cAMP-dependent phosphorylation of the tetrodotoxin-resistant voltage-dependent sodium channel SNS
cAMP-dependent phosphorylation of the tetrodotoxin-resistant voltage-dependent sodium channel SNS
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DOI：
10.1111/j.1469-7793.1999.0433v.x
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发表时间：
1999-04-15
影响因子：
5.5
通讯作者：
Moss, SJ
中科院分区：
文献类型：
--
作者：
Fitzgerald, EM;Okuse, K;Moss, SJ
1.蛋白激酶A(PKA)对河豚毒素抗性(TTX-r)电压门控性钠通道的调节可能减弱哺乳动物感觉神经元的痛敏反应。因此，我们检测了大鼠感觉神经元特异性TTX-r通道SNS的克隆a亚单位的PKA磷酸化。将SNS的磷酸化与突变通道SNS(SA)的磷酸化进行比较，在SNS(SA)中，细胞内I-II环内的所有五个PKA共识位点(RXXS)已被定点突变(丝氨酸到丙氨酸)消除。在体外对表达为谷胱甘肽-X-转移酶(GST)的突变型SNS(SA)I-II环和SNS(SA)I-II环进行的PKA磷酸化和胰酶肽谱分析证实，这5个突变的丝氨酸是SNS I-II环中的主要PKA底物。在COS-7细胞中瞬时表达SNS和SNS(XA)通道，并比较它们的电生理特性。在野生型SNS通道中，Forskolin和8-bromo cAMP产生与PKA磷酸化一致的效应。然而，突变型SNS(SA)电流不受任何一种药物的显著影响。因此，消除I-II环PKA共识位点导致野生型通道的PKA调制明显减少。在控制条件下，与SnS相比，激活SnS(XA)电流的电压依赖关系向去极化电位移动。这与SNS(XA)电流在超极化电位下失活的速度减慢有关，并提示在基础条件下野生型通道的紧张性PKA磷酸化。我们得出结论，参与SNS通道PKA功能调制的主要底物位于细胞内I-II环。
1. Protein kinase A (PKA) modulation of tetrodotoxin-resistant (TTX-r) voltage-gated sodium channels may underly the hyperalgesic responses of mammalian sensory neurones. We have therefore examined PKA phosphorylation of the cloned a-subunit of the rat sensory neurone-specific TTX-r channel SNS. Phosphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intra cellular I-II loop had been eliminated by site-directed mutagenesis (serine to alanine).2. In vitro PKA phosphorylation and tryptic peptide mapping of SNS and mutant SNS(SA) I-II loops expressed as glutathione-X-transferase (GST) fusion proteins confirmed that the five mutated serines were the major PKA substrates within the SNS I-II loop.3. SNS and SNS(XA) channels were transiently expressed in COS-7 cells and their electrophysiological properties compared. In wild-type SNS channels, forskolin and 8-bromo cAMP produced effects consistent with PKA phosphorylation. Mutant SNS(SA) currents, however, were not significantly affected by either agent. Thus, elimination of the I-II loop PKA consensus sites caused a marked reduction in PKA modulation of wild-type channels.4. Under control conditions, the voltage dependence of activation of SNS(XA) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(XA) current inactivation at hyperpolarized potentials and suggested a tonic PKA phosphorylation of wild-type channels under basal conditions.5. We conclude that the major substrates involved in functional PKA modulation of the SNS channel, are located within the intracellular I-II loop.