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Conversion of a light-driven proton pump into a light-gated ion channel

  • Scientific Reports 5, Article number: 16450 (2015)
  • doi:10.1038/srep16450
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Interest in microbial rhodopsins with ion pumping activity has been revitalized in the context of optogenetics, where light-driven ion pumps are used for cell hyperpolarization and voltage sensing. We identified an opsin-encoding gene (CsR) in the genome of the arctic alga Coccomyxa subellipsoidea C-169 that can produce large photocurrents in Xenopus oocytes. We used this property to analyze the function of individual residues in proton pumping. Modification of the highly conserved proton shuttling residue R83 or its interaction partner Y57 strongly reduced pumping power. Moreover, this mutation converted CsR at moderate electrochemical load into an operational proton channel with inward or outward rectification depending on the amino acid substitution. Together with molecular dynamics simulations, these data demonstrate that CsR-R83 and its interacting partner Y57 in conjunction with water molecules forms a proton shuttle that blocks passive proton flux during the dark-state but promotes proton movement uphill upon illumination.

Microbial rhodopsins, which are widely distributed across archaea, bacteria, and some eukarya branches, are categorized as sensory rhodopsins, ion channels, and ion pumps1,2. They are light-activated, seven-transmembrane proteins with an all-trans retinal as a chromophore. Photon absorption redistributes electrons within the protonated retinal Schiff base that promotes isomerization around the 13-cis bond followed by a cyclic cascade of proton transfer, water redistribution, and conformational changes within the protein that may be monitored by UV/Vis and infrared spectroscopy3,4,5. In the context of optogenetics, outward directed proton pumps, as well as inward directed chloride pumps, can be utilized as neural silencers or fluorescence-based voltage sensors6,7,8.

The function of all microbial proton pumps within the native environment or when applied experimentally depends absolutely on their performance at moderate to high electrochemical load. Surprisingly, our knowledge about the contribution of individual residues to proton pump strength remains very limited despite extensive research on bacteriorhodopsin (BR) since 1971. These studies have mostly been conducted by spectroscopic methods using BR in native purple membranes or BR mutants in detergent at zero voltage. Only the voltage dependence of a few pumps has been determined by electrical measurements of Xenopus laevis oocytes; however, the photocurrent amplitudes in these experiments were too small for more comprehensive studies9,10,11,12,13. Therefore, various conflicting issues regarding the transport mechanism of proton pumps remained unresolved, including the origin of the proton driving force, the background for voltage dependence of the photocycle, and the functional importance of the non-conserved proton release group.

Here we report our characterization of Coccomyxa Rhodopsin (abbreviated as CsR), a proton-pumping rhodopsin from the unicellular arctic freshwater alga Coccomyxa subellipsoidea C-169. This psychrotolerant alga resides in temperatures ranging from −50 °C to +25 °C14. CsR produces huge photocurrents in oocytes as well as in HEK-293 cells, which will enable researchers to study the molecular transport steps in greater detail than BR has ever allowed. We capitalized on this advantage by characterizing CsR-WT and various mutants. This examination identified the highly conserved CsR-R83 and its interaction partner CsR-Y57 (R82 and Y57 in BR) as the central positions for maintaining directionality and full pump strength. We demonstrate that CsRs with mutations in these residues alone exhibit low active transport forces, if any, and operate as passive light-gated proton channels already at low electrochemical load. Such light-activated proton selective channels will be of great interest for optogenetic applications in which a specific proton release is required (e.g., in lysozymes or neuronal vesicles).

Proton transport model for CsR

The amino acid sequence of CsR is 37% homologous to BR (Supplementary Fig. 1), with the regions important for transport highly conserved between the two proteins (Fig. 1a). Thus, a transport mechanism for CsR can be derived based on that generally accepted for BR15. Briefly, all-trans retinal is linked to K215 of the protein (K216 in BR) via a protonated retinal Schiff-base (RSBH+), which is stabilized by a negatively charged counterion complex consisting of D86 and D211 (D85/D212 in BR). After photo-isomerization of RSBH+ and partial rotation of the N-H dipole, the pKa of RSBH+ is dramatically decreased and the proton is transferred to D86 (D85 in BR, L to M transition)16. D86 protonation causes R83 (R82 in BR) to switch outward towards the proton release group residues E193 and E203 (E194 and E204 in BR), resulting in proton release into the extracellular medium15,17. In the second part of the photocycle, the deprotonated Schiff-base becomes accessible from the cytoplasmic site by forming a transient water chain between the retinal Schiff-base and the proton donor CsR-D97 (D96 in BR)15,18. The pK of D97 in darkness is high (>11 in BR) due to hydrogen bridging to T46 (Fig. 1a)19. Conformational changes of the protein disrupt this bond, thereby lowering the pK of D97 and allowing a proton to transit along the novel water chain to reprotonate the RSB (M to N transition). Next, CsR-D97 is reprotonated from the cytoplasmic bulk phase during the N to O transition. At the end of the photocycle, during the transition from O-state to the dark-state, the proton is transferred from D86 to the proton release group, passing or bypassing R83 and Y57 (Fig. 1a). Although the exact mechanism of this transfer has yet to be elucidated, transient protonation of D211 may be involved20.

Figure 1: Basic characterization of CsR in Xenopus laevis oocytes and HEK-293 cells.
Figure 1

(a) Proposed structure of CsR-WT with mutated residues based on the BR crystal structure (PDB: 1M0L). Brackets indicate respective positions in BR and demonstrate the high conservation of key residues. Arrows highlight the proposed proton transport pathway of CsR-WT, which is similar to BR. A dotted arrow is used to indicate that whether R83 deprotonates itself during the photocycle remains to be determined. (b) Typical photocurrents of CsR-WT measured in oocytes at extracellular pHo 7.5 after illumination with 550 ± 25 nm. Holding potentials were increased from −125 mV in 25 mV steps up to +75 mV. Separated current trace illustrates the biexponential decay after light illumination. (c) Action spectra measured in oocytes at pH 7.2 with supplementation of 5 μM all-trans retinal (ATR, n = 7) and without ATR (n = 5). Currents were normalized to the maximal stationary currents. Inset shows absolute amplitudes at 0 mV (ATR, n = 16; without ATR, n = 21). (d) Ion selectivity measured in oocytes at pHo 7.2 and 0 mV (all n = 5 except CsCl, n = 3). (e) Absolute photocurrent amplitudes of diverse microbial pumps at pHo 7.5 as measured in oocytes (all at 0 mV, illumination with 550 ± 25 nm, CsR [n = 24], Arch3 [n = 8], BR [n = 19], βHK-BR [n = 17], NpHR [n = 6]). (f) Absolute photocurrent amplitudes of CsR-WT-EGFP (n = 8) and Arch3-WT-EGFP (n = 11) measured in HEK-293 cells (0 mV, 550 ± 7 nm, pHo 7.2, pHi 7.2 after 24–30 h, 1 μM ATR). (g) Confocal images of HEK-293 cells transfected with CsR-WT-EGFP or Arch3-WT-EGFP (after 24–30 h, 1 μM ATR). Scale bars represent 25 μm. Data represent the mean ± SE.

Photocurrents of CsR-WT

CsR-expressing oocytes supplemented with all-trans retinal were illuminated for 0.5 seconds with green light of wavelength 550 ± 25 nm at holding voltages ranging from −125 mV to +75 mV (Fig. 1b). CsR exhibited active proton pumping, and photocurrents were always directed outward and graded with the membrane voltage. At negative voltages, a minor initial transient current was observed. Darkness resulted in bi-exponential decay as reported earlier for BR and Acetabularia rhodopsin12,21. The fast component was less voltage-dependent (τfast = 4 ± 1 ms), which is in contrast to the slow component that accelerated from negative (τslow = 260 ± 15 ms at −125 mV) to positive voltage (τslow = 28 ± 2 ms at +75 mV). This characteristic resembles the O- to dark-state transition used for voltage sensing with Arch3 in the neuroscience field8. Wavelength variation revealed the sensitivity maximum at 545 nm (Fig. 1c). Without supplementation of all-trans retinal, amplitudes were reduced and the action spectrum maximum was red-shifted by 10 nm, most likely due to binding of endogenous (3,4)-didehydro-retinal22,23. Exchange of extracellular cations or replacement of chloride for other anions did not affect photocurrent amplitudes or kinetics supporting the exclusive proton transport (Fig. 1d). Most surprisingly, in all conditions examined, the photocurrent amplitudes were much larger than those generated by Arch3-, NpHR-, BR-, or β-BR21 expressing oocytes (Fig. 1e). Expression and membrane targeting of CsR in HEK-293 cells was brilliant and superior to Arch3, resulting in larger photocurrents (Fig. 1f,g). This observation provided us with the opportunity to analyze the functionality of key residues in CsR with unrivaled precision.

