生物物理学
荧光
化学
荧光团
全内反射荧光显微镜
费斯特共振能量转移
电生理学
膜片钳
口腔1
离子通道
膜电位
膜
生物化学
生物
光学
神经科学
物理
受体
刺激1
作者
Joseph L. Dynes,Andriy V. Yeromin,Michael D. Cahalan
标识
DOI:10.1073/pnas.2309328120
摘要
We used electrophysiology and Ca 2+ channel tethering to evaluate the performance of jGCaMP8 genetically encoded Ca 2+ indicators (GECIs). Orai1 Ca 2+ channel–jGCaMP8 fusions were transfected into HEK 293A cells and jGCaMP8 fluorescence responses recorded by simultaneous total internal reflection fluorescence microscopy and whole-cell patch clamp electrophysiology. Noninactivating currents from the Orai1 Y80E mutant provided a steady flux of Ca 2+ controlled on a millisecond time scale by step changes in membrane potential. Test pulses to −100 mV produced Orai1 Y80E-jGCaMP8f fluorescence traces that unexpectedly declined by ~50% over 100 ms before reaching a stable plateau. Testing of Orai1-jGCaMP8f using unroofed cells further demonstrated that rapid and partial fluorescence inactivation is a property of the indicator itself, rather than channel function. Photoinactivation spontaneously recovered over 5 min in the dark, and recovery was accelerated in the absence of Ca 2+ . Mutational analysis of residues near the tripeptide fluorophore of jGCaMP8f pointed to a mechanism: Q69M/C70V greatly increased (~90%) photoinactivation, reminiscent of fluorescent protein fluorophore cis-trans photoswitching. Indeed, 405-nm illumination of jGCaMP8f or 8m/8s/6f led to immediate photorecovery, and simultaneous illumination with 405 and 488-nm light blocked photoinactivation. Subsequent mutagenesis produced a variant, V203Y, that lacks photoinactivation but largely preserves the desirable properties of jGCaMP8f. Our results point to caution in interpreting rapidly changing Ca 2+ signals using jGCaMP8 and earlier series GECIs, suggest strategies to avoid photoswitching, and serve as a starting point to produce more photostable, and thus more accurate, GECI derivatives.
科研通智能强力驱动
Strongly Powered by AbleSci AI