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

白血病抑制因子(LIF)是IL-6细胞因子家族中的一员，可诱导心肌肥大。本课题组曾报道LIF能激活被肌动蛋白激酶抑制剂PD98059阻断的L型钙通道。我们还发现，LIF与激活的ERK、CaMK-IV和钙调神经磷酸酶共同诱导心肌肥厚，其作用依赖于通过L型钙通道增加的钙电流。为确定LIF对L型钙通道功能的主要调节因子α1亚基的磷酸化作用，用LIF刺激原代培养的乳鼠心肌细胞15min，以重组A1亚基(含80 0个氨基酸的融合蛋白)为底物，用凝胶内-激酶法测定其活性。ERK1/2将含有ERK1/2基序特定丝氨酸的融合蛋白磷酸化。LiF促进免疫沉淀的α1亚单位的磷酸化，代谢标记为…更多的hP-32，显著。磷酸氨基酸分析表明，LIF仅在丝氨酸位置磷酸化了Al亚基。将兔α-1亚基靶向丝氨酸点突变体导入HEK2 93细胞。LiF显著增强野生型α-1亚基的磷酸化，但对突变体的磷酸化作用不明显。用P-32标记野生型α-1亚基，结果显示野生型MEK1亚基的磷酸化作用显著增强。用穿孔斑块法，含兔野生型α1亚基的L型钙通道在LIP给药后表现出钙电流的延长，而点突变体的钙通道无明显延长。这些结果表明，LIF在体外和体内均能磷酸化心肌L型钙通道α1亚单位的靶丝氨酸，因此ERK1/2可能是心肌细胞L型钙通道的潜在激动剂。较少

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