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Stefan Lemke |1 Stefan Lemke BIO 385 – Professor Walter 1/30/2013 Channelrhodopsin-2: Structure, Function, and Applications Photoreception in both vertebrates and invertebrates is mediated by photoreceptors called rhodopsins, consisting of seven transmembrane-helix proteins (opsins), covalently linked to retinal. Two types of rhodopsins are differentiated on the basis of primary sequence. Animal rhodopsins, expressed in animals (including humans), consist of G-protein coupled receptors. Microbial rhodopsins, on the other hand, are found in archaea, eubacteria, fungi, and algae and are direct-light-activated regulators of transmembrane ion conductance, including both light driven ion pumps and light-driven ion channels. In a seminal paper by Nagel et al. (2003), it was demonstrated that Channelrhodopsin-2 (ChR2), a microbial rhodopsin, is a directly light-gated cation-selective ion channel that can be expressed in Xenepus oocytes. This discovery began with the detection of two sequences with homology to known microbial opsins in the genome of Chlamydomonas reinhardtii, a single-cell green algae. These sequences correspond to Channelrhodopsin-1 (ChR1) and Channelrhodopsin-2 (ChR2), and are responsible for phototaxic (movement toward light) and photophobic (movement away from light) behaviors in C. reinhardtii. Nagel et al. suggested that ChR2 could be used as a tool to modulate neural activity by both increasing cytosolic Ca+ and depolarizing the cell in response to illumination. This paper aims to discuss the structure and function of ChR2, focusing on the light-gating mechanisms, electronegative pore, and similarities/differences to bacteriorhodopsin, another microbial rhodopsin. While the exact structure of ChR2 remains unclear, recent efforts have provided important information that serves to elucidate the structure and understand the function. I will first discuss the current understanding of ChR2 structure and function and then go into the application of ChR2 in optogenetics, both at its origins and today. Structure and Function Overall Muller et al. (2011) performed one of the first studies to elucidate the structure of ChR2. Twodimensional crystals were grown and flattened on electron microscopy grids, allowing for projection maps to be created, showing the surface topography of the protein. This map suggested two main findings: (1) ChR2 contains seven transmembrane helices (TMs) and (2) forms a dimer (Figure 1). An Stefan Lemke |2 SDS-PAGE gel was run to examine the dimeric nature of ChR2. As ChR2 remained dimeric in the gel despite the disruption of non-covalent interaction by SDS, it was suggested that a remarkably stable dimer is formed by ChR2 that is likely to exist in dimeric form in the membrane. The next structural data on ChR2 was provided by Kato et al. (2012). As native ChR2 has proven hard to crystalize, Kato et al. formed a chimaera (C1C2) between ChR1 and ChR2 consisting of ChR1 without its C-terminus end and with the last two TMs swapped for those from ChR2. This chimaera Figure 1. Projection map consisted of the 342 amino-terminal residues of the 737 amino acid residues of ChR2. From Muller et al. (2011) in the native channel. This was sufficient because the first 300 residues of the amino-terminal end contain the seven-transmembrane domains, extra-/intra-cellular loops and the lightgating mechanisms. A crystal structure of this chimaera at 2.3 Å was presented to provide a working model for the molecular function of ChR2 (Figure 2). The main finding for the overall structure was that the dimeric interactions between the two C1C2 protein domains were found at the N-domains, extracellular loops 1 (ECL1), and TM3s and TM4s. Figure 2. Structure of C1C2 dimer viewed parallel to membrane (left, middle) and from the extracellular side (right). N and C domain, TMs, extracellular loops (ECLs) and intracellular loops (ICLs) shown. From Kato et al. (2012). Structural comparison with BR Both Muller et al. and Kato et al. compared their structures (ChR2 and C1C2, respectively) to bacteriorhosopin (bR), another microbial rhodopsin that acts as a light-driven proton pump. Muller el al. showed that the arrangement of the seven TMs in both proteins is similar, with the greatest similarities between TM3-6 (Figure 3). In addition, Muller et al. emphasized that while ChR2 forms a dimer, bR Stefan Lemke |3 arranges itself as a trimer (Figure 3). Kato et al. found two distict differences between bR and C1C2. First, C1C2 has an additional N-terminal and C-terminal domain. It is believed that the N-terminal domain may explain the dimer formation in ChR2 while the C-terminal domain is involved in cellular localization. Second, the extracellular ends of TM1 and TM2 in C1C2 are tilted outward by 3.0 and 4.1 Å, respectively (Figure 4). As we will see, the pore of ChR2 is formed between TM1, TM2, TM3, and TM7, suggesting that this tilt may have implications on the cation permeability of ChR2. Figure 3. Superimposed projection map of ChR2 (red) on bR (gray). From Muller et al. (2011) Figure 4. Structural comparison of ChR2 and bR. From Kato et al. (2012). Light-gating mechanisms The light-gating mechanisms of ChR2 are mediated by retinal, a molecule that undergoes a conformational change in response to the absorption of energy from photons. In the “dark state” of ChR2, before any light stimulation, the retinal is found in an all-trans conformation and is covalently linked to Lys 296 on TM7 forming a Schiff base. The changes in ChR2 that occur in response to light, or the photocycle, were characterized by Ritter et al. (2008) using absorption of UV-visible light and Fourier Transform Infrared Spectroscopy (FTIR). UV-visible absorbance is used to characterize the protonation state of the retinal Schiff base. Generally, absorption below 400nm indicates a deprotonated Schiff base, whereas a red-shifted absorption above 400nm indicates a protonated form. On the other hand, FTIR allows for analysis of more discrete structural changes in the actual protein. Stefan Lemke |4 Upon light excitation, the dark state of ChR2 (D470, as it absorbs light maximally at 470nm) becomes a transient P500 intermediate, caused by the isomerization of all-trans retinal to all-cis retinal (Figure 5). The P500 quickly changes into another transient intermediate, P390, indicating the deprotonation of the Schiff base. The Schiff base quickly reprotonates, which results in the conductive state of the channel, P520. There are two main structural changes that are believed to coincide with Figure 5. Proposed photocycle of ChR2. From the reprotonation of the Schiff base and lead to the Ritter et al. (2008). opening of the channel. First, Kato et al. used their crystal structure to predict that Asp 292 is the primary proton acceptor from the Schiff base. This protonation of Asp 292 is believed to result in ionic “push” of nearby Lys 132 that moves Lys 132’s positive charge away from the channel pore and allows cations to pass (Figure 6). Secondly, Eisenhauer et al. (2012) showed, also using FTIR, that the protonated form of Glu 90 prevents cation flow through the channel pore (Figure 7). Therefore, they hypothesize that the reprotonation of the Schiff base also coincides with the deprotonation and subsequent solvation of Glu 90 which, combined with the lysine “push,” results in cation permeability. The closing mechanisms of ChR2 are less well characterized, but are believed to involve changes in the backbone of the channel, along with changes in hydrogen bonding of Glu 90 and occur on a much slower timescale. Figure 6. Interactions between the Schiff base, K292, and K132. Black lines represent hydrogen bonds. From Kato et al. (2012). Figure 7. Comparison of solvation between protonated and deprotonated Glu 90. From Eisenhauer et al. (2012) Stefan Lemke |5 Electronegative pore Channel pore properties of ChR2 have taken longer to elucidate than many other aspects of the protein. As late as 2011, Muller et al. speculated about the location of the pore channel, whether it existed at the dimer interface or within each protein domain. Two recent findings, along with knowledge of the photocycle, have made clear that a pore exists in each protein domain between TM1, TM2, TM3, and TM7. First, Kato et al. showed that polar/negative amino acid residues form an electronegative pore that exists on the extracellular side of the channel, consisting of Gln 95, Thr 98, Ser 102, Glu 122, Glu 129, Lys 132, Glu 136, Glu 140, Glu 162, Thr 285, Asp 292, and Asn 297 (Figure 8). Notably, TM2 contains most of the negative residues, and it is suggested that TM2 determines cation conductance and selectivity. The second finding was from Richards and Dempski (2012), who performed a sequence analysis of ChR2 and bR and found eight serine residues within the transmembrane domains of bR that are not present in ChR2 (C87S, G181S, G224S, and M255S located near the retinal, I197S, Q210S, and V269S located at the cytoplasmic end of the transmembrane domain, and P234S) (Figure 9). By creating single serine mutated ChR2 constructs corresponding to homologous residues in bR and expressing them in Xenopus oocytes, they were able to examine the effects of these missing residues on the channel properties of ChR2. These mutations were found to decrease cation conductance, perhaps due to the high Figure 8. Position of serine mutations. From Richards and Dempski (2012). Figure 9. Depiction of hydrophilic and electronegative pore lining surface. From Kato et al. (2012). Stefan Lemke |6 affinity for serine residues to form inter- and intra-helical hydrogen bonds, resulting