Channel-like photocurrents in CsR mutants

Active site mutants

WT and mutant CsRs were compared at different extracellular pH values (pHo) and holding voltages to determine the contribution of specific residues to active proton pumping. As expected, photocurrents of CsR-WT showed low dependence on the extracellular pH (Fig. 2a,b). Replacement of the counterion D86 with T, as found in the chloride pump halorhodopsin, blocked the stationary current completely. At pH 5 and 7.5, CsR-D86T exhibited transient currents of reduced size that were directed inward, suggesting a unique inward charge transfer (Fig. 2c). This transient current can be explained as deprotonation of RSBH+ during the photocycle with an intramolecular proton transfer towards the intracellular bulk phase. In all proton-pumping rhodopsins, D85 or its homologs stabilize the high pK of the Schiff base nitrogen during all-trans configuration of the dark-state2. In CsR-D86T, the RSBH+ was destabilized and the missing charge of D86 was only partially substituted by the secondary more distant charged residue D211, as visualized by the absorption shift from 545 to 585 nm (Fig. 2d). The concomitant drop in the RSB-pK caused deprotonation at pHo 10 and eliminated the transient photocurrents. Instead, 400 nm light evoked stable and stationary outward currents (Fig. 2e,f). At pHo 7.5 and 5.0, the fraction of CsR with deprotonated Schiff Base was small but still measurable. Moreover, the reversal voltage shifted to more positive values as expected for a passive transport. These data demonstrate that CsR-D86T with deprotonated RSB operates as a light-gated proton conductor with residual pump activity. Illumination with bright white light completely abolished residual pumping and shifted the reversal voltages to the Nernst values (Supplementary Fig. 2a). Similar currents were observed previously when the corresponding Proteorhodopsin (PR-D97T) mutant was expressed in oocytes13. For PR-D97T blue light-induced stationary currents were recorded only at pHo 7.4 and −30 mv, suggesting inward pumping without passive proton conductance. A similar phenomenon was observed for BR-D85T in experiments using black lipid membranes, but its voltage dependence was not studied24,25. These stationary currents were interpreted as chloride pumping. However, our data demonstrate that CsR-D86T is not involved in chloride pumping since substitution of chloride with gluconate did not affect the current size or current-voltage relationship (Supplementary Fig. 2b). Chloride conductance was also excluded from PR-D97T in the previous PR-study13.

Figure 2: Current traces and IStat(E) of CsR-WT, CsR-D86T, and CsR-D211N at different extracellular pH values.
Figure 2

(a) Photocurrents of CsR-WT were less dependent on pH and always outward directed (550 ± 25 nm). (b) IStat(E) of stationary photocurrents of CsR-WT (550 ± 25 nm, normalized to pH 7.5 and 0 mV, pH 10 [n = 10], pH 7.5 [n = 25], pH 5 [n = 7]). (c) Photocurrents of CsR-D86T upon illumination with green light (550 ± 25 nm). Inward-directed peak currents were observed, especially at low pHo. These are followed by outward directed peaks immediately after illumination. (d) Action spectra show two separated protein populations of CsR-D86T. Both are shifted towards CsR-WT as shown by dotted lines. Spectrum of stationary currents was measured at pH 10 and 0 mV (n = 4). Spectrum of peak currents in green was measured at pH 7.5 and 0 mV (n = 4). (e) Stationary photocurrent traces of CsR-D86T upon illumination with blue light (400 ± 25 nm). Stationary currents disappear at low pH. (f) IStat(E) of stationary currents of CsR-D86T (400 ± 25 nm, normalized to pH 10 and 0 mV, pH 10 [n = 13], pH 7.5 [n = 8], pH 5 [n = 5]). (g) Photocurrents of CsR-D211N are directed outward and inward at high and low pHo, respectively (550 ± 25 nm). (h) IStat(E) of stationary photocurrents of CsR-D211N (550 ± 25 nm, normalized to pH 7.5 and 0 mV, pH 10 [n = 6], pH 7.5 [n = 7], pH 5 [n = 6]). Data were measured in oocytes and represent the mean ± SE.

Replacement of the second counterion residue D211 with N also converted active ion pumping into passive ion transport with variable vectoriality at neutral pHo, small inward currents at low pHo, and large outward currents at high pHo (Fig. 2g,h). In this case, no residual pump activity was observed between pHo 6 and 10 (Supplementary Fig. 2a). Moreover, the RSB remained protonated under all conditions, suggesting that proton network rearrangements, not the RSB protonation state, are responsible for decoupling the pump and the observed passive proton flow.

Mutants of the proton release pathway

The proton release pathway connects RSBH+ (active site) with the extracellular surface. The main elements involved are the proton shuttle R83, the Y57 residue connecting R83 with D211, water molecules, and the proton release complex E193 and E203 (Fig. 1a). We investigated each of these critical components in CsR. Based on the assumed central role of R83 in the proton release pathway, we examined the effect of mutating this residue to Q. CsR-R83Q-mediated outward currents were larger than WT under standard conditions of pHo 7.5 and pHo 10 (Fig. 3a,b and Supplementary Fig. 3a). It as has been claimed that in the complementary mutant R82Q of BR the pK of D85 is raised from 2.7 to 7.226. Owing to the action spectra of the CsR-R83Q that are similar at pH = 5 and pH = 10 and both spectra are 5–10 nm blue shifted compared to WT (Supplementary Fig. 3d,e), deprotonation of the D86 in CsR is unlikely. At pHo 5, the currents generated by R83Q were directed inward with amplitudes twice as large as the outward currents observed at pHo 7.5. However, the current decay and photocycle kinetics were slower. These data suggest collapsed pump activity in favor of an inward rectifying passive conductance. The I(E) diagram indicates that some residual pump activity remained because the reversal potentials for all three pHo values, especially for pHo 7.5, were more negative than expected for a purely passive channel (Fig. 3b, Supplementary Fig. 3c). In the R83Q-D86N double mutant, active pumping was disrupted completely while the passive inward current remained fully intact. These experiments demonstrate again that passive proton influx is independent of proton transfer from RSBH to the acceptor residue D86 (Fig. 3c,d). Light at 400 nm only promotes small stationary inward and outward currents (Fig. 3e,f).

Figure 3: Current traces and IStat(E) of CsR-R83Q and CsR-R83Q-D86N at different extracellular pH values.
Figure 3

(a) Photocurrents of CsR-R83Q are characterized by strong inward-directed photocurrents at low pHo (550 ± 25 nm). (b) IStat(E) of stationary photocurrents of CsR-R83Q (550 ± 25 nm, normalized to pH 7.5 and 0 mV, pH 10 [n = 7], pH 7.5 [n = 10], pH 5 [n = 6]). (c) Photocurrents of CsR-R83Q-D86N (550 ± 25 nm). (d) IStat(E) of CsR-R83Q-D86N stationary photocurrents (550 ± 25 nm, normalized to pH 5 and 0 mV, pH 7.5 [n = 7], pH 5 [n = 9], pH 4 [n = 5]). Double mutant shows that inward-directed channel-like currents are independent of the primary proton donor D86. (e) Stationary photocurrent traces of CsR-R83Q-D86N upon illumination of blue light (400 ± 25 nm). Similar to D86N, low outward-directed stationary currents were observed at high pHo. (f) IStat(E) of CsR-R83Q-D86N stationary photocurrents (400 ± 25 nm, normalized to pH 10 and 0 mV, pH 10 [n = 5], pH 7.5 [n = 4], pH 5 [n = 5]). Data were measured in oocytes and represent the mean ± SE.

Y57 connects the counterion residue D211 to R83 via a single water molecule27 (Fig. 1a). To examine the role of Y57 in CsR, we mutated it into K because channelrhodopsins contain lysine at this location (e.g., K132 in C1C2)28. CsR-Y57K was an outward-directed ion channel that produced small passive inward currents at low pHo and large outward currents at high pHo with slower kinetics (τoff = 318 ± 25 ms at pH 10 and +75 mV, Fig. 4a,b). The current amplitudes generated by both CsR-R83Q and CsR-Y57K were larger than those of CsR-WT (Supplementary Fig. 3a), suggesting that the number of transported protons per photocycle varied. Interestingly, the CsR-Y57F mutant was still able to pump with moderate power in a pH-dependent manner (Supplementary Fig. 3b). The pump activity of the Y57K-R83Q double mutant was abolished completely (Fig. 4c,d), and the clean inward and outward currents seen at pHo 5 and 10 were totally passive while the conductance was quite small (Supplementary Fig. 3a,c).

