Voltage- and temperature-dependent gating of heterologously expressed channelrhodopsin-2

Abstract

Channelrhodopsins are light-activated channels originally isolated from algae that are being used increasingly as tools to non-invasively stimulate neurones. Despite their widespread use some aspects of their biophysical properties have not been fully characterised. Here we report detailed investigation of the gating kinetics and voltage-dependence of ChR2 transiently expressed in HEK-293 cells. Currents were elicited using light pulses of defined duration and intensity generated by a blue LED. Datasets were gathered both at room temperature (RT, ∼22 °C) and 37 °C. Current responses to light rose rapidly to a peak and then desensitized to a steady state plateau. When illumination was terminated currents rapidly deactivated. Recovery from desensitization at −85 mV was slow with half-times of 1.4 and 3.1 s at 37 °C and ∼22 °C, respectively. At both temperatures, the reversal potential of ChR2 responses was a few mV positive to 0 mV. Both the peak and plateau phases of ChR2 responses exhibited strong inward rectification with only small outward currents at positive membrane potentials. The rates of ChR2 activation, deactivation and desensitization were ∼2 times faster at 37 °C than at ∼22 °C. Both the activation and deactivation kinetics of ChR2 were significantly slowed by depolarization at both temperatures. Additionally, the degree of steady state desensitization was greater at more depolarized potentials. The macroscopic desensitization kinetics were not voltage-dependent, but recovery from desensitization was slowed by depolarization. These gating behaviour data provide an important basis for more detailed analysis of the properties and limitations of ChR2 use in more complex systems.