Kang Dawon, Choe Changyong, Cavanaugh Eric, Kim Donghee
Department of Physiology & Biophysics, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064, USA.
J Physiol. 2007 Aug 15;583(Pt 1):57-69. doi: 10.1113/jphysiol.2007.136150. Epub 2007 May 31.
TREK-2 (K2P10.1), a member of the two-pore domain K+ (K2P) channel family, provides the background K+ conductance in many cell types, and is a target of neurotransmitters that act on receptors coupled to Gs and Gq. We report here that TREK-2 exhibits small (TREK-2S) and large (TREK-2L) conductance phenotypes when expressed in mammalian cell lines (COS-7, HEK293, HeLa) and in Xenopus oocytes. TREK-2S phenotype shows a noisy open state with a mean conductance of 54 pS (+40 mV). TREK-2L phenotype shows a full open state (202 pS) with several short-lived sub-conductance levels. Both phenotypes were strongly activated by arachidonic acid, membrane stretch (-40 mmHg) and intracellular acidification (pH 6.4). Phosphorylation of TREK-2 produced by treatment of cells with activators of protein kinases A and C, and okadaic acid (a serine/threonine phosphatase inhibitor) decreased the current contributed by TREK-2S and TREK-2L, and caused partial switching of conductance levels from those of TREK-2S and TREK-2L to more intermediate values. Under this condition, TREK-2 exhibited six conducting levels and one closed level. TREK-2 mutants in which putative protein kinases A and C phosphorylation sites were mutated to alanines (S326A, S359A, S326A/S359A) displayed mostly TREK-2S and TREK-2L phenotypes. However, S326D and S359D mutants (as well as the double mutants) that mimic the phosphorylated state showed all six conducting levels and low channel activity. The S326A and S359A mutants did not significantly affect the intrinsic voltage dependence of TREK-2 in Mg2+-free solution. Phenotypes resembling TREK-2S and TREK-2L were also observed in cerebellar granule neurons that express TREK-2 mRNA. These results show that TREK-2 exhibits two primary modes of gating that give rise to two channel phenotypes under dephosphorylated conditions, and that its phosphorylation shifts the gating mode to include intermediate conducting levels. This represents a novel mechanism by which receptor agonists modulate the function of a K+ channel to alter cell excitability.
TREK-2（K2P10.1）是双孔结构域钾离子（K2P）通道家族的成员，在多种细胞类型中提供背景钾离子电导，并且是作用于与Gs和Gq偶联受体的神经递质的靶点。我们在此报告，当在哺乳动物细胞系（COS-7、HEK293、HeLa）和非洲爪蟾卵母细胞中表达时，TREK-2呈现出小电导（TREK-2S）和大电导（TREK-2L）表型。TREK-2S表型显示出一种嘈杂的开放状态，平均电导为54 pS（+40 mV）。TREK-2L表型显示出完全开放状态（202 pS），伴有几个短暂的亚电导水平。两种表型均被花生四烯酸、膜拉伸（-40 mmHg）和细胞内酸化（pH 6.4）强烈激活。用蛋白激酶A和C的激活剂以及冈田酸（一种丝氨酸/苏氨酸磷酸酶抑制剂）处理细胞所产生的TREK-2磷酸化，降低了TREK-2S和TREK-2L所贡献的电流，并导致电导水平从TREK-2S和TREK-2L的水平部分转变为更中间的值。在此条件下，TREK-2呈现出六个电导水平和一个关闭水平。将假定的蛋白激酶A和C磷酸化位点突变为丙氨酸的TREK-2突变体（S326A、S359A、S326A/S359A）大多表现出TREK-2S和TREK-2L表型。然而，模拟磷酸化状态的S326D和S359D突变体（以及双突变体）表现出所有六个电导水平且通道活性较低。S326A和S359A突变体在无镁溶液中对TREK-2的内在电压依赖性没有显著影响。在表达TREK-2 mRNA的小脑颗粒神经元中也观察到了类似于TREK-2S和TREK-2L的表型。这些结果表明，TREK-2呈现出两种主要的门控模式，在去磷酸化条件下产生两种通道表型，并且其磷酸化将门控模式转变为包括中间电导水平。这代表了一种新的机制，通过该机制受体激动剂调节钾离子通道的功能以改变细胞兴奋性。
