Photo-inactivation disrupts rapid fluorescence readout of jGCaMP8 calcium indicators tethered to Orai1 channels.

Abstract

We sought to optimize indicator response time in channel-tethered genetically encoded Ca2+ indicators (GECIs) using the jGCaMP8 series indicators released by the HHMI Genie Project. These indicators possess <10 ms rise times, substantially narrowing the temporal resolution gap between electrical and optical recording of Ca2+ currents. DNA encoding jGCaMP8f was inserted downstream of the Orai1 coding sequence to monitor Orai1 Ca2+ channel activity using TIRF microscopy combined with whole-cell recording. The use of non-inactivating channel currents provided a steady and electrically controlled source of Ca2+ to compare current and fluorescence readouts of Orai1 channel activity in transfected HEK 293A cells. Non-inactivating test pulses to −40 mV produced fluorescence traces that unexpectedly declined by ∼25% over 100 ms before reaching a stable plateau. Moreover, using an Orai1 Y80E mutant that lacks fast Ca2+-dependent inactivation, fluorescence traces declined by ∼50% at −100 mV. Testing of Orai1-jGCaMP8f using unroofed cells demonstrated that rapid and partial fluorescence inactivation is a property of the indicator itself, rather than channel function. Photo-inactivation spontaneously recovered over 5 minutes, and recovery was accelerated in the absence of Ca2+. Mutational analysis of residues near the tri-peptide fluorophore of jGCaMP8f pointed to a mechanism: Q69M/C70V greatly increased (∼90%) photo-inactivation reminiscent of fluorescent protein fluorophore cis-trans photo-switching. Indeed, 405 nm illumination of jGCaMP8f or 8m/8s/6f led to immediate photo-recovery, and simultaneous illumination with 405 and 488 nm light blocked photo-inactivation. Subsequent mutagenesis produced a variant that lacks photo-inactivation but largely preserves the desirable properties of jGCaMP8f. Our results point to caution in interpreting rapidly changing Ca2+ signals using jGCaMP8 indicators, suggest strategies to avoid photo-switching, and serve as a starting point to produce more photo-stable, and thus more accurate, GECI derivatives.