研究等業績 - 原著論文 - 渡邊　博之

Molecular determinants of permeation through the cation channel TRPV4.

Thomas Voets, Jean Prenen, Joris Vriens, Hiroyuki Watanabe, Annelies Janssens, Ulrich Wissenbach, Matthias Bödding, Guy Droogmans, Bernd Nilius

The Journal of biological chemistry   277 ( 37 ) 33704 - 10   2002年09月  [査読有り]

研究論文（学術雑誌）  

We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp(672) and Asp(682), as important determinants of the Ca(2+) sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca(2+) permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp(682) but not Asp(672) strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met(680), which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca(2+) permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys(675), had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway.

PubMed

L-nitro-arginine inhibits increase in endothelin binding sites induced by ischemia and reperfusion.

Takashi Saito, Etsuko Fushimi, Toshiko Tamura, Yoshimasa Fujiwara, Hiroto Miura, Hiroyuki Watanabe, Satoshi Kibira, Hitoshi Hasegawa, Mamoru Miura

Journal of molecular and cellular cardiology   34 ( 8 ) 1041 - 7   2002年08月  [査読有り]

研究論文（学術雑誌）  

We have demonstrated that ischemia and reperfusion promoted augmented contractile response to endothelin-1 (ET) in coronary arteries in the presence of polymorphonuclear leukocytes (PMN). It has been also reported that ischemia and reperfusion increase ET binding sites in cardiac membrane in isolated rat heart perfused by blood cell-free system. To determine the role of PMN and L-arginine to nitric oxide (NO) pathway in these phenomena, isolated perfused rabbit hearts were subjected to 30 min of global ischemia followed by 30 min of reflow in the absence or presence of PMN and 10(-5)M of L-nitro-arginine (LNA). PMN was prepared with Percoll density gradients from peritoneal exudate elicited by glycogen. PMN activated with 10(-6)M of phorbol myristate acetate or their supernatant were infused into the coronary perfusion circuit after 5 min of reflow. LNA was added to perfusate also after reflow. The effect of superoxide dismutase (SOD: 50 IU/ml) was also determined. After the end of protocols, membrane fraction was isolated from the hearts for (125)I-ET-1 binding assay. ET-1 binding (Bmax) showed a significant increase by ischemia and reperfusion (P<0.01 vs control). That was markedly augmented with addition of activated PMN or their supernatant (both P<0.01), but abolished either by LNA or SOD (P<0.01 and P<0.05, respectively). These results indicate that increase in ET-receptor by ischemia and reperfusion is mediated by free radicals generated via L-arginine to NO pathway.

PubMed

The intracellular tyrosine residues of the ATP-gated P2X(1) ion channel are essential for its function.

Emese Toth-Zsamboki, Cecile Oury, Hiroyuki Watanabe, Bernd Nilius, Jos Vermylen, Marc F Hoylaerts

FEBS letters   524 ( 1-3 ) 15 - 9   2002年07月  [査読有り]

研究論文（学術雑誌）  

The four highly conserved intracellular tyrosine residues of the P2X(1) ion channel were mutated into phenylalanine. Simultaneous electrophysiological and calcium measurements in transfected human embryonic kidney (HEK 293) cells indicated that Y362F and Y370F mutants were non-functional, despite their proper plasma membrane expression. The Y16F and Y363F mutants retained 2.2% and 26% of the wild-type P2X(1) activity, respectively. However, no tyrosine phosphorylation was detected on Western blots of P2X(1) immunoprecipitates derived either from HEK 293 cell lysates or from human platelets, expressing P2X(1) endogenously. Thus, Y16, Y362, Y363 and Y370 are required for the appropriate three-dimensional structure and function of the intracellular P2X(1) domains.

PubMed

ATP and nitric oxide modulate a Ca(2+)-activated non-selective cation current in macrovascular endothelial cells.

Suk H Suh, Hiroyuki Watanabe, Guy Droogmans, Bernd Nilius

Pflugers Archiv : European journal of physiology   444 ( 3 ) 438 - 45   2002年06月  [査読有り]

研究論文（学術雑誌）  

We have studied the properties of a non-selective cation current (NSC(Ca)) in macrovascular endothelial cells derived from human umbilical vein (EA cells) that is activated by an increase of intracellular Ca(2+) concentration, [Ca(2+)](i). Current-voltage relationships are linear and the kinetics of the current is time-independent. Current-[Ca(2+)](i) relationships were fitted to a Ca(2+) binding site model with a concentration for half-maximal activation of 417 +/- 76 nM, a Hill coefficient of 2.3 +/- 0.8 and a maximum current of -23.9 +/- 2.7 pA/pF at -50 mV. The Ca(2+)-activated channel is more permeable to Na(+) than for Cs(+) ( P(Cs)/ P(Na)=0.58, n=7), but virtually impermeable to Ca(2+). Current activation was transient if ATP was omitted from the pipette solution. The maximal currents at 300 and 500 nM [Ca(2+)](i) were smaller than in the absence of ATP, but were not significantly different at 2 microM. The intracellular Ca(2+) concentration for half-maximal activation of the Ca(2+)-activated current was shifted to 811 +/- 12 nM in the absence of ATP. Substitution of ATP by the non-hydrolysable ATP analogue adenylylimidodiphosphate (AMP-PNP) did not affect current activation. Sodium nitroprusside (SNP) decreased NSC(Ca) in a concentration-dependent manner. The nitric oxide (NO) donors S-nitroso- N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) also inhibited NSC(Ca). In contrast, nitro- L-arginine (NLA), which inhibits all NO-synthases, potentiated NSC(Ca), whereas superoxide dismutase (SOD), which inhibits the breakdown of NO, inhibited NSC(Ca). It is concluded that the Ca(2+)-activated non-selective action channel in EA cells is modulated by the metabolic state of the cell and by NO.

