文章基本信息

标题：A noninvasive optical approach for assessing chloride extrusion activity of the K–Cl cotransporter KCC2 in neuronal cells
本地全文：下载
作者：Anastasia Ludwig ; Claudio Rivera ; Pavel Uvarov 等
期刊名称：BMC Neuroscience
印刷版ISSN：1471-2202
电子版ISSN：1471-2202
出版年度：2017
卷号：18
期号：1
页码：23
DOI：10.1186/s12868-017-0336-5
语种：English
出版社：BioMed Central
摘要：Background Cation-chloride cotransporters (CCCs) are indispensable for maintaining chloride homeostasis in multiple cell types, but K–Cl cotransporter KCC2 is the only CCC member with an exclusively neuronal expression in mammals. KCC2 is critical for rendering fast hyperpolarizing responses of ionotropic γ-aminobutyric acid and glycine receptors in adult neurons, for neuronal migration in the developing central nervous system, and for the formation and maintenance of small dendritic protrusions—dendritic spines. Deficit in KCC2 expression and/or activity is associated with epilepsy and neuropathic pain, and effective strategies are required to search for novel drugs augmenting KCC2 function. Results We revised current methods to develop a noninvasive optical approach for assessing KCC2 transport activity using a previously characterized genetically encoded chloride sensor. Our protocol directly assesses dynamics of KCC2-mediated chloride efflux and allows measuring genuine KCC2 activity with good spatial and temporal resolution. As a proof of concept, we used this approach to compare transport activities of the two known KCC2 splice isoforms, KCC2a and KCC2b, in mouse neuronal Neuro-2a cells. Conclusions Our noninvasive optical protocol proved to be efficient for assessment of furosemide-sensitive chloride fluxes. Transport activities of the N-terminal splice isoforms KCC2a and KCC2b obtained by the novel approach matched to those reported previously using standard methods for measuring chloride fluxes.
关键词：Genetically encoded chloride sensor ; Slc12a5 gene ; KCC2 ; Inhibition ; GABA