Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration.
Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration.
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通道磷酸化和胞质 Mg2 浓度对 L 型 Ca2 电流的调节。
DOI：
10.1152/ajpcell.00579.2005
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Berlin,JoshuaR
中科院分区：
文献类型：
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作者：
Wang,Min;Berlin,JoshuaR
以往的研究表明，细胞内游离Mg ~（2+）浓度（[Mg ~（2+）]i）对L型Ca ~（2+）电流（伊卡）的抑制作用受到cAMP依赖性蛋白激酶通路激活的深刻影响。为了研究这种ICa反调节的机制，大鼠心肌细胞和表达L-型Ca 2+通道的tsA 201细胞用膜片移液管进行全细胞电压钳制，其中[Mg 2 +]（[Mg 2 +]p）由柠檬酸盐和ATP缓冲。在表达野生型Ca 2+通道（α1C/β2A/α2δ）的tsA 201细胞中，将[Mg 2 +] p从0.2 mM增加到1.8 mM使峰值ICaby降低76 ± 4.5%（n= 7）。在ATP-γ-S负载的细胞中也观察到Mg ~（2+）依赖性的ICa调节作用。用0.2 mM [Mg 2 +]p，通过移液器应用蛋白激酶A（PKA）或磷酸酶2A（PP 2A）来操纵磷酸化条件，产生了ICa幅度的较大变化;然而，用1.8 mM [Mg 2 +]p，这些相同的操纵对ICa没有显著影响。对于缺少主要PKA磷酸化位点的突变型通道（α1C/S1928 A/β2A/S478 A/S479 A/α2δ），增加[Mg 2 +] p对ICa的影响很小。当α 1C亚基截短（α1CΔ1905/β2A/S478 A/S479 A/α2δ）增加通道开放概率时，[Mg 2 +] p的增加使峰值ICa显著降低。相应地，在用移液管PP 2A进行电压钳位以使通道磷酸化最小化的心肌细胞中，当用BAY K8644促进通道开放时，增加[Mg 2 +] p产生更大的ICa降低。这些数据表明，在其生理浓度范围内，胞质Mg 2+调节通道磷酸化调节ICa的程度。这种调节不一定涉及通道磷酸化本身的变化，但更普遍地似乎取决于通道磷酸化诱导的门控动力学。
Previous studies have shown that inhibition of L-type Ca2+current (ICa) by cytosolic free Mg2+concentration ([Mg2+]i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation ofICa, rat cardiac myocytes and tsA201 cells expressing L-type Ca2+channels were whole cell voltage-clamped with patch pipettes in which [Mg2+] ([Mg2+]p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca2+channels (α1C/β2A/α2δ), increasing [Mg2+]pfrom 0.2 mM to 1.8 mM decreased peakICaby 76 ± 4.5% (n= 7). Mg2+-dependent modulation ofICawas also observed in cells loaded with ATP-γ-S. With 0.2 mM [Mg2+]p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP2A) produced large changes inICaamplitude; however, with 1.8 mM [Mg2+]p, these same manipulations had no significant effect onICa. With mutant channels lacking principal PKA phosphorylation sites (α1C/S1928A/β2A/S478A/S479A/α2δ), increasing [Mg2+]phad only small effects onICa. However, when channel open probability was increased by α1C-subunit truncation (α1CΔ1905/β2A/S478A/S479A/α2δ), increasing [Mg2+]pgreatly reduced peakICa. Correspondingly, in myocytes voltage-clamped with pipette PP2Ato minimize channel phosphorylation, increasing [Mg2+]pproduced a much larger reduction inICawhen channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg2+modulates the extent to which channel phosphorylation regulatesICa. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation.