a sensitive membrane-targeted biosensor for monitoring changes in intracellular chloride in neuronal processes一个敏感membrane-targeted生物传感器用于监视在神经元胞内氯的变化过程.pdfVIP

A Sensitive Membrane-Targeted Biosensor for Monitoring Changes in Intracellular Chloride in Neuronal Processes 1 2 1 2 Spencer D. Watts , Katherine L. Suchland , Susan G. Amara , Susan L. Ingram * 1 Department of Neurobiology, Medical School, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Department of Neurological Surgery, Oregon Health Science University, Portland, Oregon, United States of America Abstract Background: Regulation of chloride gradients is a major mechanism by which excitability is regulated in neurons. Disruption of these gradients is implicated in various diseases, including cystic fibrosis, neuropathic pain and epilepsy. Relatively few studies have addressed chloride regulation in neuronal processes because probes capable of detecting changes in small compartments over a physiological range are limited. Methodology/Principal Findings: In this study, a palmitoylation sequence was added to a variant of the yellow fluorescent protein previously described as a sensitive chloride indicator (YFPQS) to target the protein to the plasma membrane (mbYFPQS) of cultured midbrain neurons. The reporter partitions to the cytoplasmic face of the cellular membranes, including the plasma membrane throughout the neurons and fluorescence is stable over 30–40 min of repeated excitation showing less than 10% decrease in mbYFPQS fluorescence compared to baseline. The mbYFPQS has similar chloride sensitivity (k50 = 41 mM) but has a shifted pKa compared to the unpalmitoylated YFPQS variant (cytYFPQS) that remains in the cytoplasm when expressed in midbrain neurons. Changes in mbYFPQS fluorescence were induc