组成图示
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传感器类型
荧光生物传感器
检测对象
胞内氯离子（intracellular chloride, Cl-）；样品基质：培养中脑神经元（cultured midbrain neurons）胞质与突起
检测原理
mbYFPQS由YFPQS（YFP-H148Q/V163S）与神经调节蛋白N端棕榈酰化序列融合而成，表达后锚定于神经元质膜胞质侧。YFPQS的荧光素体附近位点可结合/响应Cl-，Cl-结合改变发色团pKa及质子化状态，使YFP荧光强度随胞内Cl-浓度变化：高Cl-时荧光降低，低Cl-时荧光升高。校准显示k50约41 mM。当GABAA受体被muscimol激活或DAT被amphetamine等底物刺激时，Cl-经通道/转运体跨膜通量改变胞内Cl-浓度，mbYFPQS荧光随之增强或减弱。荧光显微镜采集YFP信号，从而实时反映神经元胞体与突起的胞内氯变化。膜靶向减少全细胞膜片钳中蛋白扩散，提高同步电生理与成像稳定性。
检测灵敏度
k50: 41 ±3 mM（n=17）；R^2 = 0.993（soma）和 0.985（process）
效应效果
mbYFPQS在30–40 min重复激发下荧光下降<10%（10±5%，n=5），膜片钳下下降8±4%（n=4），优于cytYFPQS（tau 76±17 s vs 633±50 s，p<0.01）。其k50=41±3 mM，优于YFP（168 mM）和Clomeleon（160 mM），接近MQAE（2–40 mM）。pKa右移至8.0±0.2（8 mM Cl-）和8.5±0.2（30 mM Cl-），生理pH 7.5–7.2影响较小。muscimol（20 mM）诱导变化可被bicuculline（10 mM）抑制，bumetanide（25 mM）可减弱/逆转；amphetamine（1–20 mM）剂量依赖增加荧光，GBR12909（10 mM）可逆转。作者认为其适合同步全细胞膜片钳与活细胞成像，监测神经元突起内源性氯变化。
传感器的构成
- 基底/传感界面：细胞质膜（plasma membrane）与胞内环境，作为mbYFPQS定位和检测胞内Cl-的界面
- 定位修饰层：神经调节蛋白N端棕榈酰化信号肽（neuromodulin palmitoylation sequence），使蛋白锚定于膜胞质侧
- 识别/换能元件：氯离子敏感黄色荧光蛋白变体YFPQS（YFP-H148Q/V163S），通过Cl-结合改变荧光
- 融合传感蛋白：膜靶向mbYFPQS（palmitoylated YFPQS），表达于培养中脑神经元
- 信号标记物：YFPQS自身YFP荧光（EYFP/YFP），随胞内Cl-浓度变化
- 读出装置：荧光显微镜、CCD相机与YFP滤光片（Excitation 500/Emission 545），采集荧光强度
中文摘要
氯离子梯度调控是神经元兴奋性调节的重要机制，其紊乱与囊性纤维化、神经病理性疼痛和癫痫等疾病相关。由于缺乏能在生理范围内检测小胞室变化的探针，神经元突起中的氯离子调控研究较少。本研究在先前报道的敏感氯离子指示黄色荧光蛋白变体YFPQS中加入棕榈酰化序列，使其靶向培养中脑神经元质膜，得到mbYFPQS。该报告蛋白定位于细胞膜胞质侧，包括神经元整体质膜；在30–40分钟重复激发下荧光稳定，较基线下降小于10%。mbYFPQS的氯离子敏感性与未棕榈酰化的胞质型cytYFPQS相近（k50=41 mM），但pKa发生偏移。GABAA受体激动剂muscimol可诱导mbYFPQS荧光变化，且在中脑神经元胞体和突起中相似。安非他命在部分培养中脑神经元中增加mbYFPQS荧光，该变化可被选择性多巴胺转运体（DAT）抑制剂GBR12909逆转，表明mbYFPQS足以检测中脑多巴胺能神经元内源性DAT活性。结论：mbYFPQS是研究神经元突起胞内氯水平调控的敏感工具，尤其适用于同步全细胞膜片钳与活细胞成像实验。
英文摘要
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 (k(50) = 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 induced by the GABA(A) agonist muscimol and were similar in the soma and processes of the midbrain neurons. Amphetamine also increased mbYFPQS fluorescence in a subpopulation of cultured midbrain neurons that was reversed by the selective dopamine transporter (DAT) inhibitor, GBR12909, indicating that mbYFPQS is sensitive enough to detect endogenous DAT activity in midbrain dopamine (DA) neurons.
CONCLUSIONS/SIGNIFICANCE: The mbYFPQS biosensor is a sensitive tool to study modulation of intracellular chloride levels in neuronal processes and is particularly advantageous for simultaneous whole-cell patch clamp and live-cell imaging experiments.