The cardiac hypertrophic factors modulate the function of the cardiac L-type Ca channels

心脏肥大因子调节心脏 L 型 Ca 通道的功能

基本信息

批准号：
12835012
负责人：
TAKAHASHI Eiichi
金额：
$ 2.3万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12835012/
关键词：
L-type Ca^<2+> channel cardiomyocyte kinase ERK1 2 P-32 labeling LIF point mutant patch-clamp 細胞ラベルペプチドマッピング

项目摘要

The leukemia inhibitory factor (LIF) is a member of the IL-6 cytokine family that can induce cardiac hypertrophy. Our group reported that LIF can activate cardiac L-type Ca^<2+> channels blocked by PD98059 (MEK inhibitor). We also showed that LIF induces cardiac hypertrophy in concert with activated ERK, CaMK-IV and calcineurin, which are dependent on increased Ca^<2+> currents through L-type Ca^<2+>. To determine whether LIF phosphorylates the α1 subunits, the main regulator of the channel function of L-type Ca^<2+> channels, primary cultures of neonatal rat cardiomyocytes were stimulated with LIF for 15 min, and the kinase activity measured by the in-gel-kinase assay using recombinant al subunits (GST-fusion proteins containing 800 amino acids of the carboxyl terminal) as the substrate. ERK1/2 phosphorylated the fusion protein containing the certain serine of the ERK1/2 motif. LIF promoted the phosphorylation of the immuno-precipitated α1 subunits, which was metabolically labeled wit … More h P-32, significantly. Phospho-amino acid analysis showed that LIF phosphorylated the al subunits only at the serine position. The point mutant of the target serine of the rabbit α1 subunit was transfected into HEK293 cells. LIF enhanced phosphorylation of the transfected wild-type α1 subunit by significantly, but not that of the mutant. The wild-type α1 subunits which were co-transfected into HEK293 cells with the constitutively active MEK1 were labeled with P-32, and theses were showed enhanced phosphorylation significantly. By the perforated patch-clump method, the L-type Ca^<2+> channels containing the rabbit wild-type α1 subunit showed the prolongation of the Ca^<2+> current after LIP administration, but the channels of the point mutant did not showed the prolongation. These findings indicate that LIF phosphorylates the target serine of the α1 subunit of cardiac L-type Ca^<2+> channels both in vitro and in vivo, and that ERK1/2 may therefore be a potential activator of cardiac L-type Ca^<2+> channels. Less

白血病抑制因子(LIF)是IL-6细胞因子家族的成员，可以诱导心脏肥大。我们的研究小组报道，LIF可以激活被PD98059(MEK抑制剂)阻断的心脏L型Ca^2+通道。还表明LIF与激活的ERK、CaMK-IV和钙调磷酸酶协同诱导心脏肥大，这依赖于通过L型Ca ^ 2+ 增加的Ca ^ 2+ 电流。为了确定LIF是否磷酸化α1亚基（L型Ca^2+通道的通道功能的主要调节者），用LIF刺激新生大鼠心肌细胞的原代培养物15分钟，并通过in测量激酶活性。 -凝胶激酶测定使用重组α1亚基（含有羧基末端的800个氨基酸的GST融合蛋白）作为底物，ERK1/2磷酸化融合蛋白。含有 ERK1/2 基序的某些丝氨酸可显着促进免疫沉淀 α1 亚基的磷酸化，该亚基用 h P-32 进行代谢标记，磷酸氨基酸分析表明 LIF 仅磷酸化 a1 亚基。将兔α1亚基的靶丝氨酸的点突变体转染至LIF增强的磷酸化细胞中。野生型α1亚基与突变体的α1亚基显着转染，与组成型活性MEK1共转染到HEK293细胞中的野生型α1亚基被P-32标记，这些磷酸化显着增强。通过穿孔斑块法，含有兔野生型α1亚基的L型Ca^2+通道显示出延长的LIP给药后Ca ^ 2+ 有电流，但点突变体的通道没有显示延长。这些结果表明LIF磷酸化心脏L型Ca ^ 2+ 通道的α1亚基的靶丝氨酸。因此，ERK1/2 可能是心脏 L 型 Ca^2+ 通道的潜在激活剂。