Figure 2.

Intracellular acidification and slow photocurrent decline. (a) Experimental scheme of simultaneous voltage-clamp and intracellular pH measurements: HEK293 cells were loaded with BCECF free acid using the patch pipette. pH-dependent fluorescence of BCECF at 530 nm was excited by 5 ms pulses of 500 nm light during electrical recordings (bright-field and fluorescent images of a transfected HEK293 cell in the whole-cell configuration. The fluorescence intensity is represented by false colors). (b) Top: voltage and illumination protocol featuring two successive 15 s illuminations of 560 nm first at −60 mV and then at −20 mV and continuously pulsed excitation of BCECF at 500 nm. Middle: Photocurrent recording in symmetric 110 mM NaCl and pH_e/i 7.2. Bottom: Normalized BCECF fluorescence and the corresponding approximated intracellular pH_i values estimated by the BCECF calibration in supplementary Fig. 3b. (c) The same recording as in (b) with a cell first clamped at +20 mV and then at −20 mV. (d) The normalized changes in intracellular fluorescence (with approximated intracellular pH_i values) after 560 nm illumination at −60 mV (red) or +20 mV (orange) and after the second illumination at −20 mV (red/orange) in Chrimson or enhanced cyan fluorescent protein (gray) expressing cells as a control (mean ± SD, Chrimson n = 10–15 cells, eCFP n = 6 cells).