The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements. We have resolved the vibrational changes associated with the open states of the channel (P 2 390 and P 3 520 ) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane α-helices with a t 1/2 = 60 μs, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t 1/2 = 10 μs), with the latter being reprotonated by aspartic acid 156 (t 1/2 = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. Previous conclusions on the involvement of glutamic acid 90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the nonconductive P 4 480 state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions and the protein conformational changes to the gating of the cation channel.O ptogenetics provides new tools to neurophysiologists to steer cellular responses with unprecedented temporal and spatial resolution. The former takes advantage of light as an ultrashort trigger, whereas the latter is achieved by genetically encoding and directing photosensitive proteins to specific cell types. The most prominent among the optogenetics tools is channelrhodopsin (ChR), which was found to be the first light-gated ion channel of its kind (1, 2). This discovery paved the way for an exponentially growing number of neurophysiological applications, ranging from single cells to living animals (3). Light-gated ion permeation by ChR expands the various modes of action of the large family of microbial rhodopsins already comprising light-driven ion pumps and sensors (4). Among the various ChRs, which differ mostly in cation selectivity (3), ChR2 is used in the majority of optogenetic applications because of the higher expression yield in mammalian cells.A projection structure of the heptahelical ChR2 showed a dimer with the contact interface between helices C and D suggested to form the cation channel (5). More recently, a chimeric ChR (C1C2) was constructed by linking the last two helices (F and G) of ChR2 to the first five (A to E) of ChR1 and resolved by X-ray crystallography to 2.3 Å (6). The high-resolution structure confirmed the dimeric arrangement and identified an electronegative extracellular pore in each monomer framed by helices A, B, C, and G. Accompanying electrophysiological experiments on point mutants indicated r...

Edited by Richard CogdellKeywords: Optogenetic Rhodopsin Light-activated cation channel Photocycle Membrane protein EPR spectroscopy a b s t r a c tChannelrhodopsin is a cation channel with the unique property of being activated by light. To address structural changes of the open state of the channel, two variants, which contain either 1 or 2 wild-type cysteines, were derivatised with nitroxide spin label and subjected to electron paramagnetic resonance spectroscopy. Both variants contained the C128T mutation to trap the long-lived P 520 3 state by illumination. Comparison of spin-spin distances in the dark state and after illumination reflect conformational changes in the conductive P 520 3 state involving helices B and F. Spin distance measurements reveal that channelrhodopsin forms a dimer even in the absence of intermolecular N-terminal cysteines. Structured summary of protein interactions:ChR2 and ChR2bind by electron resonance (View interaction)

The interest in fructose metabolism is based on the observation that an increased dietary fructose consumption leads to an increased risk of obesity and metabolic syndrome. In particular, obesity is a known risk factor to develop many types of cancer and there is clinical and experimental evidence that an increased fructose intake promotes cancer growth. The precise mechanism, however, in which fructose induces tumor growth is still not fully understood. In this article, we present an overview of the metabolic pathways that utilize fructose and how fructose metabolism can sustain cancer cell proliferation. Although the degradation of fructose shares many of the enzymes and metabolic intermediates with glucose metabolism through glycolysis, glucose and fructose are metabolized differently. We describe the different metabolic fates of fructose carbons and how they are connected to lipogenesis and nucleotide synthesis. In addition, we discuss how the endogenous production of fructose from glucose via the polyol pathway can be beneficial for cancer cells.

A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.Channelrhodopsins are involved in phototaxis and photophobia of unicellular green algae (1). They constitute a new class of light-gated ion channels containing the seven-transmembrane helix motif and the chromophore retinal as the light-sensitive cofactor, which are both common to the other retinal-containing proteins such as bacteriorhodopsin (bR) 4 or visual rhodopsin (2). In CrChR2 the chromophore retinal is bound to Lys-257 (3). Channelrhodopsin activation via lightinduced isomerization of retinal from all-trans to 13-cis is coupled to a transient cofactor deprotonation and a functional protein structural change. In channelrhodopsin, as well as in other retinal-containing photoreceptors, subtle changes in the chromophore vicinity trigger large scale protein conformational changes remote from the chromophore binding pocket. Rearrangement of helix B is hypothesized to constitute a key element in channel opening by allowing the entry of water molecules (4 -6). A continuous water wire is a prerequisite for the formation of the ion-conducting pathway. A hydrophilic pore between helices A, B, C, and G was suggested to serve as the ion permeation pathway based on the high resolution crystal structure of the C1C2 chimera (3 10).The inner gate was hypothesized to be involved in CrChR2 activation (formation of the open conducting channel state) together with the tilt of helix B (5). Evidence for light-induced movements of helix B comes from structural studies using electron crystallography and EPR spectroscopy (double electronelectron resonance) (11-13). Together these data suggest that in contrast to bR and sensory rhod...

untransfected cells retained high levels of this drug indicating that the MRP1 protein is functional. Using fluorescence recovery after photobleach (FRAP) we also demonstrated that the 2-color MRP1 is freely diffusible within the plasma membrane. 2-color MRP1 exhibited dynamic FRET changes in response to ATP and ATP þ substrate but not by substrate alone. FRET changes were quantified as an index of MRP1 conformational changes. These FRET changes correlate well with the available crystal structures which show close interaction of NBDs in the presence of ATP. Furthermore, we showed that ATP increased FRET in a concentration dependent manner with an apparent affinity of 107 mM. The data suggested that the relative affinity of MRP1 for nucleotides was ATP > ADP >> AMP. Finally, interactions of ATP analogs (ATPgS, AMP-PNP, AMP-PCP) with MRP1 revealed their lower affinity compared to ATP, since much higher amounts were required to induce the NBD closure. Our results provide insight into the structural dynamics of the MRP1 tranporter.