Photoswitching alters fluorescence readout of jGCaMP8 Ca²⁺ indicators tethered to Orai1 channels.

ABSTRACT: We used electrophysiology and Ca²⁺ channel tethering to evaluate the performance of jGCaMP8 genetically encoded Ca²⁺ indicators (GECIs). Orai1 Ca²⁺ 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 Ca²⁺ 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 rapi

SUBMITTER: Dynes JL 

PROVIDER: S-EPMC10523504 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature