Supplementary Figure 5: Stabilization of intracellular pH strongly attenuates the adaptation of eArch3.0 photocurrents.

(a-b) Example voltage clamp traces of ion pump-expressing neurons before, during and after 5 min constant light application measured in the presence of synaptic blockers (25 µM Ap5, 10 µM CNQX). (a) Buffering of intracellular pH by acetic acid (AcOH) or L-lactate in the external solution stabilized eArch3.0 photocurrents (middle and bottom) compared with sodium-based extracellular solution (top). (b) Example trace of an eNpHR3.0 expressing neuron. Photocurrent adaptation was less pronounced than for eArch3.0. (c) Peak photocurrent amplitudes plotted against steady state photocurrent amplitudes recorded in neurons expressing eNpHR3.0 (n = 25), eArch3.0 (n = 37), or neurons expressing eArch3.0 recorded in the presence of 25 mM AcOH (n = 21) or L-lactate (n = 7). Measurements were fitted with a power law function (f(x) = a*x^b+c). The goodness of fit by R-square was 0.73, 0.89, 0.92 and 0.74 for eArch3.0, eArch3.0 in 25 mM AcOH, eArch3.0 in 50 mM L-lactate and eNpHR3.0, respectively. The fits show that with increased light power or expression level, sustained photocurrents greater than 300 pA are possible in the case of the chloride pump eNpHR3.0. However, for the proton pump eArch3.0, photocurrents above ~ 150 pA cannot be maintained over extended periods of time in physiological medium, independent of light power or expression level. This is likely due to changes in internal proton concentration caused by sustained eArch3.0 activation, as buffering of the internal pH leads to attenuation of the photocurrent adaptation. (see Supplementary Table 1 for statistics).