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

• 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

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细胞因子家族的一员，可诱导心肌肥大。本课题组报道LIF可激活被MEK抑制剂PD 98059阻断的心肌L型Ca^2+通道。我们还发现LIF与ERK、CaMK-IV和钙调神经磷酸酶的激活协同诱导心肌肥大，这些都依赖于通过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+通道功能的主要调节剂），用LIF刺激新生大鼠心肌细胞的原代培养物15分钟，并使用重组α 1亚基（羧基末端含有800个氨基酸的GST融合蛋白）作为底物，通过凝胶内激酶测定法测量激酶活性。ERK 1/2磷酸化融合蛋白，该融合蛋白含有ERK 1/2基序的特定丝氨酸。LIF促进免疫沉淀的α1亚基的磷酸化，α 1亚基被代谢标记， ...更多信息 P-32，显著。磷酸化氨基酸分析表明LIF仅在丝氨酸位置磷酸化al亚基。将兔α1亚基靶丝氨酸的点突变体转染HEK 293细胞。LIF能显著增强野生型α1亚基的磷酸化，但对突变型α1亚基的磷酸化无明显影响。将野生型α1亚基与组成型活性的MEK 1共转染HEK 293细胞，用P-32标记，发现其磷酸化水平明显增强。穿孔斑块法显示，LIP处理后，含有野生型α1亚基的L型Ca^<2+>通道的Ca ^<2+>电流延长，而点突变体的L型Ca ^<2+>通道的Ca^<2+>电流则没有延长。这些发现表明，LIF在体内和体外均可磷酸化心脏L型Ca^<2+>通道α1亚基的靶丝氨酸，因此ERK 1/2可能是心脏L型Ca^<2+>通道的潜在激活剂。少