Nature Methods：以病毒为基础的离子通道调控器筛选法

生物谷

    生物谷报道：在8月在线出版的《自然—方法学》期刊上，研究人员描述了一种以病毒为基础的对化学或基因编码的离子通道抑制器进行筛选的方法。

    离子通道是遍布在细胞膜上的蛋白质，一旦被激活就会允许离子的流入。离子通道这个大家族共有400多种蛋白质，它们在细胞功能的维持中发挥了关键作用。当抑制某种离子通道的活性被证明有益于某种医学治疗时，研究人员开发出了抑止离子通道活性的药物，但这些药物的活力范围太大而又缺乏选择性，因此也带来了许多副作用。

    Joseph  Glorioso和同事希望找到更多的选择性离子通道调节器，他们建立了以病毒为基础的筛选法，也就是用病毒作为抑制剂效力的指示剂。他们分析指出，通过病毒让细胞中的某种离子通道过度表达会对细胞产生害处，就会因此阻止病毒的复制；而离子功能抑制器的出现则有助于保持细胞的健康并允许复制。

    新方法优于其他离子抑制剂的地方是，通过共同感染编码抑制剂的第二种病毒，新方法很容易用于筛选基因编码的抑制剂，而DNA的抑制剂又很容易从病毒基因组中检索出来。

    新方法更可用于筛选表达大范围潜在离子通道调控器的整个DNA库。这些基因编码的抑制器将有助于揭开通道调控背后的生物学，并有可能导致更多的特定抑制剂。（科学时报）

原始出处:

Nature Methods

Published online: 5 August 2007; | doi:10.1038/nmeth1077

An HSV vector system for selection of ligand-gated ion channel modulators

Rahul Srinivasan1, Shaohua Huang1, Suchita Chaudhry1, Adrian Sculptoreanu2, David Krisky3, Michael Cascio1, Peter A Friedman2, William C de Groat2, Darren Wolfe1 & Joseph C Glorioso1

1 Department of Molecular Genetics and Biochemistry, 200 Lothrop Street, Biomedical Science Tower, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

2 Department of Pharmacology, 200 Lothrop Street, Biomedical Science Tower, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

3 Department of Pathology, 200 Lothrop Street, Biomedical Science Tower, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

Correspondence should be addressed to Joseph C Glorioso glorioso@pitt.edu

Pathological alterations of ion channel activity result from changes in modulatory mechanisms governing receptor biology. Here we describe a conditional herpes simplex virus (HSV) replication-based strategy to discover channel modulators whereby inhibition of agonist-induced channel activation by a vector-expressed modulatory gene product prevents ion flux, osmotic shock and cell death. Inhibition of channel activity, in this case, the rat vanilloid (Trpv1 or the glycine receptor (GlyR alpha 1), can allow selection of escape vector plaques containing the 'captured' modulatory gene for subsequent identification and functional analysis. We validated this prediction using mixed infections of a wild-type Trpv1 expression vector vTTHR and a nonfunctional 'poreless' Trpv1 subunit-expressing vector, vHP, wherein vHP was highly selected from a large background of vTTHR viruses in the presence of the Trpv1 agonist, capsaicin. The approach should be useful for probing large libraries of vector-expressed cDNAs for the presence of ion channel modulators.