Figure 4: Current traces and IStat(E) of CsR- Y57K and CsR-Y57K-R83Q at different extracellular pH values.
Figure 4

(a) Photocurrents of CsR-Y57K are characterized by strong outward-directed photocurrents at high pHo (550 ± 25 nm). (b) IStat(E) of stationary photocurrents of CsR-Y57K (550 ± 25 nm, normalized to pH 7.5 and +75 mV, pH 10 [n = 6], pH 7.5 [n = 9], pH 5 [n = 4]). (c) Photocurrents of CsR-Y57K-R83Q exhibit the properties of both single mutations but without huge amplitudes at high electrochemical loads (550 ± 25 nm). (d) IStat(E) of stationary photocurrents of CsR-Y57K (550 ± 25 nm, normalized to pH 5 and 0 mV, pH 10 [n = 9)], pH 7.5 [n = 9], pH 5 [n = 9]). Data were measured in oocytes and represent the mean ± SE.

To understand the role of the photocycle intermediate that passively conducts protons in R83Q, we combined this mutation with W182F. W182 is a highly conserved residue of the retinal-binding pocket located above the retinal C11 position and interacting with both 9-methyl and 13-methyl groups (Fig. 5a)17. The absorption of BR following mutation of W182 to F became more than 70 nm blue-shifted29, and the transition from the L to M intermediate was dramatically slower30. Surprisingly, CsR-W182F showed active outward pumping with half the amplitude, but even faster kinetics than WT with more distinct inactivation at negative voltages (Fig. 5b,c). Nevertheless, the CsR-R83Q-W182F double mutant exhibited both active pumping and passive ion conductance. Pumping predominated over passive conductance in the light, but the pump was weak and the current tended to revert at negative voltages. After light-off the current exhibited a biphasic decay in which the pump currents decayed fast and passive conductance decayed slowly. At pHo 5, however, only passive inward conductance with large amplitudes and slow off-kinetics was observed (Fig. 5d). There is clearly a slow equilibrium between a pumping and a conducting CsR with the ratio of the two activities dependent on the membrane voltage, pHo, and duration of illumination. Thus, we hypothesize that the W182F mutants perform a fast and a slow photocycle. Fast cycling is visible as active pumping whereas slow cycling does not contribute to the pumping process but rather to passive currents in cases where the CsR has been made conductive.

Figure 5: Inward-directed photocurrents produced by CsR-R83Q are channel-like.
Figure 5

(a) Position W182 is highly conserved in microbial rhodopsins and is located close to the methyl groups of the retinal. (b) Photocurrents of CsR-W182F are characterized by a large transient outward peak compared to the stationary currents. (c) IStat(E) of stationary photocurrents of CsR-W182F are always directed outward but curved compared to CsR-WT (550 ± 25 nm, normalized to pH 7.5 and 0 mV, pH 10 [n = 4], pH 7.5 [n = 6], pH 5 [n = 5]). (d) Similar to CsR-R83Q, photocurrents of CsR-R83Q-W182F are inward-directed, especially at low pHo. Additionally, the currents show drastically increased decay times. Data were measured in oocytes and represent the mean ± SE.

Next, we mutated the extracellular E193 and E203 residues (seen in Fig. 1) (Supplementary Fig. 4a–c). The corresponding residues E194 and E204 have been considered in several publications as part of the extracellular proton release machinery of BR31,32,33,34,35 without drawing any conclusion about the relevance of these residues for proton pumping.

Our studies show that the two release group residues are relatively unimportant for pumping activity and pump force of CsR. In both single mutants CsR-E193Q and CsR-E203Q, as well as in the CsR-E193Q-E203Q double mutant, pump activity at pHo 10 was almost completely retained while current amplitudes were slightly reduced at pHo 7.5 and 5. This was especially evident at negative voltages. These data suggest that proton release was less efficient at high extracellular proton concentration but the influence is relatively minor.

Comparison with Bacteriorhodopsin (BR)

We identified two residues, R83 and Y57, as critical elements for active proton pumping of CsR. To determine whether these residues also play major roles in other proton pumps, we examined the effect of similar mutations on BR proton pump activity. Our data demonstrate that pumping by the BR-R82Q and BR-Y57K mutants was severely disrupted albeit to a different extent from CsR. Both still pumped weakly with moderate outward-directed currents at pHo 10 and 7.5; however, at pHo 5, the currents were passive and inward (Supplementary Fig. 5a–c). These results show that, like CsR, R82 and Y57 are important residues for active pumping in BR, with the nature of substitutions determining the degree and size of passive conductance.

Proton uptake pathway of CsR

As explicitly shown for BR, the cytoplasmic (CP) channel of a light-driven pump is responsible for reprotonation of the RSB via a transiently formed water chain18. In BR, D96 is the key residue of the CP channel and is considered as the primary proton donor for reprotonation of the RSB during the M to N transition36,37,38,39,40. In Green-Proteorhodopsin and Gloeobacter rhodopsin, this aspartate is replaced by a glutamate that accelerates the reprotonation step but at the cost of leakiness in the pump at high load, i.e. low pHo and negative voltage41. Taking a cue from these earlier results, we investigated the effect of mutating the analogous position, D97, in CsR. The CsR-D97E mutant was functional with reduced photocurrent amplitudes without inward-directed leak currents at any load (Supplementary Fig. 4d). Because the pK of D96 in BR is tuned by hydrogen bridging to T4618,42, similar results were expected for CsR (Fig. 1a). In CsR-T46N, photocurrents were enlarged at negative voltages and the kinetics were accelerated, indicating that this variant is even more effective and powerful than WT (Supplementary Fig. 6a,b). Promotion of the driving force for T46N was also visible in combination with R83Q. For CsR-T46N-R83Q, the passive conductance at low pH was reduced compared to the single mutant R83Q and the reversal voltages were more negative between pHo 5 and 10 (Supplementary Fig. 6c,d).

MD simulations

To achieve a better understanding of the differences between WT pumps and R83/Y57 mutants, we calculated the structural differences between dark states and O-intermediates based on previously published BR structures43,44. During the BR dark-state, R82 is in direct contact with D212 (Fig. 6a), disrupting the water density between the active site and the H+-release residues E194 and E204. During the O-state, R82 faces outward to facilitate water invasion between R82 and D212, thereby forming a possible proton transfer pathway from D212 to E204 (Fig. 6b). The situation is quite different in the R82Q mutant where Q82 is oriented outward in both the dark- and O-states, allowing water to enter a channel between the active site and PRG. This structure forms a water wire in the dark-state and a proton transfer pathway that may involve Y57 in the O-state. The situation is more complicated for Y57K mutants. In the Y57K dark-state, K57 flips away from the counterions, leading to a very high water density in the active site, which is connected to the PRG. In the Y57K O-state model, however, K57 forms a salt bridge with D212, thereby neutralizing the charge of this counterion.

Figure 6: Molecular dynamics simulation and prediction of water molecule distribution of dark states and O-intermediates of BR.
Figure 6

(a) The dark state of BR-WT with separated areas of mobile water. (b) In the O-state of BR-WT, the downward (extracellular side) movement of the guanidine side chain of R82 is depicted. The hydrogen bond connecting the guanidine group with Y57 and D212 was interrupted due to this downward movement. (c) Mutation of R82 by the smaller and neutral Q82 induced a large space with an extended water cluster. (d) Extended water cluster was still present in the O-state of R82Q, but direct accessibility to the primary proton donor D85 was interrupted. (e) Mutation of Y57 by the positively charged K57 promoted a salt bridge between D212 and K57. The dark state of K57Y was also characterized by an increased water cluster. (f) The O-state of Y57K lacks an interaction between R82 and E204. The huge cavity of mobile water molecules between K57, R82, D85, and D212 caused accessibility to the Schiff Base. Downward movement of the guanidine side chain of R82 was also observed.

Throughout the years, numerous spectroscopists, crystallographers, and theoreticians have investigated the three elements of light-driven pumps: active site, proton release pathway, and Schiff base reprotonation. Unfortunately, in most cases except wild type BR, these studies did not evaluate the electrochemical gradients of the pumps in vivo. We demonstrated above quantitatively how the force, that a pump requires to overcome electrical or electrochemical gradients, depends on all three elements. Modification of any one element is sufficient to completely disrupt active pumping or convert the pump into a light-gated proton channel. However, the three elements have very different functions, as discussed below.

