iGABASnFR2: Improved genetically encoded protein sensors of GABA

Kolb, I.; Hasseman, J. P.; Matsumoto, A.; Arthur, B. J.; Zhang, Y.; Tsang, A.; Reep, D.; Tsegaye, G.; Zheng, J.; Patel, R.; Looger, L. L.; Marvin, J. S.; Korff, W. L.; Yonehara, K.; Turner, G. C.

doi:10.1101/2025.03.25.644953

bioRxiv · 10.1101/2025.03.25.644953

iGABASnFR2: Improved genetically encoded protein sensors of GABA

Kolb, I.·Hasseman, J. P.·Matsumoto, A.·Arthur, B. J.·Zhang, Y.·Tsang, A.·Reep, D.·Tsegaye, G.·Zheng, J.·Patel, R.·Looger, L. L.·Marvin, J. S.·Korff, W. L.·Yonehara, K.·Turner, G. C.

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Abstract

Monitoring GABAergic inhibition in the nervous system has been enabled by development of an intensiometric molecular sensor that directly detects GABA. However, the first generation iGABASnFR exhibits low signal-to-noise and suboptimal kinetics, making in vivo experiments challenging. To improve sensor performance, we targeted several sites in the protein for near-saturation mutagenesis and evaluated the resulting sensor variants in a high throughput screening system using evoked synaptic release in primary cultured neurons. This identified a sensor variant, iGABASnFR2, with 4.1-fold improved sensitivity and 30% faster rise time, and binding affinity that remained in a range sensitive to changes in GABA concentration at synapses. We also identified sensors with an inverted response, decreasing fluorescence intensity upon GABA binding. We termed the best such negative-going sensor iGABASnFR2n, which can be used to corroborate observations with the positive-going sensor. These improvements yielded a qualitative enhancement of in vivo performance when compared directly to the original sensor. iGABASnFR2 enabled the first measurements of direction-selective GABA release in the retina. In vivo imaging in somatosensory cortex revealed that iGABASnFR2 can report volume-transmitted GABA release following whisker stimulation. Overall, the improved sensitivity and kinetics of iGABASnFR2 make it a more effective tool for imaging GABAergic transmission in intact neural circuits.

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Kolb, I., Hasseman, J. P., Matsumoto, A., Arthur, B. J., Zhang, Y., Tsang, A., Reep, D., Tsegaye, G., Zheng, J., Patel, R., Looger, L. L., Marvin, J. S., Korff, W. L., Yonehara, K., Turner, G. C.. 2025-03-26. iGABASnFR2: Improved genetically encoded protein sensors of GABA. https://doi.org/10.1101/2025.03.25.644953

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