Properties of BR-WT stationary and transient photocurrents.

<p>Photocurrents of BR-WT expressed in <i>Xenopus</i> oocytes are shown, which were evoked either by continuous illumination with green light (A) and/or blue laser flashes (C,D,G). (A) BR-WT photocurrents induced by illumination with green light (grey bar) at 0 mV (red) and −100 mV (blue). (B) Current-voltage plots of normalized stationary photocurrents of BR-WT evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed line is simply drawn to guide the eye. (C, D, G) Green light-induced stationary and blue laser flash-induced transient currents of BR-WT recorded at 0 mV (C), −30 mV (D) and −100 mV (G), green light illumination is indicated by a grey bar above the current traces. According to the illumination scheme above panel (C), the signals shown in (C,D,G) are superpositions of 12 recordings, from which the first is drawn in red color. In each sweep of the protocol, two blue laser flashes (indicated as #1, #1′… #12, #12′ for the 12 traces) were applied: The first blue flash was given at time Δt = 25 ms after the start of illumination with green light, the second at Δt = 25 ms after illumination stop. From sweep to sweep, Δt increased by 25 ms up to 300 ms. (E) and (F) show the transient currents in response to blue laser flash #1 (trace1) and #12 (trace 12) during illumination with green light from panel (C) in higher magnification.</p

3D structure of BR.

<p>Cartoon representation of the 3D structure of bacteriorhodopsin according to the coordinates in PDB structure entry 1C3W by Luecke et al. (1999) prepared with PyMol 1.0 software. The retinal chromophore (magenta) is covalently linked via a Schiff base to Lys-216 in helix G (orange), which - together with the primary proton acceptor (Asp-85) and proton donor group (Asp-96) - is depicted in ball-and-chain representation (oxygen atoms: red, carbon atoms: green, nitrogen atoms: blue). Also shown are Phe-171 and Phe-219, which, together with Asp-96, were mutated herein.</p

Photocurrents of mutants BR-F171C and BR-F219L.

<p>Photocurrents of BR-F171C (A) and BR-F219L (E) induced by illumination with green light (grey bar) at 0 mV (red) and −100 mV (blue). (B,F) Current-voltage plots of normalized stationary photocurrents of BR-F171C (B) and BR-F219L (F) evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed lines connecting the data points are drawn to guide the eye; for comparison, the corresponding WT curve from Fig. 2B is included as dotted line. (C,D,G,H) Green light-induced stationary and blue laser flash-induced transient currents of BR-F171C at 0 mV (C) and −100 mV (D), and BR-F219L at 0 mV (G) and −100 mV (H). Green light illumination is indicated by grey bars. The shown signals are superpositions of 12 recordings according to the illumination protocol from Fig. 2C. In each sweep, the first blue flash was given at Δt = 100 ms after start and the second at Δt = 100 ms after the end of illumination with green light. From sweep to sweep, Δt increased by 100 ms up to 1200 ms.</p

Properties of BR wild-type and mutants.

*<p>n.d. - not determined.</p

Photocurrents of mutants BR-D96N and BR-D96G.

<p>Photocurrents of BR-D96N (A) and BR-D96G (F) induced by illumination with green light (grey bar) at 0 mV (red) and −100 mV (blue). (B,G) Current-voltage plots of normalized stationary photocurrents of BR-D96N (B) and BR-D96G (G) evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed lines connecting the data points are drawn to guide the eye; for comparison, the corresponding WT curve from Fig. 2B is included as dotted line. (C,H) Photocurrents of BR-D96N (C, same cell as in panel A) and BR-D96G (H, same cell as in panel F) after addition of 50 mM azide. (D,E,J,K) Green light-induced stationary and blue laser flash-induced transient currents of BR-D96N at 0 mV (D) and −100 mV (E), and of BR-D96G at 0 mV (J) and −100 mV (K). Green light illumination is indicated by grey bars. The shown signals are superpositions of 12 recordings according to the illumination protocol from Fig. 2C. In each sweep, the first blue flash was given at Δt = 100 ms after start and the second at Δt = 100 ms after the end of illumination with green light. From sweep to sweep, Δt increased by 100 ms up to 1200 ms.</p

Structural details during the BR photocycle.

