Identification of the large-conductance background K+ channel in mouse B cells as TREK-2.
Mouse B cells and their cell line (WEHI-231) express large-conductance background K(+) channels (LK(bg)) that are activated by arachidonic acids, characteristics similar to TREK-2. However, there is no evidence to identify the molecular nature of LK(bg); some properties of LK(bg) were partly different from the reported results of TREK type channels. In this study, we compared the properties of cloned TREK-2 and LK(bg) in terms of their sensitivities to ATP, phosphatidylinositol 4,5-bisphosphate (PIP(2)), intracellular pH (pH(i)), and membrane stretch. Similar to the previous findings of LK(bg), TREK-2 showed spontaneous activation after membrane excision (i-o patch) and were inhibited by MgATP or by PIP(2). The inhibition by MgATP was prevented by wortmannin, suggesting membrane-delimited regulation of TREKs by phosphoinositide (PI) kinase. The same was observed with the property of LK(bg); the activation of TREK-2 by membrane stretch was suppressed by U73122 (PLC inhibitor). As with the known properties of TREK-2, LK(bg) were activated by acidic pH(i) and inhibited by PKC activator. Finally, we confirmed the expression of TREK-2 in WEHI-231 by using RT-PCR and immunoblot analyses. The amplitude of background K(+) current and the TREK-2 expression in WEHI-231 were commonly decreased by genetic knockdown of TREK-2 using small interfering RNA. The downregulation of TREK-2 attenuated Ca(2+)-influx induced by arachidonic acid in WEHI-231. As a whole, these results strongly indicate that TREK-2 encodes LK(bg) in mouse B cells. We also newly suggest that the low activity of TREK-2 in intact cells is due to the inhibition by intrinsic PIP(2).
Zheng
Haifeng
H
Dept. of Physiology, Seoul National Univ. College of Medicine, 103 Daehangno, Jongno-gu, Seoul 110-799, Korea.
Nam
Joo Hyun
JH
Pang
Bo
B
Shin
Dong Hoon
DH
Kim
Ji Seon
JS
Chun
Yang-Sook
YS
Park
Jong-Wan
JW
Bang
Hyowon
H
Kim
Woo Kyung
WK
Earm
Yung E
YE
Kim
Sung Joon
SJ
D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001402:B-Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D048430:Cell Shape; D003001:Cloning, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D040542:Mechanotransduction, Cellular; D008564:Membrane Potentials; D051379:Mice; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D024683:Potassium Channels, Tandem Pore Domain / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D051381:Rats; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
C503445:Kcnk10 protein, mouse; C503444:Kcnk10 protein, rat; D019269:Phosphatidylinositol 4,5-Diphosphate; D010726:Phosphodiesterase Inhibitors; D024683:Potassium Channels, Tandem Pore Domain; D047428:Protein Kinase Inhibitors; C103742:potassium channel protein TREK-1; D016718:Arachidonic Acid; D000255:Adenosine Triphosphate; D019870:1-Phosphatidylinositol 4-Kinase; D010738:Type C Phospholipases; D011188:Potassium; D002118:Calcium