PubMed

Ca2+ overload evokes a transient outward current in guinea-pig ventricular myocytes.

Ichirota Nakajima, Hiroyuki Watanabe, Kenji Iino, Takashi Saito, Mamoru Miura

Circulation journal : official journal of the Japanese Circulation Society   66 ( 1 ) 87 - 92   2002年01月  [査読有り]

研究論文（学術雑誌）  

There are 2 types of transient outward currents (Ito) in the hearts of various mammals: a 4-aminopyridine (4-AP) sensitive K+ current and a 4-AP resistant Ca2+ activated current, carried by Cl-, (referred to as I(to1) and I(to2), respectively). However, the I(to) has been considered to be absent in guinea-pig ventricular myocytes and so this study tested the hypothesis that I(to1) is generally absent in guinea-pig ventricular myocytes, but I(to2) appears under the condition of Ca2+ overload. Membrane currents were recorded by the whole-cell patch-clamp technique and Ca2+ overload was achieved by adding internal, and eliminating external, Na+ with subsequent enhancement of Ca2+ influx via the Na+-Ca2+ exchange. Under physiological conditions, I(to) could not be elicited by 300 ms-test pulse from -70 mV to 0 mV (n=32). However, under Ca2+ overload, a biphasic current resulting from the overlap of the L-type Ca2+ channel current and Ito was elicited (n=38). This I(to) was resistant to 4-AP (3 mmol/L, n=30) but sensitive to both anthrancene-9-carboxylic acid (9-AC, 3 mmol/L, n=8) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (100 micromol/L, n=3). Replacing K+ with Cs+ on both sides of the membrane failed to abolish I(to) (n=38). I(to) disappeared by lowering the external Cl- (n=3). The amplitude of I(to) was dependent on that of the L-type Ca2+ channel current (n=4). Because Ca2+ release from the sarcoplasmic reticulum was prevented by caffeine (5 mmol/L), I(to) was negligible (n=6). These results suggest that I(to1) is absent, but Ca2+ overload evokes I(to2) in guinea-pig ventricular myocytes.

PubMed

Activation of TRPV4 channels(hVRL2/mTRP12) by phorbol derivatives.

Watanabe H et. Al

J Biol Chem   277 ( 16 ) 13569 - 13577   2002年01月  [査読有り]

研究論文（学術雑誌）   国内共著

Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells.

Watanabe H et. al

J Biol Chem   277 ( 49 ) 47044 - 47051   2002年01月  [査読有り]

研究論文（学術雑誌）   国内共著

CaT1 and the Calcium Release-activated Calcium Channel Manifest Distinct Pore Properties

Thomas Voets, Jean Prenen, Andrea Fleig, Rudi Vennekens, Hiroyuki Watanabe, Joost G.J. Hoenderop,René J.M. Bindels,Guy Droogmans, Reinhold Penner, Bernd Nilius

JOURNAL OF BIOLOGICAL CHEMISTRY   276 ( 51 ) 47767 - 47770   2001年12月  [査読有り]

研究論文（学術雑誌）  

The calcium release-activated calcium channel (CRAC) is a highly Ca2+-selective ion channel that is activated on depletion of inositol triphosphate (IP3)-sensitive intracellular Ca2+ stores. It was recently reported that CaT1, a member of the TRP family of cation channels, exhibits the unique biophysical properties of CRAC, which led to the conclusion that CaT1 comprises all or part of the CRAC pore (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705–709). Here, we directly compare endogenous CRAC with heterologously expressed CaT1 and show that they manifest several clearly distinct properties. CaT1 can be distinguished from CRAC in the following features: sensitivity to store-depleting agents; inward rectification in the absence of divalent cations; relative permeability to Na+ and Cs+; effect of 2-aminoethoxydiphenyl borate (2-APB). Moreover, CaT1 displays a mode of voltage-dependent gating that is fully absent in CRAC and originates from the voltage-dependent binding/unbinding of Mg2+ inside the channel pore. Our results imply that the pores of CaT1 and CRAC are not identical and indicate that CaT1 is a Mg2+-gated channel not directly related to CRAC.

DOI

Congestive Heart Failure Associated with Portasystemic Shunt by Small-Bowel Varices

KIBIRA Satoshi, ISHIDA Hideaki, WATANABE Hiroyuki, IZUMI Chikako, OOMOTO Naoki, OGAWA Yasuhiko, SAITO Takashi, MIURA Mamoru

日本臨床生理学会雑誌   30 ( 1 ) 59 - 64   2000年02月  [査読有り]

研究論文（学術雑誌）  

Validity of the right ventricular Doppler index for assessment of severity of congestive heart failure in patients with dilated cardiomyopathy

C Izumi

Heart and vessels   14 ( 5 ) 232 - 239   1999年09月  [査読有り]

研究論文（学術雑誌）  

DOI

Calcium channel blocking properties of NKY-722, a highly hydrophilic dihydropyridine derivative, in single ventricular cells of guinea-pig hearts

Watanabe H, Nishio M, Iijima T

Japanese Heart Journal   35   451 - 452   1994年  [査読有り]

研究論文（学術雑誌）