期刊
JOURNAL OF NEUROSCIENCE METHODS
卷 170, 期 1, 页码 67-76出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jneumeth.2007.12.016
关键词
fluorescent proteins; real-time optical detection; patch-clamp; glycine receptor
Chloride (Cl) is the most abundant physiological anion. Abnormalities in Cl regulation are instrumental in the development of several important diseases including motor disorders and epilepsy. Because of difficulties in the spectroscopic measurement of Cl in live tissues there is little knowledge available regarding the mechanisms of regulation of intracellular Cl concentration. Several years ago, a CFP-YFP based ratiometric Cl indicator (Clomeleon) was introduced [Kuner, T., Augustine, G.J. A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 2000; 27: 447-59]. This construct with relatively low sensitivity to C1 (K-app similar to 160 mM) allows ratiometric monitoring of C1 using fluorescence emission ratio. Here, we propose a new CFP-YFP-based construct (Cl-sensor) with relatively high sensitivity to Cl (K-app similar to 30 mM) due to triple YFP mutant. The construct also exhibits good pH sensitivity with pK(alpha) ranging from 7.1 to 8.0 pH units at different Cl concentrations. Using Cl-sensor we determined non-invasively the distribution of [Cl](i) in cultured CHO cells, in neurons of primary hippocampal cultures and in photoreceptors of rat retina. This genetically encoded indicator offers a means for monitoring Cl and pH under different physiological conditions and high-throughput screening of pharmacological agents. (c) 2008 Elsevier B.V. All rights reserved.
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