Many studies have investigated the role of the active site. Our photocurrent measurements of CsR-D86T are consistent with a model in which the primary proton acceptor is absent and photoexcitation causes release of the Schiff base (SB) proton to the intracellular side of the medium as previously proposed45. We expect that in this case deprotonation occurs late during the photocycle when the water chain between the SB and internal bulk phase is established. The fact that stationary outward currents are observed for CsR-D86T with a deprotonated dark-state chromophore at high pHo (Fig. 2d–f) may imply that, after photoisomerization of deprotonated all-trans retinal into 13-cis, the RSB retrieves a proton from the intracellular compartment and releases it to the extracellular side during thermal reisomerization.

The fact that D211N also promotes passive currents even though the RSB remains protonated in the dark-state favors a model in which disturbance of the proton network during one of the late photocycle intermediates (N or O) is important and not the protonation state of the counter ion. Moreover, the passive currents of CsR-D86T and CsR-D211N are small (Supplementary Fig. 2c), suggesting that these mutants retained a 1:1 coupling between photoisomerization and proton translocation and do not promote a channel-like conductance. The reduced currents observed with CsR-D86T compared to CsR-D211N can be explained by the lower quantum efficiency of deprotonated RSB chromophores46.

The effects of CsR-W182F (Fig. 5) were unexpected. The homologous mutants in BR and Sensory Rhodopsin II (SRII) have been described as mutants with slow photocycle and a particularly slow L to M and M to N conversion29,47,48 which seemed at a first glance inconsistent with our results of CsR-W182F that produced significant pump currents. However, CsR-R83Q-W182F revealed a dual function for this W182F mutation with fast pump currents and slow passive conductance, suggesting that the two photocycles may represent two populations that operate in an independent manner. Weidlich, et al. proposed that retinal isomerization is distorted in BR-W182F47. Consistent with this argument, it is conceivable that two photocycles, one with 13-cis,15-anti M-state and the other with at,15-syn, coexist as photoproducts from at,15-anti (trans-cycle) and 13-cis,15-syn (cis-cycle) dark-states, respectively. Such a phenomenon has been reported for dark-adapted BR (1:1)49 and channelrhodopsin (0.7:0.3)50.

The extracellular proton release pathway has also been the subject of intense research. We calculated the reversal voltages Erev for WT and mutant CsRs, as well as compared the values with the theoretical expected Erev for passive proton flow according to the Nernst equation (Fig. 7 and Supplementary Figs 2a and 3c). In contrast to Nernst, Erev for WT changes only weakly with the pHo from −190 mV at pHo 10 to −150 mV at pHo 4, similar to BR. Considering the power of the CsR pump relative to the proton motive force (Δp), it increases from −40 mV at pH 10 up to −360 mV at pH 4, revealing that, in principle, the pump is very strong. However, at higher pH, this power cannot be exploited for reasons that are unknown (ΔΨ-pump). This is unlikely a thermodynamic limitation but rather due to modification of the hydrogen bonding network or protonation stages of essential residues that are responsible for this limitation. But, it was totally unexpected that modification of the pK of CsR-D97 by replacement of its hydrogen bond partner T46 with N (Fig. 7 and Supplementary Fig. 6) the power of the Δ Ψ−pump increased over the CsR-WT pump by almost −70 mV.

Figure 7: Reversal voltages of Δp- and ΔΨ-type light-driven proton pumps.
Figure 7

Reversal voltage (Erev) of Gloeobacter rhodopsin (GR), CsR-WT, CsR-T46N, CsR-R83Q, and CsR-R83Q-T46N are plotted against the extracellular pHo. The black line represents the theoretical expected Erev of free proton flow calculated by the Nernst equation based on an intracellular pHi of 7.4. Reversal potentials below this line indicate proton pump activity. Values from −125 mV up to +75 mV were determined directly. Other values are determined by extrapolation. CsR-WT (550 ± 25 nm, pH 10 [n = 8], pH 9 [n = 3], pH 7.5 [n = 26], pH 6 [n = 5], pH 5 [n = 8], pH 4 [n = 6]) exhibited high resistance against low pHo whereas GR (400–600 nm, pH 7.5 [n = 23], pH 6 [n = 6], pH 5 [n = 9], pH 4 [n = 10]) was shifted −260 mV in parallel to the calculated potentials. Mutation of CsR-R83 (550 ± 25 nm, pH 10 [n = 7], pH 9 [n = 4], pH 7.5 [n = 11], pH 6 [n = 4], pH 5 [n = 6], pH 4 [n = 4]) into Glu converts Coccomyxa rhodopsin into a Gloeobacter-like proton pump in relation to the Erev. CsR-T46N (550 ± 25 nm, pH 10 [n = 4], pH 7.5 [n = 8], pH 5 [n = 4], pH 4 [n = 4]). CsR-T46N-R83Q (550 ± 25 nm, pH 10 [n = 4], pH 7.5 [n = 5], pH 5 [n = 5], pH 4 [n = 2]). Data were measured in oocytes and represent the mean ± SE.

In contrast, the R83Q mutant was able to pump against a −100 mV electrochemical gradient (proton motive force Δp) over the whole pHo range from 4 to 10 (Δp-pump). At any condition exceeding this Δp value, the current switches to passive inward transport (Fig. 7). Thus, the characteristics of this pump are completely different from CsR-WT but similar to Gloeobacter rhodopsin, which also possesses a strong dependence on pH at a pumping power of −260 mV at all pH values typical for a Δp-pump (Fig. 7).

The fact that proteorhodopsins do not contain E194 and E204 in the PRG and in parallel exhibit a higher pK of the D85 counterion prompted researchers to conclude that these two glutamates are the parameters that allow ΔΨ-pumps including BR to remain insensitive to the external pH51. However, the small reduction of currents produced by the PRG-deficient mutant at pHo 10, and the equal pumping observed at pHo 5 and 7.5, also questions the importance of these two residues for pH-insensitivity. In contrary, studies in BR demonstrate that replacement of BR-R82 with Q increases the pK of the SB D85 counterion from 2.5 to 7.2 at 150 mM NaCl26,52. FTIR measurements of BR-WT and R82A led to the suggestion that R82 is deprotonated during M-formation and is reprotonated via D85 late in the photocycle during O to BRdark transition53. However, this hypothesis is not generally accepted15. MD simulations, including those presented here, favor formation of a proton-conducting water chain that is formed during N to O transition (Fig. 6b). Further, our MD simulations support the conclusion that in the CsR-R83Q and Y57K mutants such a water chain between RSB and the extracellular bulk phase already exists in darkness and probably during the entire photocycle. Thus, when the intracellular water chain is established during the M to N transition, the mutants become capable of conducting protons.

Cloning, heterologous expression, and mutagenesis of CsR

A human codon-adapted COCSUDRAFT_42526 gene (Gene ID: 17040130, protein accession number I0YUS5) was synthesized by Geneart (Germany) and cloned into the pGEMHE vector. Site-directed mutagenesis was performed using the QuikChange Kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions.

Electrophysiology with Xenopus laevis oocytes

Preparation of Xenopus laevis oocytes54 and expression of microbial rhodopsins were performed as described previously41. Briefly, 25–35 ng capped-mRNA were injected per oocyte and incubated at 18 °C in ORI solution supplemented with 5 μM all-trans retinal (Sigma, St. Louis, MO). Oocytes were measured 4–5 days after injection.

A two-electrode voltage clamp was performed with an amplifier, Turbo Tec-10×, without transient compensation (NPI Electronic GmbH, Tamm, Germany). Continuous light was provided by a 75 W Xenon lamp, XBO75/2 (Karl Zeiss GmbH, Jena, Germany). The light passed through broadband filters (K55 filter for 550 nm light, K40 filter for 400 nm light, Optics Balzers AG, Lichtenstein) with an intensity of 12 mW/cm2. For action spectra measurement, polychrome II (Till photonics, Munich, Germany) was used as a light source and currents were normalized to the corresponding photon flux at each wavelength. The resistances of microelectrodes were kept between 0.4 and 1.2 MΩ. Data acquisition and light triggering were controlled with pCLAMP 9.0 software via the DigiData 1322A interface (Molecular Devices, Sunnyvale, CA, USA). Currents were recorded at 10 kHz and filtered to 1 kHz by built-in circuits. Shown current traces were additionally filtered to 500 Hz (Gauss-lowpass cutoff filter using pCLAMP). The composition of standard extracellular buffer solutions was as follows: 100 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 5 mM MOPS (pHo 7.5, adjusted with 1 M NMG), 5 mM glycine (pHo 10.0, adjusted with 1 M NMG), and 5 mM citric acid/Na-citrate (pHo 5.0, adjusted with citric acid/Na-citrate). Buffers with different compositions than these are described in the appropriate section.

Culturing, confocal microscopy, and patch-clamp experiments on HEK-293 cells

Culturing, confocal microscopy, and patch-clamp experiments on HEK-293 cells were performed as described previously41. Cells were supplemented with 1 μM all-trans retinal (Sigma) and analyzed 24–30 hours post-transfection.

Molecular dynamics (MD) simulation

To gain insight into the structural and dynamic properties of BR, we performed 50 ns molecular dynamics (MD) simulations. The x-ray structure of BR at 1.55 Å resolution (PDB ID: 1C3W)43 was used as the starting point for dark states. Since a crystal structure for the wild type O-state has yet to be published, we constructed the O-state structure based on its main characteristics, namely all-trans retinal, neutral D85, and deprotonated proton release group (E194, E204). The MMTSB tool set55 was used to simulate the point mutations R82Q and Y57K. The protein was embedded into a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer and explicit TP3P56 water molecules were used as solvent. The complex was neutralized with chloride anions and was described with the Charmm36 classical force field57. The production simulations employed a time step of 2 fs, and the lincs algorithm was applied to keep all covalent bonds constrained at their respective reference distances. Regarding the treatment of non-bonded interactions, a cut-off of 1.2 nm was used for the van der Waals interactions, while the particle-mesh Ewald implementation58 was used to evaluate the point-charge electrostatics. All MD simulations are performed using Gromacs version 4.6.159.

Computation of water distribution and complex structure

The VolMap plug-in implemented in VMD 1.9.1 was used to compute the water distribution as the mass weighted atomic densities were averaged over all frames over a 50 ns trajectory. The 3D grid resolution was set at 1 Å and the atom size value was set to 1 and the default cutoffs for the water densities were used. The resulting OpenDX voxels of the average water densities were then analyzed using dxTuber, a grid-based cavity detection method60, with default settings to derive the PDB format output encoded as formal b-factors. The MD trajectories were aligned to the starting structure of the production simulations based on Cα atoms prior to VolMapping to eliminate protein drift in the simulation box. The most likely structures61 of the complexes were derived utilizing the gromacs tool g_mlstr, in which the position of the side chain atoms is determined by dihedral angle analysis while the backbone atoms are averaged.

Additional Information

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Download references

We thank Andreas Herrmann and Thomas Korte for their support with confocal microscopy, and Maila Reh for technical support and oocyte preparation. This work was supported by the Leibniz Graduate School of Molecular Biophysics (A.V.) and by the German Research Foundation DFG, SFB1078, B2 (P.H.), FOR1279 (P.H. and M.E.).

Author information

Author notes

    • S. P. Tsunoda

    Present address: Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.

  1. Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany

    • A. Vogt
    • , S. P. Tsunoda
    •  & P. Hegemann
  2. Institute of Physical Chemistry, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany

    • Y. Guo
    •  & M. Elstner
  3. Department of Biochemistry, University of Delhi South Campus, New Delhi, India

    • S. Kateriya

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All presented electrophysiological experiments were performed and analyzed by A.V. Identification and design of mutants with channel-like photocurrents was performed by A.V. Coccomyxa rhodopsin was originally identified and cloned by S.K. Basic experiments of CsR-WT in oocytes were conducted by S.T. MD simulations were produced and interpreted by Y.G. and M.E. The manuscript was written by P.H. and A.V.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to P. Hegemann.

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Record number of panther deaths in 2014

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A record number of Florida panthers were killed in 2014 -- all right here in Southwest Florida.  Now in the New Year, there are new efforts to help save the endangered animal.

Florida panthers are a statement to our area.  But wildlife officials are becoming concerned with the number of panthers dying on our roads.  A total of 33 panthers were killed last year.  All of those deaths happened in Collier, Lee and Hendry counties.

It's the most panther deaths ever recorded.  For the first time, more than 30 panthers have died in one year.

We're told most of the deaths happened from territorial fights with other panthers or crashes on our roadways.  Twenty-five of the 33 panthers were hit and killed by vehicles last year.

Wildlife officials estimate only about 180 Florida panthers are living in the wild -- meaning every loss is significant.

"When you think about it in the big picture, the loss of this many animals in one year due to causes that we can try to reduce is pretty significant," said Amber Crooks from the Conservancy of Southwest Florida.

Wildlife officials with the Conservancy of Southwest Florida say the increase in deaths is a result of an increased loss of land for the panthers.

In 2015, they have a team working to comment on proposed communities being built near panther habitats to try to move builders away from developing on their land.  They are also working to comment on road project, and get more underpasses built for panthers to safely cross roadways.

To put it in perspective for you, the 33 panther deaths last year is compared to just 20 deaths in 2013.


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Arrest made in case of Lehigh teen beaten up for headphones

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An arrest has been made in the case of a Lehigh teen that was nearly beaten to death for his headphones on October 20. He had brain surgery after a group of kids jumped him in the middle of the Lowe's parking lot in Fort Myers.

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"It's a lot harder to sleep. I can't really fall asleep at night," said Damian Warner, who's only a few days removed from his stay at Lee Memorial Hospital. "I think when I got hit... That's when I can't remember. From until I woke up in the hospital, so there's kind of a time gap between them."

James Cuttino, 17, was arrested and charged with felony battery and robbery.

Despite Damian's blackout, he said three more teens are at fault.

"I was glad they found him at least...but there are still more," said Damian.

"My son almost lost his life," said Damian's mother Tonya Stoner, who has been fighting back the tears since it happened. "[Cuttino] gets put behind bars and he's only there an hour. He was only there hour and he got bonded out."

Both Tonya Stoner and her son hope Cuttino is tried as an adult.

"This kid should not be out. He should not be roaming the roads. He's a danger to society," said Tonya.

Damian, trying find his way back to normal, wishes the man who nearly killed him would be charged with attempted murder.

"I guess I almost died, so I don't think it's enough."

Damian was supposed to start his GED program Monday, just two days after he was sent to the hospital.

Doctors said physically he will recover, but mentally, he may never be the same.


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NEW YORK -- "Star Wars: The Force Awakens" had the best opening weekend in box office history, and it looks like the Disney film will be a gift that keeps on giving.

Industry analysts project that "The Force Awakens" will bring in between $150 million and $180 million at the box office during the upcoming holiday weekend.

If the film hits those numbers, it would be the biggest second weekend of any movie in history, and by a large margin. The previous record belongs to Universal's "Jurassic World," which made $106.6 million in its second weekend of release in June.

But analysts note that the huge popularity of "Star Wars," as well as intense competition from new films, makes it harder to produce an accurate forecast.

"Star Wars" will be joined at the box office this weekend by Jennifer Lawrence's "Joy," Will Smith's "Concussion," and "Daddy's Home," a comedy featuring Will Ferrell and Mark Wahlberg. Leonardo DiCaprio's "The Revenant" and Quentin Tarantino's western "The Hateful Eight" also open in select cities.

Fresh competition or not, the box office galaxy likely still belongs to Disney's "The Force Awakens."

"Even with a slew of new releases hitting theaters this weekend, the big story will remain 'Star Wars' for quite some time," said Shawn Robbins, senior analyst at BoxOffice.com. "Crowded theaters and continued sellouts will benefit other movies, though."

The U.S. box office may be heading for a record itself as annual ticket sales approach $11 billion, a number that would make 2015 the biggest year in box office history.

But even if the annual sales record stands, it appears that Hollywood will have a happy holiday season.

"With what looks to be a film chasing down the biggest box office revenues in history, this may easily become the busiest Christmastime box office on the books," Robbins said.

The-CNN-Wire: By Frank Pallotta
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Ratke suggests Allen partly responsible for national debt - NBC12 - WWBT - Richmond, VA News On Your Side

Politifact Virginia: Ratke suggests Allen partly responsible for national debt

RICHMOND, VA (WWBT) - In a year and a half, Virginia will vote for a senator. But the race for the seat is already heating up in a big way.

Tea Party activist Jamie Radtke is going after former senator George Allen, both of whom are vying for the Republican nomination. Radtke accuses Allen of being part of the problem when he previously served in Washington and argues a vote for her will be a vote for real change.

In a statement she wrote, "During Senator Allen's six years in office, he voted for budgets adding $3.1 trillion to the national debt. That's $16,400 for every second Senator Allen was in office."

Is that possible? If so, it would be the national debt went up the price of a brand new Chevy Cobalt every second Allen was in office.

According to the reporters at Politifact Virginia, Radtke's claim is in fact true. In fact, they say she was a little too generous with Allen's record.

The treasury department says national debt rose more than $3 trillion during Allen's senate term. It was nearly $6 trillion when Allen took office in 2001 and more than $9 trillion when he left six years later.

The math shows debt went up almost $17,000 every second Allen was in office.