in a reduction in pore size. The authors concluded that the cation selectivity of the channel does not solely exist in the extracellular pore, but further along the channel, and that these missing serines play a large role. ChR2 and bR We can now appreciate the differences between ChR2 and bR and ask ourselves, why is ChR2 permeable to cations while bR is a proton pump? Three main differences can help us preliminarily explain this difference, further work will be needed to affirm and extend these conclusions. First of all, the light-gated structural changes are much greater in ChR2 compared to bR. As mentioned above, Asp 292 is the proton acceptor from the deprotonation of the Schiff base in ChR2. This leads to a cascade of structural changes that allow cation permeability. However, in bR the primary proton acceptor is a water molecule, which does not result in the same structural changes. Incidentally, this also accounts for ChR2’s blue-shifted absorption spectrum (max absorbance at a wavelength of 470), as compared to that of BR (max absorbance at a wavelength of 568 nm). The absorption spectrum is determined by the energy difference between the ground and first excited state of retinal. This is determined by the distance between the protonated Schiff base and its counter ion, which for ChR2 is 1 Å closer to the Schiff base compared to BR. The second difference accounting for cation permeability in ChR2, is the outward shifted TM1 and TM2. As emphasized by Kato et al., TM2 is important in cation permeability due to the presence of negative amino acid residues. An outward shift of these TMs enlarges the pore and is likely to influence cation permeability. Lastly, the missing serines studied by Richards and Dempski are a distinct difference between ChR2 and bR that have been shown to prevent cation permeability if reintroduced. These differences can begin to explain the differences in permeability between ChR2 and bR. However, due to the strong homology between ChR2 and bR, investigations about a possible proton pump mechanism in ChR2 were undertaken. Feldbauer et al. (2009) were the first to characterize the function of ChR2 as both a light-driven proton pump and cation channel. They reconstituted ChR2 in a planar membrane with no ion gradient or electrical potential difference. In response to illumination, no current flow is expected. However, small outward photocurrents were observed, providing evidence for the proton-pumping ability of ChR2. Nack et al. (2012) further elucidated the kinetics of this mechanism. Nack et al. expressed ChR2 in yeast and examined proton currents using spectrophotometry and an optical pH indicator. They found that proton release and uptake in ChR2 matches the rise and Stefan Lemke |7 decay of the P520 intermediate (Figure 10). As the P520 state represents the cation permeable state of ChR2, an intimate mechanistic link between the two functions of ChR2 is suggested. Interestingly, proton release by ChR2 occurs with the re-protonation of the retinal Schiff base (rise of the P520 state) and not with the deprotonation as is seen in BR, suggesting a mechanistic difference between the two proteins. Therefore, it seems as if ChR2 is a leaky proton pump, meaning that it allows cation flow, in addition to pumping protons, in Figure 10. Proton pumping one of its photointermediates. This suggests a similar function to bR. photocycle of ChR2. From Nack et al.the (2012). However, we must keep in mind the mechanistic differences seen between proteins, in regards to both cation conductance and proton pumping. Application Optogenetics The application of ChR2 to modulate neuronal activity, termed optogenetics, began with Boyden et al. (2005). By injecting adeno-associated virus (AAV), containing ChR2 and yellow fluorescent protein in rat CA3/CA1 hippocampal neurons, illumination of ChR2-positive neurons with blue light induced rapid depolarizing currents. These exciting results showed consistency both in the same neuron and across neurons. Additionally, spike frequency was dependent on light intensity, allowing for unprecedented control of neural activity (Figure 11). Since its genesis, optogenetics has evolved in incredible ways. The advances in the functional use of ChR2 far outreach its structural discoveries. We will look at two recent papers showing how far optogenetics has come, and some of the incredible applications it posseses. Figure 11. Data from some of the first optogenetic experiments. From Boyden et al. (2005). Stefan Lemke |8 Stuber et al. (2011) used optogenetics to investigate neural circuits implicated in reward, specifically the Nucelus Accumbens (NAc), part of the mesolimbic dopamine pathway, thought to be involved in the rewarding effects of drugs of abuse. Stuber et al. were able to stimulate specificly glutamatergic afferent projections