Effect of Voltage Sensitive Fluorescent Proteins on Neuronal Excitability
Effect of Voltage Sensitive Fluorescent Proteins on Neuronal Excitability
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DOI：
10.1016/j.bpj.2009.02.046
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发表时间：
2009-05-20
影响因子：
3.4
通讯作者：
Knopfel, Thomas
中科院分区：
文献类型：
--
作者：
Akemann, Walther;Lundby, Alicia;Knopfel, Thomas
荧光蛋白电压传感器是利用荧光的电压依赖性调节机制设计为膜电位的遗传编码细胞探针的重组蛋白。包括VSFP2.3和VSFP3.1在内的几种此类蛋白最近被报道在哺乳动物细胞中具有可靠的功能。它们被设计为含有荧光报告结构域的磷酸酶的玻璃海鞘电压传感器的电压传感器的分子融合物。这些蛋白质在细胞膜中的表达伴随着额外的动态膜电容或“感测电容”，对靶细胞的天然电响应性具有反馈效应。我们使用VSFP2.3和VSFP3.1的传感电流和荧光响应的记录来推导这些蛋白质的电压依赖性信号传导的动力学模型。使用计算神经元模拟，我们定量研究了两个中枢神经元模型，小脑浦肯野和第5层锥体神经元的输入/输出关系的感知电容的扰动效应。探针诱导的传感电容表现为动作电位的时移和体细胞动作电位启动的突触输入阈值增加，并与探针的膜密度呈线性依赖关系。而荧光信号/噪声随着探针的表面密度的平方根而增长，感测电容的增长是线性的。我们分析了最小化的传感电容和信号/噪声的光学读出取决于动力学性质和细胞分布的探针之间的权衡。仿真结果提出了在给定的信号/噪声水平下减少电容效应的方法。然而，模拟表明，仍然需要显着改善现有的探针，以报告在单个试验中哺乳动物脑组织中的单个神经元的动作电位。
Fluorescent protein voltage sensors are recombinant proteins that are designed as genetically encoded cellular probes of membrane potential using mechanisms of voltage-dependent modulation of fluorescence. Several such proteins, including VSFP2.3 and VSFP3.1, were recently reported with reliable function in mammalian cells. They were designed as molecular fusions of the voltage sensor of Ciona intestinalis voltage sensor containing phosphatase with a fluorescence reporter domain. Expression of these proteins in cell membranes is accompanied by additional dynamic membrane capacitance, or "sensing capacitance", with feedback effect on the native electro-responsiveness of targeted cells. We used recordings of sensing currents and fluorescence responses of VSFP2.3 and of VSFP3.1 to derive kinetic models of the voltage-dependent signaling of these proteins. Using computational neuron simulations, we quantitatively investigated the perturbing effects of sensing capacitance on the input/output relationship in two central neuron models, a cerebellar Purkinje and a layer 5 pyramidal neuron. Probe-induced sensing capacitance manifested as time shifts of action potentials and increased synaptic input thresholds for somatic action potential initiation with linear dependence on the membrane density of the probe. Whereas the fluorescence signal/noise grows with the square root of the surface density of the probe, the growth of sensing capacitance is linear. We analyzed the trade-off between minimization of sensing capacitance and signal/noise of the optical read-out depending on kinetic properties and cellular distribution of the probe. The simulation results suggest ways to reduce capacitive effects at a given level of signal/noise. Yet, the simulations indicate that significant improvement of existing probes will still be required to report action potentials in individual neurons in mammalian brain tissue in single trials.