MOLECULAR FUNCTION OF CHIMERIC Ca-Na IONIC CHANNELS

嵌合 Ca-Na 离子通道的分子功能

• 批准号: 07807008
• 负责人: ONO Katsushige
• 金额: $ 1.54万
• 依托单位: OITA MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07807008/
• 关键词: COS CELL; PATCH CLAMP; Ca CHANNEL; alpha1 SUBUNIT; CONDUCTANCE; beta SUBUNIT; Na CHANNEL; α_1サブユニット

BACKGROUND and OBJECTIVERecent electrophysiological studies combined with molecular biology provided evidence that the kinetics of the L-type Ca^<2+> channel derives largely from the structure of the pore-forming subunit of the channel (alpha_<1c> subunit), however, the molecular events underlying the formation of subconductance levels are still under investigation. To understand the nature and structural requirements for subconductance, we have expressed cardiac alpha_<1c> L-type Ca^<2+> channels with or without coexpression of the beta_2 subunit in COS cells or in Chinese Hamster Fibroblast (CHW) cells to determine if alpha_<1c> channels and alpha_<1c>/beta_2 channels display different single channel subconductance levels.METHODSTransfection of COS- and CHW-cells : COS cells transiently transfected with cDNA encoding the alpha_1 subunit of the cardiac L-type calcium channel (alpha_<1c>), and CHW cells stably transfected with alpha_1 subunit alone or with alpha_<1c> plus cardiac beta … More subunits (alpha_<1c>/beta_2), were maintained in cell culture (see below). Electrophysiology : Small (conventional) patch pipettes with resistances between 4-5 MOMEGA allowed recording of alpha_<1c>/beta_2 channels. Large (macropatch) pipettes with resistances between 0.2-0.3 MOMEGA allowed recording of alpha_<1c> channels. Patches were depolarized for 195 ms at every 0.5-1 seconds or for 395ms at every 5 seconds from a holding potential (V_H) of -80mV.In ramp clamp experiments, slope ratio of -0.1V/sec was applied at every 3 seconds.RESULTS(1) Structural requirements for the formation of subconducting state (s) of the L-type Ca^<2+> channel were studied in cardiac alpha_<1c> channels in COS- and CHW-cells with or without coexpression of beta_2-subunit.(2) Similar four conductance levels were detected in alpha_<1c> alone and in alpha_<1c>/beta_2 channels : 3.4<plus-minus>0.2pS,6.4<plus-minus>0.2pS,14.5<plus-minus>0.2pS and 22.2<plus-minus>0.2pS(3) Existence of pore-forming alpha_<1c> subunit alone fulfillls the condition to form multiple conducting states.(4) Coexpression of beta_2-subunit leads to a higher probability of channel openings at three subconductance levels as well as full conducting level of 22 pS,but may not be prerequisite for production of sublevel openings in cardiac L-type Ca^<2+> channels. Less

背景和目的最近的电生理学研究结合分子生物学证明，l型Ca^<2+>通道的动力学主要源于通道的成孔亚基（α _<1c>亚基）的结构，然而，亚电导水平形成的分子事件仍在研究中。为了了解心脏α _<1c> l型Ca^<2+>通道亚导的性质和结构要求，我们在COS细胞和中国仓鼠成纤维细胞（CHW）中分别表达了α _<1c>通道和α _<1c>/beta_2通道是否具有不同的单通道亚导水平。方法转染COS-和CHW细胞：COS细胞短暂转染了编码心脏l型钙通道α _1亚基（α _<1c>）的cDNA， CHW细胞稳定转染了α _1亚基或α _<1c>加心脏β _2 （α _<1c>/ β _2）的亚基（见下）。电生理：电阻在4-5 MOMEGA之间的小型（传统）贴片移液器允许记录α _<1c>/ β _2通道。电阻在0.2-0.3 MOMEGA之间的大型（macropatch）移液器允许记录α _<1c>通道。在保持电位（V_H）为-80mV的条件下，斑块每0.5-1秒去极化195ms或每5秒去极化395ms。斜坡钳实验中，每3秒施加-0.1V/sec的斜率比。结果(1)研究了COS-和chw细胞α <1c>通道中β 2亚基共表达或不共表达时l型Ca^<2+>通道亚传导态形成的结构条件。(2) α <1c>通道与α <1c>/beta_2通道的电导水平相似，分别为3.4<正负>0.2pS、6.4<正负>0.2pS、14.5<正负>0.2pS和22.2<正负>0.2pS。(3)单独存在成孔α <1c>亚基，满足了形成多导态的条件。(4) β 2-亚基的共表达导致3个亚电导水平的通道开放和22 pS的全导水平的通道开放的可能性更高，但可能不是心脏l型Ca^<2+>通道产生亚电导水平开放的先决条件。少

项目成果

Ono, K et al.: "Single channel kinetics of L-type calcium channels expressed in COS cells" Japanese Circulation Journal. 60(SI). 124 (1995)

Ono, K 等人：“COS 细胞中表达的 L 型钙通道的单通道动力学”日本循环杂志。