to the NAc from both the basolateral amygdala (BLA) and the medial prefrontal cortex Figure 12. Optogenetic (mPFC). To do this, an AAV dependent on a Camk2a promoter, was technique depiction. From injected in either the BLA or the mPFC of adult mice, leading to the Stuber et al. (2011). specific trasfection of gluatamatergic neurons in either the BLA or the mPFC. Subsequent applications of light to the BLA only depolarized projections from either the BLA or the mPFC (Figure 12). The mice were then underwent one hour self-stimulation sessions in which active “nosepokes” provided 5ms of photostimulation to projections from the BLA or mPFC. Selective activation of BLA, but not mPFC, glutamatergic inputs to the NAc promoted nosepoking (Figure 13). This is consistent with the hypothesized role of BLA inputs in facilitating responding to cues, and of mPFC inputs in suppressing inappropriate actions. Furthermore, it was also shown that selective activation of projections from the BLA led to EPSC with greater amplitude. This may suggest that BLA projections release glutamate, while mPFC projections do not (Figure 13). Figure 13. Comparison of nose pokes and EPSC from BLA-to-NAc and mPFC-to-NAc. From Stuber et al. (2011). Packer et al. (2012) used two photon optogenetics to push the limits of neuronal modulation with light. By activating membrane opsins with two lower-energy, longer-wavelength photons, incredible precision, on the level of single dendrites and dendritic spines is possible, as lower-wavelength photons scatter less. Unfortunately, ChR2 has a small single-channel conductance and fast kinetics, meaning that when using two-photon activation of a neuron with ChR2, very high ChR2 expression would be required Stefan Lemke |9 to create action potentials. To make two-photon microscopy and optogenetics commensurable, Packer et al. created a chimeric opsin by combining ChR1 and VchR1 (from multicellular algae Volvox carteri). This chimaera, C1V1, has a red-shifter photon absorption (over 1000nm) and slow channel kinetics, allowing for the use of two-photon optogenetics to induce neural firing. Transfection of this chimaera, in conjunction with yellow fluorescent protein, allowed Packer et al. to make three incredible “gestals.” First, they were able to achieve selective activation of cellular processes down to the dendrite and dendritic spine (Figure 14). Second, they demonstrate the potential of two-photon optogenetics to map synaptic circuits. Stimulating single neurons allows for an investigation of the functional relationship (e.g. excitatory or inhibitory synapse) between neurons. Specifically, one can patch clamp a neuron and stimulate various surrounding neurons and look for a response in the patched neuron (Figure 15). Third, Packer et al. examined the use of two-photon spatial light modulation (SLM)-based microscopy. This method allows for the simultaneous activation of up to 15 neurons with consisted firing patterns. These two papers exemplify both the current work and the incredible potential for optogenetics as a tool to modulate neural activity. Future efforts to bridge our knowledge of the structure of ChR2 and other microbial rhodopsins with the incredible new developments in optogenetics will provide immense benefits to both fronts and allow for even greater variations and applications of ChR2 going forward. Figure 14. Two-photon stimulation of cellular processes (top), including individual dendrites and spines (bottom). From Packer et al. (2012). Figure 15. Mapping synaptic circuits with two-photon optogenetics. Photostimulation of neuron i shows a response in patch clamped neuron ii. From Packer et al. (2012). Stefan Lemke | 10 References Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8:1263-1268. Eisenhauer K, Kuhne J, Ritter E, Berndt A, Wolf S, Freier E, Bartl F, Hegemann P, Gerwert K (2012) In channelrhodopsin-2 Glu-90 is crucial for ion selectivity and is deprotonated during the photocycle. J Biol Chem 287:6904-6911. Feldbauer K, Zimmermann D, Pintschovius V, Spitz J, Bamann C, Bamberg E (2009) Channelrhodopsin-2 is a leaky proton pump. Proc Natl Acad Sci U S A 106:12317-12322. 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Packer AM, Peterka DS, Hirtz JJ, Prakash R, Deisseroth K, Yuste R (2012) Two-photon optogenetics of dendritic spines and neural circuits. Nat Methods 9:1202-1205. Richards R, Dempski RE (2012) Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2. PLoS One 7:e50018. Ritter E, Stehfest K, Berndt A, Hegemann P, Bartl FJ (2008) Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy. J Biol Chem 283:35033-35041. Stuber GD, Sparta DR, Stamatakis AM, van Leeuwen WA, Hardjoprajitno JE, Cho S, Tye KM, Kempadoo KA, Zhang F, Deisseroth K, Bonci A (2011) Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking. Nature 475:377-380.