Modulating the Voltage-sensitivity of a Genetically Encoded Voltage Indicator

Abstract

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.

A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice

“…1F ). Given the empirical nature for the voltage ranges of GEVIs 14 , 24 , 25 , 200 mV depolarizations were also performed to identify potential probes in need of voltage tuning. All of the constructs showed only a slight increase in the optical signal for 200 mV depolarizations, eliminating the need to further optimize the VSD.…”

Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity

“…By changing VSP concentrations, PIP concentrations can now be more finely tuned, targeting only 5-phosphates instead of both 3- and 5-phosphate. In addition, VSP domains have been used to generate biosensors to noninvasively track biologically relevant signaling processes within live cells ( Dimitrov et al, 2007 ; Lundby et al, 2008 ; Jung et al, 2017 ; Lee et al, 2017 ). For many years, making a sensor to track membrane voltage was stymied because the sensors were not properly trafficked to the plasma membrane.…”

Voltage and Calcium Imaging of Brain Activity

“…Furthermore, the range of their voltage responses can be adjusted along the voltage axis (41) and thus they can be selectively sensitive to the membrane potential ranges that occur during action potentials or during inhibition. Furthermore, there is preliminary evidence that the steepness of the signal versus voltage relationship can be modified by inhibiting the movement of S4 in one direction (69) or by altering the charge in the linker region between the voltage-sensing domain and the FP (B. J. Yi, S. Braubach, and B. J. Baker, unpublished data).…”