"Much of the debate around congress centers around spending and how to curb in deficits and the record deficits that we are taking in right now. Radkte is a Tea Party organizer and her signature issue is curbing government and reining it in," said Warren Fiske of the Richmond Times-Dispatch.

Now, to be fair, the situation has gotten worse since Allen left D.C. The debt has gone up $54,000 a second during Jim Webb's term.

Politifact points out that is more like a new luxury SUV than a Chevy Cobalt. And you can see all of the information behind this report on Politifact's web site. Just log on to www.PolitifactVirginia.com. And you can see all of our past Politifact Virginia reports on www.DecisionVirginia.com.

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L. Neil Smith's
Number 371, June 11, 2006

"A lot of controversy"

Sports and the Free Society
by Christopher Awuku

Reprinted by The Libertarian Enterprise

As a libertarian, I often discuss my political views with family members, friends, acquaintances, co-workers and other people I interact with on a regular basis. On numerous occasions we would go back and forth on topics including the war on drugs, non-interventionist foreign policies, ending state-run education or ceasing the welfare state. Still, there is one question which even I, a hardened libertarian, admittedly struggle to answer in the course of my conversations. How shall a libertarian society cater for and successfully fund sports? I would acknowledge that it's a worthy question in itself and certainly has given me plenty of food for thought.

Sports are an aspect of life that many people enjoy. A lot of people take pleasure from observing and actively participating in them. Apart from the evident physical benefits, sports are also beneficial for emotional and mental health. Whenever the Olympics come around, we all desire our country to do well and win as many medals as possible. Sports are often central to a country's sense of pride.

I live in the United Kingdom, which is the birthplace of several major world sports, such as soccer, tennis, rugby union & league, cricket, etc. In our country, football (as we call it) is by far the most popular sport. The most prominent, and certainly richest, football league in the UK is the Barclays English Premiership.

There is no reason why the Premiership shouldn't prosper within a libertarian society. Since it remains the highest profile league in the country, there will naturally be a demand for coverage of its games, either on TV, radio or other media. Broadcasting companies such as British Sky Broadcasting presently provide the supply to cater for this demand, by showing Premiership matches on the satellite and cable network. The money that British Sky Broadcasting shall pay for the rights to host Premiership games financially benefits all of the 20 football clubs in the league. This enables them to secure players of the quality of Thierry Henry, Wayne Rooney, Frank Lampard and Steven Gerrard and compete with the other top football leagues in the world, such as La Liga in Spain. In this sense, football would adequately be financed in a free society.

A similar rationale applies to other major sports in Britain. Let us take cricket as an example. People would be excited to see England play Australia for the Ashes, hence creating a demand for media coverage of such a Test Series. Broadcasting companies would compete fiercely to secure the broadcasting rights for the Ashes, hence aiding the financing of cricket. In rugby union, the annual Six Nations Championship would draw natural attention, for the same reasons as the Ashes.

Fine, so large sports would remain largely unaffected in a libertarian society. But what about the so-called "minority sports"? Currently, in many first world nations, sports are heavily subsidised by the state. Governments would provide the capital for sports centres, athletic tracks, swimming pools, etc. to be built. Of course, in a libertarian society, government would only be restricted to protecting rights to the person and property. Funding for smaller sports would have to be secured from other sources.

Well, in a libertarian society, there would be no income tax or direct taxation of any kind. Without state-sanctioned theft and force, people would possess more funds that could be used to donate to their favourite causes. These could be a sports centre for their local community, or equipment for a martial arts club, or boats for a rowing club. In general, the enlarged charitable/voluntary sector in a libertarian society would cater for the financing of smaller sports. Local communities could all pitch in to purchase playing fields. Maybe such funding could be a condition of moving to a new neighbourhood. Who really is to say what scenarios would occur if the free market funded minority sports?

The governmental funding of sports can also be described as "welfare for the rich". In the United States, there are some examples of wealthy individuals (some of whom are billionaires) receiving government money to construct stadiums. Qwest Field, the home stadium of the Seattle Seahawks in the USA, was partly financed by taxation. This happened even though the owner of the Seattle Seahawks, Paul Allen, is one of the world's richest men and is a billionaire. Clearly, there was little need for government funding in this instance. Allen did provide 30% of the financing for the stadium, nevertheless the wealthy should have little need for governmental aid. Wembley Stadium's re-construction is being partly funded by the Department for Culture, Media and Sport. At the time of writing, the date of opening for Wembley Stadium is unclear even though the stadium was due to be completed in May 2006. This simply demonstrates that government programmes and policies seldom turn out as intended, no matter how benevolent the intentions of politicians are. One can only look at a lack of tangible enhancement in the NHS, for example, as proof of this. Government isn't even needed to construct stadia. During the 2005/06 Premiership season, Manchester United increased the capacity of their Old Trafford ground. Government expenditure wasn't necessary for such construction. In the summer of 2006, Arsenal will move into the new Emirates Stadium, which is totally being financed by the private sector. Private bodies have an incentive to constantly update and upgrade their stadia, since they are catering for the comfort and pleasure of their supporter base.

Sports are a fun aspect of the lives of many people, right across the world. Due to this fact, one shouldn't worry about the financial upkeep of sports. Generally speaking, human beings like to preserve things they cherish or are fond of. I believe this aspect of human nature also relates to sports.

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Number 494, November 23, 2008

"Any government will grow until it
claims power over absolutely everything."

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On a Clear Day You Can See Bulgaria—But Who Wants to Look?
by L. Neil Smith

Special to The Libertarian Enterprise

I've been a science fiction writer for 17 or 27 years, depending on how you look at it. During that time, it seems like there's always been some editor or agent on the phone, whining into my ear about how bad business is lately.

Of course you have to accept some of this as a bargaining strategy on the part of editors—or excuses on the part of agents—for why writers should be happy to accept less money. That sort of thing's been going on since the first copper stylus got mashed into the first moist clay tablet. From an editor or an agent's point of view (and they are essentially the same, no matter what they claim to the contrary) writers should always be happy to accept less money.

But there's a particle of truth here, too: the same period has indeed been characterized by shrinking rack space for science fiction in grocery stores and drugstores, incongruously occupied by offerings with dragons, dwarves, enchanted swords, bazookas, and armored hovercraft on their covers. Where I differ with the editors and agents—to whom I've vainly attempted to communicate this point for virtually every one of those 17 or 27 years—is in my belief that science fiction is dying from self-inflicted injuries. Furthermore, I believe that I'm uniquely qualified to pontificate on this subject because in many respects, I'm the only writer in the whole wide world still writing the stuff.

Historically, science fiction has almost always been driven by some variety of Utopianism: stirring visions of the wonderful new universe that will "inevitably" result from practicing whatever it is the writer has to preach.

Almost always it has been some variety of socialism.

On rare occasions it has been right-wing socialism, a little- understood intellectual phenomenon in which the central idea is that the life, liberty, and property of the average individual should be sacrificed (or at least temporarily dragooned) for the sake of achieving certain collective goals—like constructing a base on the Moon, slaughtering pesky aliens, wiping out interplanetary drug pushers, or simply moving Antarctic icebergs to thirsty tropical consumers—goals such as those traditionally advocated by conservatives (or even outright fascists) ranging from E.E. "Doc" Smith to Dr. Jerry Pournelle.

But most often it was left-wing socialism, in which the central idea is that the life, liberty, and property of the individual should be sacrificed for the sake of achieving certain collective goals—like national healthcare or universal weapons confiscation—traditionally advocated by liberals or even outright communists. These unworthies have dominated science fiction since its inception, although 20 or 30 years ago they grudgingly made room for a few token right-wing socialists because the real goal of both camps (like the viewpoints of editors and agents) is essentially the same: sacrificing the life, liberty, and property of the individual for its own sake, whatever the excuse.

Sometimes I think the lefties moved over and made room for the righties because they became absolutely terrified of what else might be bearing down on them.

Well, not me, exactly, but somebody like me.

Only a lot worse.

Ayn Rand scared the living shit out of these people. An Evgeny Zamiatin or a Robert LeFevre or even an Ira Levin they could suppress or dismiss for one reason or another, which is why so few readers have ever heard of We, Lift Her Up Tenderly, or This Perfect Day. But little old Alice Rosenbaum was always right there in their nasty collectivist faces, her literary fists clenched, challenging their most fundamental assumptions in the very language (skiffy, the correct pronunciation of "sci-fi") that socialists of both stripes thought they had invented, stubbornly refusing to be dismissed or suppressed.

But what scared the lefties even more was the actual new universe that seemed to be resulting—inevitably, as it turned out—from the practice (by Joseph Stalin, Mao Tse Tung, Lyndon Baines Johnson, and Pol Pot, among others) of what they had been preaching since the time of H.G. Wells and Edward Bellamy.

