iGABASnFR2: Improved genetically encoded protein sensors of GABA

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bioRxiv. 2025 Mar 25;. doi: 10.1101/2025.03.25.644953

iGABASnFR2: Improved genetically encoded protein sensors of GABA GENIEScientific Computing Software

Kolb I, Hasseman JP, Matsumoto A, Arthur BJ, Zhang Y, Tsang A, Reep D, Tsegaye G, Zheng J, Patel R, Looger LL, Marvin JS, Korff WL, Yonehara K, Turner GC

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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 4.2-fold improved sensitivity and 20% faster kinetics, 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, enabling us to make the first measurements of direction selective GABA release in the retina and confirm a longstanding hypothesis for how sensitivity to motion arises in the visual system.

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iGABASnFR2: Improved genetically encoded protein sensors of GABA

Abstract

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