Intramolecular proton transfer in channelrhodopsins

Abstract

Channelrhodopsins serve as photoreceptors that control the motility behavior of green flagellate algae and act as light-gated ion channels when heterologously expressed in animal cells. Here, we report direct measurements of proton transfer from the retinylidene Schiff base in several channelrhodopsin variants expressed in HEK293 cells. A fast outward-directed current precedes the passive channel current that has the opposite direction at physiological holding potentials. This rapid charge movement occurs on the timescale of the M intermediate formation in microbial rhodopsins, including that for channelrhodopsin from Chlamydomonas augustae and its mutants, reported in this study. Mutant analysis showed that the glutamate residue corresponding to Asp(85) in bacteriorhodopsin acts as the primary acceptor of the Schiff-base proton in low-efficiency channelrhodopsins. Another photoactive-site residue corresponding to Asp(212) in bacteriorhodopsin serves as an alternative proton acceptor and plays a more important role in channel opening than the primary acceptor. In more efficient channelrhodopsins from Chlamydomonas reinhardtii, Mesostigma viride, and Platymonas (Tetraselmis) subcordiformis, the fast current was apparently absent. The inverse correlation of the outward proton transfer and channel activity is consistent with channel function evolving in channelrhodopsins at the expense of their capacity for active proton transport.

Figure 1

Photoelectric signals generated by the wild-type proton pump AR-3 (above zero line) and its D95N mutant (below zero line) in E. coli suspensions (dashed lines) and HEK293 cells (solid lines) in response to a 6-ns laser flash (532 nm). The signals recorded in E. coli suspensions were normalized to those measured in HEK cells. The arrows show a shift of the peak times and a decrease in the relative amplitude of the negative phase caused by a lower time resolution of measurements in HEK cells compared to those in E. coli suspensions.

Figure 2

(A) Electrical currents generated by CaChR1 expressed in an HEK293 cell in response to a laser flash at the holding potential applied in 20-mV increments from −60 to 20 mV (bottom to top). (B and C) Decomposition of the net signals recorded at 20 mV (B) and −20 mV (C) in two components. Shown are the recorded traces (black lines), the properly scaled fast positive current, obtained by measuring the signal at 0 mV (red lines), and the channel current, revealed by subtraction of the fast current from the net signal (blue lines). The smooth lines are the results of fitting of each of the two components with two exponentials.

Figure 3

Voltage dependence of the mean current measured between 50 and 150 μs (open squares) and between 0.75 and 1.55 ms (open circles) after the flash, and of the amplitude of the fast positive current (solid squares), corrected for contribution of channel current. Data are represented as the mean ± SE of experiments in 6 cells.

Figure 4

(A) Spectral dependence of flash-induced absorbance changes in detergent-purified wild-type CaChR1. (B) Absorbance changes monitored at 390 nm in wild-type CaChR1 (black), CaChR1_E169Q (red), and CaChR1_D299N (green) in response to a 6-ns laser flash (532 nm).The traces in the main figure were arbitrarily scaled for better visualization of changes in the rates of the M intermediate accumulation. (Inset) Relative yields of the M formation.

Figure 5

Electrical currents recorded from wild-type CaChR1 (black lines) and its E169Q (red lines) and D299N (green lines) mutants expressed in HEK293 cells at the reversal potential for channel currents to minimize their contribution to the signal kinetics (A), and at −60 mV holding potential (B).

Figure 6

(A) Electrical currents generated by VcChR1 expressed in an HEK293 cell in response to a laser flash at the holding potential applied in 20-mV increments from −60 to 40 mV. (B and C) Electrical currents recorded from wild-type VcChR1 (black lines) and its E118Q (red lines) and D248N (green lines) mutants expressed in HEK293 cells at the reversal potential for channel currents to minimize their contribution to the signal kinetics (B), and at −60 mV holding potential (C).

Figure 7

(A) Electrical currents generated by DsChR1 expressed in a HEK293 cell in response to a laser flash at the holding potentials indicated in the figure legend. (B and C) Electrical currents recorded from wild-type DsChR1 (red lines) and its A178E (black lines) and A178E_E309Q (green lines) mutants expressed in HEK293 cells at the reversal potential for channel currents to minimize their contribution to the signal kinetics (B), and at −60 mV holding potential (C).

Figure 8

Electrical currents recorded from CrChR2, MvChR1, and PsChR in HEK293 cells in response to a laser flash (solid lines). For comparison, traces from CaChR1 are shown (dashed line). Traces were recorded at the reversal potential for channel currents to minimize their contribution to the signal kinetics (A), and at −60 mV holding potential (B).

Figure 9

The effect of neutral residue substitution of the Asp⁸⁵ (A) and Asp²¹² (B) homologs on peak proton transfer currents (hatched bars) and the ratio between peak channel currents and peak proton transfer currents (black bars) in CaChR1, VcChR1, and DsChR1. The channel currents were measured at −60 mV and the proton transfer currents at the V_r for channel currents. The data are mean values (n = 3–8 cells) measured upon neutralization of the corresponding carboxylate residues normalized to those in the wild-type for CaChR1 and VcChR1, and in the A178E mutant for DsChR1.

Figure 10

The relationship between the amplitudes of fast and channel currents generated by wild-type channelrhodopsins. The channel currents were measured at −60 mV and the fast currents at the V_r for channel currents. The data are mean values (n = 3–8 cells).