It wasn't simply that the ideas of left-wing socialism weren't working, although that was certainly bad enough. Invariably they seemed to culminate in the deaths—from causes ranging from starvation to firing-squad—of tens of millions of the proletarians they'd been intended to benefit in the first place. And even worse, under the stress-testing of harsh reality (this was before the Evil Empire collapsed in an unprecedented, although not exactly unpredictable, manner) they sometimes—often—mutated into right-wing socialism.

Which is how it came to be that all those lonely, toothless, quakey-voiced old-timers (of all ages) still eking out their existences in the philosophical badlands and political ghost-towns that Left-Wing Utopia has become—and even those lucky enough to be living in far greater luxury off the tailings of the statist mother-lode they once helped mine—have nothing but bad news for us now. They're mistaking the failure of their ideas for a failure of reality.

As a consequence, many of them have simply given up and become whining nihilists. Those who are more successful tell us tales today of interstellar super-states with the unquestioned power to quarantine whole sectors of the galaxy because, in their infinite wisdom, they've decided it would be bad for unsupervised individuals of differing species to meet and freely exchange ideas and articles of trade with one another. At the same time, they speak of local authorities with the technological ability to search individuals for concealed weapons or other contraband at a distance—a practice nobody in these stories ever resists or even complains about on humane or Constitutional grounds.

This kind of thing used to be a staple of negative Utopias like 1984 and Brave New World, furnishings for cautionary tales about a regrettable loss of liberty. Now it's taken for granted as inevitable, and probably even desirable, whatever other possibilities the future may present. The writers never seem to notice that nobody is listening—at least not to that part of the story.

So what happens to a community of timeworn left-wing Utopian writers who for decades have continued to insist on seeing a future that demonstrably—to anyone who isn't tenured, working for television, or living in Sri Lanka—doesn't work? Enter J.R.R. Tolkien, along with what seemed at the time like thousands of blatant imitators, sucked into the world-swallowing vacuum in the science fiction market created by the implosion of Marxoid idealism. Enter the dragons, the dwarves, and the enchanted swords. Bazookas and armored personnel carriers came later, and when they did, it seemed like a breath of fresh air.

And so, as irrationality and magic began to displace reason and science as the motivating epistemology, and as the genre began looking backward to feudalism and the Middle Ages (for all its socialism, science fiction had been a forward-looking literature of limitless perspectives) and as readers began to tire of narrowed horizons (not to mention the same old thing re- rewritten over and over), the rack space—"inevitably" once again—began to diminish.

It was the exceptions (and don't you hate it when this happens?) that proved the rule—and still do today. The books that kept the rack space open for all those parasitic and reactionary dragons, dwarves, enchanted swords, bazookas, and armored personnel carriers, the only books that didn't gradually decrease in number, were those with spaceships, aliens, and ringed planets still on their covers, those whose subtitles now always seemed to include the word "star", accompanied either by the word, "wars" or the word, "trek".

There is some truth in the idea that Star Wars succeeded partly by co-opting medievalism. And it's equally true that Star Trek and its progeny have remained as unabashedly, old-fashionedly socialistic as The Shape of Things to Come steadfastly (and this is an important secret of their success) refusing to acknowledge the utter demise of socialism in every other branch of the cosmos.

But both displayed a future (yes, I know, Star Wars claims to be set "a long time ago in a galaxy far, far away", but who believes it?) featuring individualistic causes, violent adventures, and technology almost anyone could look forward to. Hence their remarkable success in a period when science fiction generally lay dying, killed by the bankruptcy of its underlying ideas and a craven retreat from a future it knew it could no longer predict, create, or control.

But the question is, do we really want to grant to Lucasian neofeudal mysticism or to Roddenberrian military socialism a monopoly on the future by default? Lucas and Roddenberry did their job, they held the line for science fiction, and preserved a remnant of that precious and dwindling rack space. But, to put the question a different way, how do we go about expanding it again?

The one realistic answer will be a bitter pill for all the right-wing socialists, the obsolete leftists, the dwarfmongers, and the bazookists to swallow. Science fiction died because its self-contradictory dreams died first. Its one hope is to usher in an alternative literature of a credible yet fantastic future worth believing in, worth working for, and therefore worth reading about.

That literature doesn't have to be created, it already exists. I'm very proud to say that I had a hand in its creation over 15 years ago, along with a dozen other novelists of my approximate age and outlook. Even better, I know of at least a dozen more science fiction manuscripts by other, mostly younger writers with the same viewpoint as ours, languishing now for lack of proper editorial attention. I predict that if New York publishing doesn't make a place for them soon, they will make a place for themselves, and on their own terms.

I can't bring myself to believe that New York really wants that. The last time something like that happened, they got Rush Limbaugh. Do they actually want a dozen Limbaughs, with both halves of their brains fully operational (which automatically makes them libertarians rather than conservatives) occupying the intellectual void science fiction has made of itself?

It may already be too late; New York may no longer have a choice.

I hope so.

[see also the following article]

Four-time Prometheus Award-winner L. Neil Smith has been called one of the world's foremost authorities on the ethics of self-defense. He is the author of 25 books, including The American Zone, Forge of the Elders, Pallas, The Probability Broach, Hope (with Aaron Zelman), and his collected articles and speeches, Lever Action, all of which may be purchased through his website "The Webley Page" at

Ceres, an exciting sequel to Neil's 1993 Ngu family novel Pallas was recently completed and is presently looking for a literary home.

Neil is presently working on Ares, the middle volume of the epic Ngu Family Cycle, and on Roswell, Texas, with Rex F. "Baloo" May.

The stunning 185-page full-color graphic-novelized version of The Probability Broach, which features the art of Scott Bieser and was published by BigHead Press has recently won a Special Prometheus Award. It may be had through the publisher, or at

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New Figures - British Parade Ground

June 29 2012 at 8:33 AM
Dan Taylor  (Login Dan-Taylor)
Missing-Lynx members
from IP address

Hello All
Whilst trying to finish the QLR, a number of little jobs have inserted themselves into my plans! I'll be back to that soon but, in the mean time, here are some new 1/76-1/72 figure sets just added to my website. I actually made them a while ago and Milicast have been marketing them but it's about time I did a little more to promote them.

The theme, as the title suggests, is British Soldiers a parade ground/inspection setting. There are three sets - 'Atten-Shun'; 'Stand Easy' and 'Officers-on-Parade'. Each has five figures in various appropriate poses. The attached pictures show figures from the 'Atten-shu' and 'Officers..' sets. If you would like to see further pictures, please go to my site -http://www.dantaylormodelworks.com/british-parade-ground-41-c.asp.
[linked image]

[linked image]

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Thanks for looking. Any questions, please let me know.

Best wishes
Dan Taylor

 Respond to this message

  1. Nice! - Tomas Jakl on Jun 29, 2012, 1:41 PM
  3. Re: New Figures - British Parade Ground - Neil Craig on Jun 29, 2012, 3:39 PM
  5. British Figures - Charles Martin on Jun 29, 2012, 10:46 PM
  7. Re: New Figures - British Parade Ground - T-izumi on Jun 29, 2012, 11:46 PM
  9. Thanks and paint colours - Dan Taylor on Jul 1, 2012, 4:54 AM

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Home / Digital News / Six Strategies for Growing Your Cannabis Brand

Six Strategies for Growing Your Cannabis Brand

With the emerging cannabis industry extending its reach across the country, many marketers are venturing into this new frontier with questions about how to best make an impact. In a field this new, which saw revenues of nearly $7 billion in 2016, there are no established rules to guide you (although there are laws — but we’ll get to that in a moment).

HOW can you market your cannabis products for success? Here are a few strategies that will expand your appeal and position you as a successful pioneer in this yet-uncharted territory.

1. Think “sophisticated” rather than “stoner”

The days of the stoner stereotype are behind us, so leave the smiley faces, 420, and Rasta flags out. Today, cannabis brands are seeking to widen their appeal to a broad range of consumers, not just giggling hippies.

Take Leafs by Snoop, the cannabis line that is owned and promoted by rapper Snoop Dogg. Leafs has a clean, classy appeal that looks like it could be sold at any luxury boutique or spa; it would fit in a gift basket alongside fine soaps, candies, or perfumes.

Think of it this way: you want your cannabis brand to have an aesthetic that would look right on a shelf at Whole Foods rather than behind the counter at the 7-Eleven.

2. Elevate the language

Part of escaping the old stoner trope involves lending cannabis a more refined lexicon. That’s why we’re calling it the “cannabis” industry rather than the “pot” or, even, the “marijuana” industry.

