Naylor J, Milligan C J, Zeng F, Jones C, Beech D J
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Br J Pharmacol. 2008 Oct;155(4):567-73. doi: 10.1038/bjp.2008.283. Epub 2008 Jul 7.
Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family.
A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca(2+) measurement.
Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5-10 min. Ca(2+) entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response.
The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers.
亚型特异性离子通道阻滞剂在药物研发的靶点验证中很有用，可为新型治疗药物提供依据，并有助于确定离子通道的生理功能。本研究的目的是开发一种人TRPM3通道的特异性阻滞剂，作为帮助研究TRP阳离子通道家族这一成员的工具。
制备了针对TRPM3第三个细胞外环（E3）保守肽段的多克隆抗体（TM3E3），并测试其结合能力和功能效应。通过全细胞平面膜片钳和fura-2细胞内Ca²⁺测量研究通道活性。
TRPM3介导的离子电流在5 - 10分钟内被TM3E3部分抑制。TM3E3还以肽段特异性方式部分抑制表达TRPM3的细胞中的Ca²⁺内流，且与用于激活TRPM3的激动剂类型无关。TM3E3对TRPC5、TRPV4、TRPM2或内源性ATP反应无影响。
数据表明成功开发了一种特异性TRPM3抑制剂，并进一步证明以E3为靶点是生产亚型特异性离子通道阻滞剂的一种方法。