<p>Three-dimensional ball and stick representations of the retinal chromophore (yellow), coupled via a Schiff base to Lys-216 (nitrogen atom in red) are shown and distances from the Schiff base nitrogen to the carboxyl oxygens (black) of Asp-85 are indicated by green dashed lines according to the following structural coordinates: (A) BR ground state structure (PDB structure entry 1C3W) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073338#pone.0073338-Sass1" target="_blank">[9]</a>, (B) BR ground state structure (PDB structure entry 1FBB) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073338#pone.0073338-Subramaniam3" target="_blank">[41]</a>, (C) structure of the M intermediate (PDB structure entry 1C3W) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073338#pone.0073338-Sass1" target="_blank">[9]</a>, (D) Ground state structure of the triple mutant BR-D96G/F171C/F219L (PDB structure entry 1FBK) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073338#pone.0073338-Subramaniam2" target="_blank">[11]</a>.</p

Comparison of characteristic properties of photocurrent signals.

<p>Photocurrents in response to continuous green light are characterized by five parameters: the amplitude of the transient current peak at the beginning of illumination (a), a stationary current amplitude (b), an initial amplitude of the slow phase of current decay after the end of illumination (c), a relaxation time τ₁ for the decrease from the initial peak current to the stationary level and τ₃ for the slow current decay after the end of illumination. The time constants for the initial current increase (τ₀) at the beginning and for the initial current decrease after light switch-off (τ₂) are not resolved due to the limited time resolution in TEVC experiments. In each panel, the parameters for the shown signals are included for comparison.</p

Photocurrents of mutant BR-D96G/F171C/F219L (BR-tri).

<p>(A) Photocurrents of BR-tri induced by illumination with green light (grey bar) at 0 mV (red) and −100 mV (blue). (B) Current-voltage plot of normalized stationary photocurrents evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed line connecting the data points are drawn to guide the eye; for comparison, the corresponding WT curve from Fig. 2B is included as dotted line. (C) Photocurrents of BR-tri (same cell as in panel A) after addition of 50 mM azide. (D,G) Green light-induced stationary and blue laser flash-induced transient currents of BR-tri at 0 mV (D) and −100 mV (G). Green light illumination is indicated by grey bars. The shown signals are superpositions of 12 recordings according to the illumination protocol from Fig. 2C. In each sweep, the first blue flash was given at Δt = 100 ms after start and the second at Δt = 100 ms after the end of illumination with green light. From sweep to sweep, Δt increased by 100 ms up to 1200 ms. (E,F) Transient photocurrents of BR-tri (E) and BR-WT (F) in response to blue laser flashes in higher magnification. Blue laser flashes were either applied during continuous illumination with green light (left signals in E,F) or without illumination in the dark (right signals in E,F).</p

Active properties of iNGNs in long-term culture.

<p>(A) Representative current-clamp traces showing action potentials from 7d, 28d, and 56d iNGNs, in response to a 500 ms pulse of current injection from a starting membrane potential of -75 mV. (B) Firing properties of iNGNs from 4 to 70 days in response to current injections of different amplitudes. The numbers of APs were summed over a 500 ms stimulus starting from a membrane potential of -75 mV. Numbers at the top of the histogram bars show the number of cells recorded at each age. (C) Average numbers of APs from 7d, 28d, and 56d iNGNs, in response to current injections up to +60 pA. (D) Action potential properties of iNGNs from 7 to 70 days. Average AP peak amplitudes (top histogram), average AP width at half the maximal peak (half-width, middle histogram), and average AP threshold (bottom histogram) in iNGNs from 7 to 70 days age. Error bars denote SEM and numbers next to the errors bars show the number of cells recorded at each age.</p

iNGNs mature in long-term culture.

<p>(A) Cell culture protocol for deriving long-term cultures of iNGNs. Astrocytes (ACs) were plated onto coverslips between 1 and 3 days before iNGNs. (B) Representative DIC microscope images of iNGNs were taken over the course of long-term co-culture with astrocytes, from 1–112 days. Ages (in days) are labeled in the upper left corners of the images. The uppermost left image shows iNGNs that are undifferentiated (no Dox). (C) Intrinsic properties of iNGNs from 4 to 70 days were measured in whole-cell patch-clamp recordings, and averaged measurements are shown for cell capacitance, resting membrane potential, and input resistance. Error bars denote SEM and numbers next to the errors bars show the number of cells recorded at each age.</p