Use language that pushes your products into a more refined space. “Buds” become “flowers.” “Hits” are now “doses.” And so forth.

Forget the slang, and speak to a higher conversation.

3. Hit on the health factors

The modern consumer is more concerned with health than ever. She eats organic, practices yoga, and has quit smoking. He traded coffee for tea, grows his own food, and uses a fitness app to track his workouts.

Bring this thinking to cannabis. Not only does it fit naturally into this lifestyle, but we’re learning more and more about the health benefits of cannabis all the time.

Leverage this, and let people know that your cannabis product is an integral part of any health routine.

4. Join the conversation and the community

Cannabis is more than a product — it’s a movement. Whether we’re talking about medicinal applications, legal issues, or simply the enjoyment value, remember that there is a larger culture around your product.

Your brand needs to be a driving force in that culture. That means joining the conversation and getting involved. Create a blog that provides the latest industry news. Participate in industry events and expos. Support relevant causes.

Make your company synonymous with the cannabis community.

5. Know the law, and follow it

Different states and localities have widely varying laws pertaining to cannabis marketing. Educate yourself and stick to the rules lest you end up facing penalties that can span fines to losing your business license, or worse.

For example, be careful when it comes to making cartoons or videos. Any marketing that could be construed as being directed at minors could land you in hot water.

So study up, and stick to the rules.

6. Innovate

When it comes to the cannabis industry, there is no beaten path, which means it is a space that is rife with opportunity for innovation. Not only that, but cannabis consumers tend to be somewhat more open-minded, creatively inspired folks.

Have fun. Don’t think you have to do what everyone else is doing. Take risks. Experiment and make mistakes.

In the end, it’s important to remember that you’re on the forefront of an entirely new industry, and that you’re positioned to set trends rather than follow them.

So think different. Think new. Think forward.

After all, isn’t that kind of what cannabis is all about in the first place?

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Home / Digital News / The Future of the Internet Is at Stake With Next-Generation Ad Blocking

The Future of the Internet Is at Stake With Next-Generation Ad Blocking

The internet is creating an open-source revolution — a bottoms-up change in human habits across the globe, driven by hundreds of thousands of technologists, content creators, and business leaders to create nearly unlimited entertainment, news, information, commerce and services unimaginable in other eras.

But something insidious has begun to threaten the free web as we know it.

Armed with powerful tools that block advertising from appearing on websites, new and old players in the ad-blocking and mobile operator industry are threatening to strip ads at the “network level” from websites, in some cases seeking revenue-sharing agreements from the very companies whose ads are being blocked.

What does that mean for consumers? Simple. Ad blocking at the network level undercuts consumers’ ability to control the content they see and don’t see, which is a bedrock principle of the world wide web.

Shine Technologies, backed prominently by Horizons Ventures and based in Israel, offers ad-blocking technology that allows companies to block ads at the network level. And last September, Jamaica-based mobile carrier Digicel announced that it would begin to implement Shine’s technology on its network, blocking ads across all mobile display, apps and mobile video. Although there is an opt-out mechanism, by using Shine, Digicel is indiscriminately blocking ads for all customers by default, without offering a real choice or any transparency into their actions. Exactly how Shine determines what consumers can and cannot see is not available for consumers to understand.

Digicel has been sharply criticized by ECTEL, the Eastern Caribbean Telecommunications Authority, which sent correspondence to Digicel reiterating its position on net neutrality, which states that “service providers should treat all data on the internet the same, not intercepting, interrupting, blocking, degrading, discriminating or charging differentially,” and that ECTEL is “therefore advising Digicel to adhere to the stated principles of net neutrality and to refrain from deploying its ad control technology within ECTEL member states.”

The ultimate hypocrisy? After framing all of this as favorable to consumers, Digicel has approached major media companies for revenue-sharing agreements. In Digicel’s vision of the future, the network will resemble a pay-to-play toll booth, charging consumers and publishers for access to each other.

Online ads power the open internet and preserve the value exchange that lets consumers enjoy unlimited content for free by simply indulging a few seconds of advertising. Yes, some consumers are choosing to install ad blockers, and many websites are coping with this by explaining the value exchange and even offering leaner ad experiences to strike a balance for consumers. But if a consumer chooses to use an ad blocker, it should be his or her choice to decide what to see and what not to see.

Digital publishers are committed to improving the user experience — but regrettably, it looks like this won’t stop the unilateral, industrial ISP-level ad blockers. In February of this year, in advance of Mobile Congress World in Barcelona, Three UK and Three Italy announced plans to implement Shine’s technology on their mobile networks, paving the way for other brands in the Three Group to follow.

Another new entrant in the ad-blocking field is Brave, launched by former Mozilla CEO Brendan Eich. Brave’s business model not only strips ads from publishers’ pages — it replaces them with its own for-profit ads. Earlier this month, 17 member companies of the Newspaper Association of America (representing more than 1,200 newspapers in the U.S.) sent a letter to Brave arguing that its actions are unethical and raise legal questions about copyright infringement and unfair competition.

This type of ad blocking raises a host of issues that should worry casual internet users, American businesses and proponents of an open internet. These bad actors are stealing from publishers, subverting freedom of the press, operating a business model predicated on censorship of content, and ultimately forcing consumers to pay more money for less and less diverse information. Worse, there is no distinction between good ads and bad ads — it is all or nothing. At the end of the day, this will do nothing to create a better media ecosystem and will only contribute to the end of the open and free web as users know and enjoy it today. It also raises concerns about anti-competitive behavior and freedom of expression.

There is a way out of this conundrum. It requires new operating standards — consumer-friendly rules of the road that regulate how we will operate our sites, our advertising, and our delivery. The IAB and IAB Tech Lab are doing their part in working with online publishers and advertisers, but everyone who values a free and open internet should unite in opposition to this looming threat in Latin America and the Caribbean — and even here in the U.S.

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Celebrating 2012 through art

Published Tuesday 1 May 2012

Come along and visit the Red House Museum in Christchurch to celebrate this culturally significant year.

Budding artists, painters, photographers and sculptors were challenged by the National Park Authority and Forestry Commission to create innovative works of art using the colours gold, silver and bronze.

These works of art, along with a joint exhibition with New Forest District Council celebrating New Forest athletes of the future, will be on display until Saturday 19 May.

Open art competition winners:

  • First prize: Christine Hughes with her picture of a New Forest Pony.
  • Second prize: Susan Rolfe with her poem ‘My New Forest stroll’.
  • Third prize: Barry Fry with his painting of Mark Ash Wood.

Barry Whitcher, Judge and New Forest photographer, said: ‘This year’s winners and runners-up have been creative and imaginative; showing the New Forest at its very best. Art enables you to stop and take in your surroundings. You may have visited a place many times before but stopping to think about where you are often shows you something new and interesting.’

In particular the judges were impressed by the originality, creativity and imagination shown by many of the entrants.

Hilary Marshall, fellow Judge and Manager of the New Forest Centre, said about the winners:

‘New Forest ponies – This photograph really challenges our notion of Forest landscape and the ambiguity of the image causes the viewer to think again about our most loved icon. In playing with scale and form it almost hints at other worldliness and is both creative and charming.

‘My New Forest Stroll – What I love about this poem is the way that it captures a moment in time. A casual stroll through the Forest becomes an exploration of the senses and the concise and accessible structure of the poem allows us to join in that experience.

‘Golden light – This painting is on one hand a representation of Forest landscape and on the other, an exploration of light and texture. The glorious colours and patterning of the canopy and ground cover create a remarkably strong visual statement.

Both these exhibitions will also be at the New Forest Centre from 14 July – 2 September 2012.

Notes to Photo Editor:

Photograph of winning picture by Christine Hughes attached. Higher-res picture is available on request.

Notes to Editors:

Red House Museum opening times: Tuesday to Friday 10am-5pm; Saturday 10am-4pm. Temporary closures to some areas of the museum (not the art gallery) from 6 May -19 May.

Protect - Enjoy - Prosper

The New Forest National Park Authority’s statutory purposes are to:

  • Conserve and enhance the natural beauty, wildlife and cultural heritage of the Park - Protect
  • Promote opportunities for understanding and enjoyment of its special qualities – Enjoy.

We also have a duty to:

  • Seek to foster the social and economic well-being of local communities within the Park – Prosper.

The New Forest National Park was designated in March 2005. Its unique landscape has been shaped over the centuries by grazing ponies, cattle and pigs which roam free. Majestic woodlands, rare heathland and a spectacular coastline provide fabulous opportunities for quiet recreation, enjoyment and discovery.

More articles in the news archive.

New Forest National Park Authority news feed

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