Fig. 5.

An ATP-insensitive light-gated cation channel drives synaptic activity and action potential firing in neurons. (A) The K69A/I328C mutant tethered to MEA-TMA responds to light (Left), but not to ATP (Right, 100 μM) in HEK cells. (B) Current-voltage curves for the same construct recorded in different extracellular solutions in HEK cells (Man, mannitol; Na-Ise, sodium isethionate; Ca, calcium). Light-gated currents were obtained after subtracting photocurrents recorded at 525-nm light (intensity was set to 4.5 mW/mm²) to those obtained at 365-nm light (for NMDG, current shown was recorded 60 s after light switching). (C) Whole-cell current from two example recordings (with moderate and robust synaptic activities) evoked by light in hippocampal neurons expressing the K69/I328C mutant following brief application of 100 μM MEA-TMA (solid lines). (D) Bar plot summarizing the recorded current densities (mean ± SEM for the indicated number of experiments). (E) Extracellular recording from hippocampal neurons expressing the K69/I328C mutant showing action-potential (AP) firing triggered by 525-nm light illumination. Cells were labeled with MEA-TMA (30 μM) under 365-nm light to maintain channels in nonconducting states. (F) Bar plot summarizing AP rates, defined as the number of AP per second, recorded under 365-nm light (violet bar) and 525-nm light (green bar) in experiments similar to that shown in E (n = 4, mean ± SEM). Rates, calculated every 5 s, were collected just before 525-nm excitation, and at the peak, within the first minute of the 525-nm excitation. Measurement of statistical significance is based on paired Student t tests; *P < 0.05.