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. Author manuscript; available in PMC: 2014 Sep 17.
Published in final edited form as: Nat Methods. 2011 Dec 18;9(2):159–172. doi: 10.1038/nmeth.1808

Figure 5.

Figure 5

Properties of hyperpolarizing tools. (a) NpHR is an inward chloride pump (halorhodopsin type; HR), whereas Arch, ArchT, and Mac are outward proton pumps (bacteriorhodopsin type; BR). The 3.0 versions include the endoplasmic reticulum export sequence (ER) after the fluorophore (which constitutes the 2.0 version) as well as a trafficking sequence (TS) between opsin and fluorophore. (b) Confocal images of 1.0 (the originally described version of the molecule) and 3.0 versions (green) expressed in culture and immunolabeled with an ER marker (KDEL; red). Scale bar, 25 µm. (c) Representative traces and raw photocurrents in response to 1 s light for 1.0 (open bars) versus 3.0 versions (closed bars) for Arch (n = 15–19), ArchT (n = 14–16) and Mac (n = 8–12). Vertical and horizontal scale bars represent 500 pA and 500 ms, respectively. Photocurrents were normalized to eNpHR3.0 values from within the same experiment to enable direct comparisons across opsins (n = 8–35). (d) Action spectra for 3.0 versions (n = 7–20) alongside ChR2 (black). (e) τon and τoff (n = 7–35). Vertical and horizontal scale bars represent 200 pA and 5 ms, respectively. (f) EPD50 values for all hyperpolarizing opsins (n = 5–14). Raw photocurrent versus light power density plotted alongside within-experiment eNpHR3.0 (n = 5–14). Population data are plotted as mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001. Unless otherwise indicated, eNpHR3.0 was activated with 590-nm light, and all other tools were activated with 560-nm light, both at ∼5 mW mm−2.