Multisite phosphorylation of voltage-gated sodium channel alpha subunits from rat brain.
Multisite phosphorylation of voltage-gated sodium channel alpha subunits from rat brain.
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DOI：
10.1021/pr901171q
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发表时间：
2010-04-05
影响因子：
4.4
通讯作者：
Trimmer, James S.
中科院分区：
文献类型：
--
作者：
Berendt, Frank J.;Park, Kang-Sik;Trimmer, James S.
关键词：
离子通道的可逆磷酸化是哺乳动物神经元细胞可塑性的基础。电压门控钠或Nav通道是动作电位起始和传播、树突兴奋性和神经元兴奋性的许多其他方面的基础。已经提出各种蛋白激酶磷酸化Nav通道的初级α亚基，影响通道功能的各个方面。先前对Nav α亚基磷酸化的研究已经鉴定了一小部分磷酸化位点，这些位点在Nav通道功能的调节方面很重要。在这里，我们使用纳流液相色谱串联质谱（nano-LC MS/MS）从大鼠脑中亲和纯化的Nav α亚基，用两种不同的单克隆抗体鉴定了Nav 1.2上的15个磷酸化位点，其中12个以前没有报道过。我们还发现了3个新的磷酸化位点的Nav1.1。一般来说，常用的磷酸化位点预测算法不能准确预测这些新的体内磷酸化位点。我们的研究结果表明，从大鼠脑中分离的特定Nav α亚基是高度磷酸化的，并表明在哺乳动物脑中Nav通道活性的广泛调节。利用基于单克隆抗体的免疫纯化和质谱鉴定磷酸化位点是确定哺乳动物脑中Nav通道和重要膜蛋白磷酸化状态的有效方法。
Reversible phosphorylation of ion channels underlies cellular plasticity in mammalian neurons. Voltage-gated sodium or Nav channels underlie action potential initiation and propagation, dendritic excitability, and many other aspects of neuronal excitability. Various protein kinases have been suggested to phosphorylate the primary α subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav α subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in meditating aspects of Nav channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nav α subunits affinity-purified from rat brain with two distinct monoclonal antibodies to identify 15 phosphorylation sites on Nav1.2, 12 of which have not been previously reported. We also found 3 novel phosphorylation sites on Nav1.1. In general, commonly used phosphorylation site prediction algorithms did not accurately predict these novel in vivo phosphorylation sites. Our results demonstrate that specific Nav α subunits isolated from rat brain are highly phosphorylated, and suggest extensive modulation of Nav channel activity in mammalian brain. Identification of phosphorylation sites using monoclonal antibody-based immunopurification and mass spectrometry is an effective approach to define the phosphorylation status of Nav channels and important membrane proteins in mammalian brain.
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影响因子：
56.9
作者：
NUMANN, R;CATTERALL, WA;SCHEUER, T
通讯作者：
SCHEUER, T
DOI：
10.1083/jcb.200805169
发表时间：
2008-12-15
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bréchet A;Fache MP;Brachet A;Ferracci G;Baude A;Irondelle M;Pereira S;Leterrier C;Dargent B
通讯作者：
Dargent B
影响因子：
4.8
作者：
Miloushev, Vesselin Z.;Levine, Joshua A.;Palmer, Arthur G., III
通讯作者：
Palmer, Arthur G., III
DOI：
10.1073/pnas.84.23.8682
发表时间：
1987-12-01
影响因子：
11.1
作者：
GORDON, D;MERRICK, D;CATTERALL, WA
通讯作者：
CATTERALL, WA
影响因子：
5.3
作者：
Rasband, MN;Peles, E;Shrager, P
通讯作者：
Shrager, P