Fig. 2.
Photostationary state determination by NMR and spectral sensitivity of photoresponses. (a) Fraction of MAG-1 in the cis form determined from NMR spectroscopy. Maximal wavelengths for cis and trans populations are 380 and 500 nm, respectively. Values are given as mean ± SEM (n = 3). (b) Wavelength dependence of photoresponses of iGluR6-L439C channels expressed in HEK293 cells and conjugated to MAG-1. Currents were measured by whole-cell patch clamping in the voltage clamp mode at Vh = −60 mV, with desensitization blocked by Con A. The current vs. time traces and corresponding wavelength step protocol used to record action spectra are indicated. The first set of steps (activation spectrum) starts at the wavelength of maximal deactivation (500 nm) and spans UV illuminations of increasing wavelength. The second set of steps (deactivation spectrum) starts at the wavelength of maximal activation (380 nm) and spans visible illuminations of increasing wavelength. (c) Each temporal trace can be fitted with a single exponential function whose amplitude and time constant is used to build the action spectra. (d) The activation spectrum (“ON”) is centered on 380 nm and falls off rather steeply at higher and lower wavelengths. The deactivation spectrum (“OFF”) is wider, with maximal amplitude between 460 and 560 nm. Values are mean ± SEM (n = 5). (e) Wavelength dependence of photoswitch rate. Time constants τON and τOFF from fits of traces in b are represented as switch rates (1/τ). Values are mean ± SEM (n = 5).