Fig. 2: Functional characterization of eErNaR in HEK293T cells.

a Representative confocal images of HEK293T cell expressing eErNaR. Fluorescence of EYFP, fused to eErNaR, is shown in green; the plasma membrane stain CellMask - in magenta; nucleus stain Hoechst 33342 - in gray. The co-localization of EYFP and CellMask appears in white. Scale bars, 10 µm. The localization was similar in at least 6 representative confocal images from 2 independent transfections. b Representative photocurrents of eErNaR recorded from HEK293T cells at 130 mM (left), 20 mM (middle) and 0 mM (right) intracellular [Na⁺]_i and pH_i 7.5. c Voltage dependence of the stationary photocurrents of eErNaR at intracellular [Na⁺]_i 130 mM (left), 20 mM (middle) and 0 mM (right) and different pH_i values (LJP-corrected; normalized to respective cell capacitance; mean ± SEM; n  =  9 cells at [Na⁺]_i 130 mM, pH_i 5.0, 7.5 and 9.0; n = 7 at [Na⁺]_i 20 mM, pH_i 5.0, 7.5, 9.0 and at [Na⁺]_i 0 mM, pH_i 9.0; n = 6 at [Na⁺]_i 0 mM, pH_i 5.0 and 7.5). d Stationary photocurrents of eErNaR at +60 mV, normalized to respective cell capacitance (mean ± SEM and individual data points). Data were extracted from the recordings at different intracellular [Na⁺]_i and pH_i described in (c). Normalized currents were analyzed using two-way ANOVA with two Turkey’s multiple comparisons tests – for the effect of pH_i at fixed [Na⁺]_i (depicted on the graph, ns is not shown) and for the effect of [Na⁺]_i at fixed pH_i (inset table). e Kinetics of photocurrent decay upon light-off at 130 mM intracellular [Na⁺]_i and different pH_i (mean ± SEM and individual data points of n = 5, 7, and 9 cells at pH_i 5.0, 7.5 and 9.0, respectively). The time constant (t_off) was determined by monoexponential fit of photocurrent decay at holding voltage +80 mV. Data were analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons test (all ns). b–e All patch-clamp experiments were conducted at 110 mM extracellular [Na⁺]_e, pH_e 7.5; LED light with maximum at 550 nm was applied for 1 s at 34.3 mW/mm² irradiance. ns – not significant.