MOLECULAR FUNCTION OF CHIMERIC Ca-Na IONIC CHANNELS

近年来，电生理学和分子生物学的研究表明，L-型Ca^&lt;2+&gt;通道的动力学主要来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机制仍在研究之中。为了解亚电导的性质和结构要求，我们<1c>在COS细胞和中国人成纤维细胞（CHW）中表达了有或无β_2亚基共表达的心脏α_L型Ca^&lt;2+&gt;通道，以确定α_<1c>通道和α_<1c>/β_2通道是否显示不同的单通道亚电导水平。方法转染COS细胞和CHW细胞：瞬时转染编码心脏L型钙通道α_1亚基（α_）的cDNA<1c>，以及稳定转染α_1亚基或α_+ β_2亚基的CHW细胞<1c>。 ...更多信息 亚基（α_<1c>/β_2）维持在细胞培养物中（见下文）。电生理学：电阻在4-5 M Ω之间的小（常规）贴片移液管允许记录α_<1c>/β_2通道。电阻在0.2-0.3 M Ω之间的大型（macropatch）移液器允许记录α<1c>通道。结果（1）在<1c>COS-和CHW-细胞中，β 2-亚基共表达或不共表达的情况下，研究了L型Ca^&lt;2+&gt;通道亚传导态形成的结构要求。(2)在单独的α_<1c>通道和α_<1c>/β_2通道中检测到类似的四个电导水平：3.4 <plus-minus>± 0.2pS、6.4 <plus-minus>± 0.2pS、14.5 <plus-minus>± 0.2pS和22.2 ± <plus-minus>0.2pS。（3）单独的成孔α_<1c>亚基的存在满足了形成多个电导状态的条件。(4)β_2-亚基的共表达导致在三个亚电导水平以及22 pS的全电导水平上通道开放的可能性更高，但可能不是心脏L型Ca^&lt;2+&gt;通道产生亚电导水平开放的先决条件。少

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