Chotoo Cavita K, Silverman Gary A, Devor Daniel C, Luke Cliff J
Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
PLoS One. 2013 Sep 10;8(9):e75869. doi: 10.1371/journal.pone.0075869. eCollection 2013.
In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying.
在小鼠的神经系统中,小电导钙激活钾(SK)通道通过产生动作电位后超极化中期的一个成分来调节神经元兴奋性。在人类中,动作电位发放频率不规则是共济失调、癫痫、精神分裂症和帕金森病等疾病的基础。由于在哺乳动物神经系统中研究蛋白质功能的复杂性,我们试图在秀丽隐杆线虫中鉴定一种SK通道同源物KCNL-2,秀丽隐杆线虫是一种遗传上易于处理的系统,其中单个神经元的谱系从其早期发育阶段就已绘制出来。KCNL-2蛋白的序列分析表明,其六个跨膜结构域、钾选择性孔和钙调蛋白结合结构域与哺乳动物同源物高度保守。我们使用宽视野和共聚焦荧光成像表明,在转基因品系中KCNL-2与绿色荧光蛋白(GFP)的融合构建体在秀丽隐杆线虫的神经系统中表达。我们还表明,KCNL-2基因敲除菌株kcnl-2(tm1885)表现出轻度的产卵缺陷表型,该表型以KCNL-2依赖的方式得到挽救。相反,我们表明过表达KCNL-2的转基因品系表现出产卵过度活跃的表型。在本研究中,我们表明转基因雌雄同体的阴门由神经元突起以及表达GFP标记的KCNL-2的VC4和VC5神经元高度支配。我们提出KCNL-2在秀丽隐杆线虫的神经系统中发挥作用以调节产卵速率。