首页> 外文期刊>Biochimica et biophysica acta. Molecular cell research >Maitotoxin activates an endogenous non-selective cation channel and is an effective initiator of the activation of the heterologously expressed hTRPC-1 (transient receptor potential) non-selective cation channel in H4-IIE liver cells
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Maitotoxin activates an endogenous non-selective cation channel and is an effective initiator of the activation of the heterologously expressed hTRPC-1 (transient receptor potential) non-selective cation channel in H4-IIE liver cells

机译:Maitotoxin激活内源性非选择性阳离子通道,是激活H4-IIE肝细胞中异源表达的hTRPC-1(瞬时受体电位)非选择性阳离子通道的有效引发剂

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The structures and mechanisms of activation of non-selective cation channels (NSCCs) are not well understood although NSCCs play important roles in the regulation of metabolism, ion transport, cell volume and cell shape. It has been proposed that TRP (transient receptor potential) proteins are the molecular correlates of some NSCCs. Using fura-2 and patch-clamp recording, it was shown that the maitotoxin-activated cation channels in the H4-IIE rat liver cell line admit Ca~(2+), Mn~(2+) and Na~+, have a high selectivity for Na~+ compared with Ca~(2+), and are inhibited by Gd~(3+) (half-maximal inhibition at 1 μM). Activation of the channels by maitotoxin was inhibited by increasing the extracellular Ca~(2+) concentration or by inclusion of 10 mM EGTA in the patch pipette. mRNA encoding TRP proteins 1, 2 and 3 at levels comparable with those in brain was detected using reverse transcriptase-polymerase chain reaction in poly(A)~+ RNA prepared from H4-IIE cells and freshly-isolated rat hepatocytes. In H4-IIE cells transiently transfected with cDNA encoding hTRPC-1, the expressed hTRPC-1 protein was chiefly located at intracellular sites and at the plasma membrane. Cells expressing hTRPC-1 exhibited a substantial enhancement of maitotoxin-initiated Ca~(2+) inflow and a modest enhancement of thapsigargin-initiated Ca~(2+) inflow (measured using fura-2) and no enhancement of the highly Ca~(2+)-selective store-operated Ca~(2+) current (measured using patch-clamp rocording). In cells expressing hTRPC-1, maitotoxin activated channels which were not found in untransfected cells, have an approximately equal selectivity for Na~+ and Ca~(2+), and are inhibited by Gd~(3+) (half-maximal inhibition at 3 μM). It is concluded that in liver cells (i) maitotoxin initiates the activation of endogenous NSCCs with a high selectivity for Na~+ compared with Ca~(2+); (ii) TRP proteins 1, 2 and 3 are expressed; (iii) maitotoxin is an effective initiator of activation of heterologously expressed hTRPC-1 channels; and (iv) the endogenous TRP-1 protein is unlikely to be the molecular counterpart of the maitotoxin-activated NSCCs nor the highly Ca~(2+)-selective store-operated Ca~(2+) channels.
机译:尽管非选择性阳离子通道(NSCCs)在代谢,离子转运,细胞体积和细胞形状的调节中起着重要作用,但人们对非选择性阳离子通道(NSCCs)的激活的结构和机制尚不十分了解。已经提出,TRP(瞬时受体电位)蛋白是一些NSCC的分子相关性。使用fura-2和膜片钳记录,表明H4-IIE大鼠肝细胞系中被毒素毒素激活的阳离子通道允许Ca〜(2 +),Mn〜(2+)和Na〜+具有与Ca〜(2+)相比,对Na〜+的选择性高,并且被Gd〜(3+)抑制(在1μM时抑制最大一半)。通过增加细胞外Ca〜(2+)浓度或在贴片移液器中加入10 mM EGTA来抑制麦芽毒素对通道的激活。使用逆转录酶-聚合酶链反应,在由H4-IIE细胞和新鲜分离的大鼠肝细胞制备的poly(A)〜+ RNA中,检测到了与脑部水平相当的编码TRP蛋白1、2和3的mRNA。在用编码hTRPC-1的cDNA瞬时转染的H4-IIE细胞中,表达的hTRPC-1蛋白主要位于细胞内位点和质膜上。表达hTRPC-1的细胞显示出由人毒素引发的Ca〜(2+)流入的显着增强和由毒胡萝卜素引发的Ca〜(2+)流入的适度增强(使用fura-2测得),而高度Ca〜2的增强却没有。 (2 +)-选择性存储操作的Ca〜(2+)电流(使用膜片钳rocording测量)。在表达hTRPC-1的细胞中,未转染的细胞中未发现的人毒素激活通道,对Na〜+和Ca〜(2+)的选择性近似相等,并被Gd〜(3+)抑制(半数最大抑制)。在3μM下)。结论:在肝细胞中(i)与Ca〜(2+)相比,人毒素启动了内源性NSCC的活化,对Na〜+的选择性高。 (ii)表达TRP蛋白1、2和3; (iii)毒素是激活异源表达的hTRPC-1通道的有效引发剂; (iv)内源性TRP-1蛋白不太可能是人毒素激活的NSCC的分子对应物,也不可能是高Ca〜(2 +)-选择性存储操作的Ca〜(2+)通道。

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