Saturation mutagenesis was performed on a single position in the voltage-sensing domain (VSD) of a genetically encoded voltage indicator (GEVI). The VSD consists of four transmembrane helixes designated S1-S4. The V220 position located near the plasma membrane/extracellular interface had previously been shown to affect the voltage range of the optical signal. Introduction of polar amino acids at this position reduced the voltage-dependent optical signal of the GEVI. Negatively charged amino acids slightly reduced the optical signal by 33 percent while positively charge amino acids at this position reduced the optical signal by 80%. Surprisingly, the range of V220D was similar to that of V220K with shifted optical responses towards negative potentials. In contrast, the V220E mutant mirrored the responses of the V220R mutation suggesting that the length of the side chain plays in role in determining the voltage range of the GEVI. Charged mutations at the 219 position all behaved similarly slightly shifting the optical response to more negative potentials. Charged mutations to the 221 position behaved erratically suggesting interactions with the plasma membrane and/or other amino acids in the VSD. Introduction of bulky amino acids at the V220 position increased the range of the optical response to include hyperpolarizing signals. Combining The V220W mutant with the R217Q mutation resulted in a probe that reduced the depolarizing signal and enhanced the hyperpolarizing signal which may lead to GEVIs that only report neuronal inhibition.

中文翻译：

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调制基因编码电压指示器的电压灵敏度。

在遗传编码电压指示器（GEVI）的电压感应域（VSD）中的单个位置上进行了饱和诱变。VSD由四个称为S1-S4的跨膜螺旋组成。先前已显示位于质膜/细胞外界面附近的V220位置会影响光信号的电压范围。在该位置引入极性氨基酸会降低GEVI的电压依赖性光信号。带负电荷的氨基酸将光信号稍微降低了33％，而在此位置带正电荷的氨基酸将光信号降低了80％。出人意料的是，V220D的范围与V220K的范围相似，但光学响应朝负电位方向偏移。相比之下，V220E突变体反映了V220R突变的反应，表明侧链的长度在确定GEVI的电压范围中起作用。在219位的带电突变都表现出相似的轻微变化，使光学响应向更多的负电位移动。221位的带电突变表现为不稳定，表明与VSD中的质膜和/或其他氨基酸发生相互作用。在V220位置引入大量氨基酸会增加光学响应的​​范围，以包括超极化信号。将V220W突变体与R217Q突变相结合，产生的探针可减少去极化信号并增强超极化信号，这可能导致仅报告神经元抑制作用的GEVIs。