM, respectively. At 1 mM Mg2+, binding was lower than at 3 microM Mg2+. 3. [3H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg2+ concentrations of 3 microM and 1 mM, gave KD values of 1.8 and 3.4 nM and BMAX values of 876 and 698 fmol mg(-1), respectively. 4. In competition experiments, openers inhibited [3H]-P1075 binding with potencies similar to those determined in rings of rat aorta. 5. Glibenclamide inhibited [3H]-P1075 binding with Ki values of 0.35 and 2.4 microM at 3 Mm and 1 mM free Mg2+, respectively. Glibenclamide enhanced the dissociation of the [3H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas. 6. It is concluded that an MgATP site on SUR2B with microM affinity must be occupied to allow opener binding whereas Mg2+ concentrations > or = 10 microM decrease the affinities for openers and glibenclamide. The properties of the [3H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

ATP-Binding Cassette Transporters↗
PubMed1998

[ATP-sensitive K+ channels of the cardiovascular system].

ATP-sensitive K+ (KATP) channels are inhibited by intracellular ATP and activated by intracellular nucleoside diphosphates, and thus provide a link between cellular metabolism and excitability. They are widely distributed in various tissues including heart and vasculature, and thus may play essential regulatory roles in the cardiovascular system. Furthermore, KATP channels are the targets of two important classes of drugs, i.e., the antidiabetic sulfonylureas which block the channels, and a series of vasorelaxants called "K+ channel openers" which tend to maintain the channels in an open conformation. Recently, the molecular structure of KATP channels has been clarified in various tissues including cardiovascular system to be a complex of at least two subunits, i.e. SUR and Kir6.0. The KATP channels in heart and vascular smooth muscle now appear to be the complexes of SUR2A/Kir6.2 and SUR2B/Kir6.1, respectively. Further works are now in progress to understand the molecular mechanisms responsible for the control of KATP channel function by intracellular nucleotides and drugs.

Adenosine Triphosphate↗
PubMed1998

Inwardly rectifying K+ channel in retinal Müller cells: comparison with the KAB-2/Kir4.1 channel expressed in HEK293T cells.

Inwardly rectifying K+ (Kir) channels are considered to play the major role in the spatial buffering of glial cells. We have examined the electrophysiological properties of Kir channels in isolated rabbit Müller cells (retinal glial cells). Although a previous study reported that three kinds of Kir channels with different conductance and rectification properties were expressed in distinct regions of rabbit Müller cell membrane, we could record only a single population of Kir channels from the distal end to the endfoot in 205 successful cell-attached patches. The identified Müller cell Kir channel had a unitary conductance of 25 pS in the inward direction with symmetrical 153 mM K+ condition. The conductance and gating properties of the Müller cell Kir channels were identical to those of the KAB-2/Kir4.1 heterologously expressed in a mammalian cultured cell line, HEK293T cells. Thus KAB-2/Kir4.1 was the predominant glial Kir channel not only in the brain, but also in the retina. Because its rectification is intermediate, this Kir channel may contribute to both the intrusion and the extrusion of K+ ions across glial cell membrane and may be the major pathway for redistribution of extracellular K+ ions in the central nervous system.

Animals↗
PubMed1998

Identification and functional analysis of sulfonylurea receptor 1 variants in Japanese patients with NIDDM.

The sulfonylurea receptor 1 (SUR1) is an essential regulatory subunit of the beta-cell ATP-sensitive K+ channel (K[ATP]). The possible role of SUR1 gene mutation(s) in the development of NIDDM remains controversial as both a positive association and negative linkage results have been reported. Therefore, we examined the SUR1 gene at the single nucleotide level with single strand conformation polymorphism analysis in 100 Japanese NIDDM patients. We identified a total of five amino acid substitutions and 17 silent mutations by examining all 39 exons of this gene. Two rare novel mutations, D811N in exon 20 and R835C in exon 21, were identified in the first nucleotide-binding fold (NBF), a functionally important region of SUR1, in one patient each, both heterozygotes. To analyze possible functional alterations, we reconstituted the mutant K(ATP) by coexpressing beta-cell inward rectifier (BIR) (Kir 6.2), a channel subunit of K(ATP), and mutant SUR1 in HEK293T and COS-7 cells. As demonstrated by the patch clamp technique and rubidium (Rb+) efflux studies, neither mutation alters the properties of channel activities. Two other rare missense mutations, R275Q in exon 6 and V560M in exon 12, were also identified. The R275Q substitution was not found in 67 control subjects, and V560M was present in three control subjects. Neither of these substitutions appeared to cosegregate with NIDDM in the probands' families. A previously reported S1370A substitution located in the second NBF was also common in the Japanese subjects (allelic frequency 0.37), and was found at an equal frequency in nondiabetic control subjects. In conclusion, SUR1 mutations impairing K(ATP) function do not appear to be major determinants of NIDDM susceptibility in Japanese.

ATP-Binding Cassette Transporters↗
PubMed1998

Chimeras of Kir6.1 and Kir6.2 reveal structural elements involved in spontaneous opening and unitary conductance of the ATP-sensitive K+ channels.

The ATP-sensitive K+ (KATP) channels, e.g. in heart and pancreatic beta-cells, open spontaneously in the absence of intracellular ATP (ATPi). Their unitary conductance is approximately 80 pS with 150 mM extracellular K+. These features are shared by the K+ channels composed of various sulfonylurea receptors (SURs) and Kir6.2, whereas SUR/Kir6.1 channels have a smaller conductance (approximately 35 pS) and do not open spontaneously in the absence of ATPi. To identify the structural elements in Kir6.0 subunits which determine these properties, we analyzed the properties of functional K+ channels composed of SUR2A, the cardiac type SUR, and various chimeras of Kir6.1 and Kir6.2 heterologously expressed in HEK (human embryonic kidney) 293T cells. The analyses indicate that the extracellular linker domain between the two putative membrane-spanning regions is responsible for the difference in the single channel conductance between SUR2A/Kir6.1 and SUR2A/Kir6.2 channels. The cytosolic N-terminal domain of Kir6.2 was mandatory for spontaneous channel opening in the absence of ATPi, although a part of C-terminus was also involved. These results implicate specific regions of Kir6.0 in the spontaneous opening and the single channel conductance.

Adenosine Triphosphate↗
PubMed1997

Expression and clustered distribution of an inwardly rectifying potassium channel, KAB-2/Kir4.1, on mammalian retinal Müller cell membrane: their regulation by insulin and laminin signals.

Inwardly rectifying potassium (K+) channels (Kir) in Müller cells, the dominant glial cells in the retina, are supposed to be responsible for the spatial buffering action of K+ ions. The molecular properties and subcellular localization of Müller cell Kir channels in rat and rabbit retinas were examined by using electrophysiological, molecular biological, and immunostaining techniques. Only a single population of Kir channel activity, the properties of which were identical to those of KAB-2/Kir4.1 expressed in HEK293T cells, could be recorded from endfoot to the distal portion of Müller cells. Consistently, Northern blot, in situ hybridization, and RT-PCR analyses indicated expression of Kir4. 1 in Müller cells per se. The Kir4.1 immunoreactivity was distributed in clusters throughout Müller cell membrane. The Kir4.1 expression in Müller cells disappeared promptly after culturing. When the dissociated Müller cells were cultured on laminin-coated dishes in the presence of insulin, Kir4.1 immunoreactivity was detected in a clustered manner on the cell membrane. Because insulin and laminin exist in the surrounding of Müller cells in the retina, these substances possibly may be physiological regulators of expression and distribution of Kir4.1 in Müller cells in vivo.

Animals↗