Margatoxin binds to a homomultimer of K(v)1.3 channels in Jurkat cells. Comparison with K(v)1.3 expressed in CHO cells
Margatoxin binds to a homomultimer of K(v)1.3 channels in Jurkat cells. Comparison with K(v)1.3 expressed in CHO cells
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DOI：
10.1021/bi962351p
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发表时间：
1997-03-25
期刊：
影响因子：
2.9
通讯作者：
Slaughter, RS
中科院分区：
文献类型：
--
作者：
Helms, LMH;Felix, JP;Slaughter, RS
电压门控性钾通道（K-V）在人外周血T淋巴细胞的静息电位和激活级联反应中起关键作用。Margatoxin（MgTX）是一种来自Centruroides margaritatus的39个氨基酸的肽，是淋巴细胞Ky通道的有效抑制剂。单碘酪氨酸玛格毒素（[I-125]MgTX）与Jurkat细胞（一种人白血病T细胞系）或稳定转染Shaker型电压门控K+通道（K（V）1.3）的CHO细胞制备的质膜的结合已被用于研究淋巴细胞Ky通道的性质。将这些数据与[I-125]MgTX结合到大鼠脑突触质膜中的异四聚体Ky通道[Knaus，H. G.，等人（1995）Biochemistry 34，13627-13634]。在Jurkat或CHO/K（V）1.3膜中，[I-125]MgTX的亲和力为100-200 fM，受体密度在Jurkat膜中为20-120 fmol/mg，在CHO/K（V）1.3膜中为1000 fmol/mg。与大鼠脑相反，[I-125]MgTX与Jurkat和CHO/K（V）1.3膜的结合显示出对K+的绝对需求，通过[I-125]MgTX结合的免疫沉淀或通过使用序列-PCR的Western印迹分析确定，Nai K（V）1.3不增强结合，是CHO/K（V）1.3或Jurkat质膜中存在的唯一K（V）1系列通道。针对K（V）1家族成员制备的特异性抗体。一系列肽基Ky通道抑制剂抑制[I-125]MgTX与Jurkat、CHO或大鼠脑细胞膜结合的相对效力和阻断Rb-86（+）从CHO/K（V）1.3细胞流出的相对效力基本相同，除了cl-树枝毒素在阻断与大鼠脑细胞膜结合方面比其他试验更有效。[I-125]MgTX结合的特征、抗体谱和肽基Ky抑制剂的作用均表明Jurkat淋巴细胞中的[I-125]MgTX受体由K（V）1.3的同多聚体组成，这与受体的异多聚体排列不同。大鼠大脑。
Voltage-gated potassium (K-V) channels play key roles in setting the resting potential and in the activation cascade of human peripheral T lymphocytes. Margatoxin (MgTX), a 39-amino acid peptide from Centruroides margaritatus, is a potent inhibitor of lymphocyte Ky channels. The binding of monoiodotyrosinyl margatoxin ([I-125]MgTX) to plasma membranes prepared from either Jurkat cells, a human leukemic T cell line, or CHO cells stably transfected with the Shaker-type voltage-gated K+ channel, K(V)1.3, has been used to investigate the properties of lymphocyte Ky channels. These data were compared with [I-125]MgTX binding to heterotetrameric Ky channels in rat brain synaptic plasma membranes [Knaus, H. G., et al. (1995) Biochemistry 34, 13627-13634]. The affinity for [I-125]MgTX is 100-200 fM in either Jurkat or CHO/K(V)1.3 membranes, and the receptor density is 20-120 fmol/mg in Jurkat membranes or 1000 fmol/mg in CHO/K(V)1.3 membranes. In contrast to rat brain, [I-125]MgTX binding to Jurkat and CHO/K(V)1.3 membranes exhibits an absolute requirement for K+, with no potentiation of binding by Nai K(V)1.3 was the only K(V)1 series channel present in either CHO/K(V)1.3 or Jurkat plasma membranes as determined by immunoprecipitation of [I-125]MgTX binding or by Western blot analyses using sequence-specific antibodies prepared against members of the K(V)1 family. The relative potencies of a series of peptidyl Ky channel inhibitors was essentially the same for inhibition of [I-125]MgTX binding to Jurkat, CHO, or rat brain membranes and for blocking Rb-86(+) efflux from the CHO/K(V)1.3 cells, except that cl-dendrotoxin was more potent at blocking binding to rat brain membranes than in the other assays. The characteristics of [I-125]MgTX binding, the antibody profiles, and the effects of the peptidyl Ky inhibitors all indicate that the [I-125]MgTX receptor in Jurkat lymphocytes is comprised of a homomultimer of K(V)1.3, unlike the heteromultimeric arrangement of the receptor in rat brain.