Figure 3: roNaV2 senses light-induced chemical modification.
(a) Current recordings (bottom) from a HEK 293 cell expressing roNaV2 in response to a train of depolarizing pulses to −20 mV from a holding potential of −120 mV (top). The blue bar marks illumination of the cell with 470-nm light via a 63x oil immersion objective. The red curve superimposed to the current traces illustrates a data fit of the time course of light-induced loss of channel inactivation, i.e. increase in steady-state current. Note that the start of the fit is delayed with respect to the light signal because of pertinent channel inactivation (see below). (b) Time constants of 470-nm light-induced loss of inactivation for the indicated channel types. (c) Mean time course of light-induced loss of inactivation (increase in RI) of roNaV2 (red) and subsequent recovery in the dark (gray) with superimposed single-exponential fits. (d) Mean fluorescence ratio of HEK 293 cells expressing eGFP:147U-204C, i.e. a variant of roGFP2 where one of the disulfide bridge partners is a selenocysteine. The blue bar indicates continuous illumination with 470 nm, the gray bar marks recovery in the dark. (e) Mean time constants of light-induced loss of inactivation of roNaV2 (“On”) and recovery in the dark (“Off”) comparing experiments performed in whole-cell mode (“WC”) and perforated patch mode (“PP”). Also shown are time constants of fluorescence ratio changes of eGFP:147U-204C (“UC”). Data in (b–e) are mean ± s.e.m. with n indicated in parentheses. ***P < 0.001; **P < 0.01; n.s., P > 0.05 for a two-sided t-test; in (b) IFU and IFC vs. IFM.
