Walerczyk M, Fabczak H, Fabczak S
Department of Cell Biology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
Protoplasma. 2006 May;227(2-4):139-46. doi: 10.1007/s00709-005-0143-1. Epub 2006 May 3.
Immunoblotting and immunocytochemical assays were employed to identify and localize a channel protein activated by cyclic GMP (cGMP) in the protozoan ciliate Stentor coeruleus. Analysis of whole-cell homogenate with antibodies raised against the alpha-subunit of the cGMP-activated channel protein from bovine rod outer segments and against cGMP revealed four major protein bands with molecular masses of 40 kDa, 63 kDa, and over 120 kDa, which bound cGMP. However, only a cGMP-binding protein of 63 kDa, corresponding to the alpha-subunit of the cGMP-activated ion channel protein from bovine rod outer segments, was found in the ciliate cortex fraction. The functional cGMP-activated channel protein was also shown to be present in the cortex fraction of S. coeruleus by patch-clamp measurements of artificial liposomes. Incorporation of the cortex fraction into liposomes resulted in the appearance of ion channel activity related to cGMP. The reconstituted protein channels were strongly inhibited by l-cis-diltiazem, a known potent blocker of many types of cyclic-nucleotide-activated channels. The results presented here are the first demonstration of the existence and localization of a putative cGMP-activated channel protein in the ciliate S. coeruleus. Cyclic-nucleotide-activated channel proteins are nonspecific cation channels which mediate the receptor potentials in photoreceptor cells and in cells of the olfactory epithelium. On the basis of these data, we suggest that the 63 kDa protein identified in Stentor coeruleus is also a cGMP-activated ion channel and that it may be involved as an effector in the photosensory transduction pathway leading to the motile photophobic response in this ciliate protist.
采用免疫印迹和免疫细胞化学分析方法，鉴定并定位了原生动物蓝喇叭虫中一种由环磷酸鸟苷（cGMP）激活的通道蛋白。用针对牛视杆外段cGMP激活通道蛋白α亚基以及cGMP产生的抗体对全细胞匀浆进行分析，发现了4条主要蛋白条带，分子量分别为40 kDa、63 kDa和超过120 kDa，它们能结合cGMP。然而，在纤毛虫皮层组分中仅发现一种63 kDa的cGMP结合蛋白，它对应于牛视杆外段cGMP激活离子通道蛋白的α亚基。通过对人工脂质体进行膜片钳测量，还表明功能性cGMP激活通道蛋白存在于蓝喇叭虫的皮层组分中。将皮层组分掺入脂质体导致出现了与cGMP相关的离子通道活性。重构的蛋白通道受到l-顺式地尔硫䓬的强烈抑制，l-顺式地尔硫䓬是许多类型环核苷酸激活通道的已知强效阻滞剂。本文给出的结果首次证明了在纤毛虫蓝喇叭虫中存在一种假定的cGMP激活通道蛋白并对其进行了定位。环核苷酸激活通道蛋白是非特异性阳离子通道，介导光感受器细胞和嗅觉上皮细胞中的受体电位。基于这些数据，我们认为在蓝喇叭虫中鉴定出的63 kDa蛋白也是一种cGMP激活离子通道，并且它可能作为效应器参与导致这种纤毛虫原生生物产生运动性避光反应的光感觉转导途径。
