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Dysfunction and diseases of the gastrointestinal (GI) tract are a major driver of medical care. The vagus nerve innervates and controls multiple organs of the GI tract and vagus nerve stimulation (VNS) could provide a means for affecting GI function and treating disease. However, the vagus nerve also innervates many other organs throughout the body, and off-target effects of VNS could cause major side effects such as changes in blood pressure. In this study, we aimed to achieve selective stimulation of populations of vagal afferents using a multi-contact cuff electrode wrapped around the abdominal trunks of the vagus nerve. Four-contact nerve cuff electrodes were implanted around the dorsal (N = 3) or ventral (N = 3) abdominal vagus nerve in six ferrets, and the response to stimulation was measured via a 32-channel microelectrode array (MEA) inserted into the left or right nodose ganglion. Selectivity was characterized by the ability to evoke responses in MEA channels through one bipolar pair of cuff contacts but not through the other bipolar pair. We demonstrated that it was possible to selectively activate subpopulations of vagal neurons using abdominal VNS. Additionally, we quantified the conduction velocity of evoked responses to determine what types of nerve fibers (i.e., Aδ vs. C) responded to stimulation. We also quantified the spatial organization of evoked responses in the nodose MEA to determine if there is somatotopic organization of the neurons in that ganglion. Finally, we demonstrated in a separate set of three ferrets that stimulation of the abdominal vagus via a four-contact cuff could selectively alter gastric myoelectric activity, suggesting that abdominal VNS can potentially be used to control GI function.

Objective. Epidural spinal cord stimulation (SCS) is a potential intervention to improve limb and autonomic functions, with lumbar stimulation improving locomotion and thoracic stimulation regulating blood pressure. Here, we asked whether sacral SCS could be used to target the lower urinary tract (LUT) and used a high-density epidural electrode array to test whether individual electrodes could selectively recruit LUT nerves. Approach. We placed a high-density epidural SCS array on the dorsal surface of the sacral spinal cord and cauda equina of anesthetized cats and recorded the stimulation-evoked activity from nerve cuffs on the pelvic, pudendal and sciatic nerves. Main results. Here we show that sacral SCS evokes responses in nerves innervating the bladder and urethra and that these nerves can be activated selectively. Sacral SCS always recruited the pelvic and pudendal nerves and selectively recruited both of these nerves in all but one animal. Individual branches of the pudendal nerve were always recruited as well. Electrodes that selectively recruited specific peripheral nerves were spatially clustered on the arrays, suggesting anatomically organized sensory pathways. Significance. This selective recruitment demonstrates a mechanism to directly modulate bladder and urethral function through known reflex pathways, which could be used to restore bladder and urethral function after injury or disease.

INTRODUCTION AND OBJECTIVE:Lower urinary tract (LUT) dysfunction arising from injury and disease remain a challenge in the clinic due to paucity of pharmacological treatments. Peripheral neuromodul...

IUCrJ 5(1), 103 - 117 (2018). doi:10.1107/S2052252517017043, Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals., Published by IUCr, Chester

Photosynthetic purple bacteria convert solar energy to chemical energy with near unity quantum efficiency. The light-harvesting process begins with absorption of solar energy by an antenna protein called Light-Harvesting Complex 2 (LH2). Energy is subsequently transferred within LH2 and then through a network of additional light-harvesting proteins to a central location, termed the reaction center, where charge separation occurs. The energy transfer dynamics of LH2 are highly sensitive to intermolecular distances and relative organizations. As a result, minor structural perturbations can cause significant changes in these dynamics. Previous experiments have primarily been performed in two ways. One uses non-native samples where LH2 is solubilized in detergent, which can alter protein structure. The other uses complex membranes that contain multiple proteins within a large lipid area, which make it difficult to identify and distinguish perturbations caused by protein-protein interactions and lipid-protein interactions. Here, we introduce the use of the biochemical platform of model membrane discs to study the energy transfer dynamics of photosynthetic light-harvesting complexes in a near-native environment. We incorporate a single LH2 from Rhodobacter sphaeroides into membrane discs that provide a spectroscopically amenable sample in an environment more physiological than detergent but less complex than traditional membranes. This provides a simplified system to understand an individual protein and how the lipid-protein interaction affects energy transfer dynamics. We compare the energy transfer rates of detergent-solubilized LH2 with those of LH2 in membrane discs using transient absorption spectroscopy and transient absorption anisotropy. For one key energy transfer step in LH2, we observe a 30% enhancement of the rate for LH2 in membrane discs compared to that in detergent. Based on experimental results and theoretical modeling, we attribute this difference to tilting of the peripheral bacteriochlorophyll in the B800 band. These results highlight the importance of well-defined systems with near-native membrane conditions for physiologically-relevant measurements., United States. Department of Energy. Office of Basic Energy Sciences (Award DE-SC0018097)

Contains fulltext : 154849.pdf (Publisher’s version ) (Open Access) Channelrhodopsins (ChRs) from green flagellate algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs to bioengineer their properties for specific optogenetic applications such as elucidating the function of specific neurons in brain circuits. While most studies have used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), in this work low-temperature Fourier transform infrared-difference spectroscopy is applied to study the conformational changes occurring during the primary phototransition of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1). Substitution with isotope-labeled retinals or the retinal analogue A2, site-directed mutagenesis, hydrogen-deuterium exchange, and H2(18)O exchange were used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to that of CrChR2, almost exclusively an all-trans to 13-cis isomerization of the retinal chromophore, as in the primary phototransition of bacteriorhodopsin (BR). In addition, significant differences are found for structural changes of the protein and internal water(s) compared to those of CrChR2, including the response of several Asp/Glu residues to retinal isomerization. A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1, which reflects along with amide I bands alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2, indicating a more heterogeneous environment. Mutations involving residues Glu169 and Asp299 (homologues of the Asp85 and Asp212 Schiff base counterions, respectively, in BR) lead to the conclusion that Asp299 is protonated during P1 formation and suggest that these residues interact through a strong hydrogen bond that facilitates the transfer of a proton from Glu169.

Channelrhodopsins (ChRs), which form a distinct branch of the microbial rhodopsin family, control phototaxis in green algae. Because ChRs can be expressed and function in neuronal membranes as light-gated cation channels, they have rapidly become an important optogenetic tool in neurobiology. While channelrhodopsin-2 from the unicellular alga Chlamydomonas reinhardtii (CrChR2) is the most commonly used and extensively studied optogenetic ChR, little is known about the properties of the diverse group of other ChRs. In this study, near-infrared confocal resonance Raman spectroscopy along with hydrogen-deuterium exchange and site-directed mutagenesis were used to study the structure of red-shifted ChR1 from Chlamydomonas augustae (CaChR1). These measurements reveal that (i) CaChR1 has an all-trans-retinal structure similar to those of the light-driven proton pump bacteriorhodopsin (BR) and sensory rhodopsin II but different from that of the mixed retinal composition of CrChR2, (ii) lowering the pH from 7 to 2 or substituting neutral residues for Glu169 or Asp299 does not significantly shift the ethylenic stretch frequency more than 1-2 cm(-1) in contrast to BR in which a downshift of 7-9 cm(-1) occurs reflecting neutralization of the Asp85 counterion, and (iii) the CaChR1 protonated Schiff base (SB) has stronger hydrogen bonding than BR. A model is proposed to explain these results whereby at pH 7 the predominant counterion to the SB is Asp299 (the homologue to Asp212 in BR) while Glu169 (the homologue to Asp85 in BR) exists in a neutral state. We observe an unusual constancy of the resonance Raman spectra over the broad range from pH 9 to 2 and discuss its implications. These results are in accord with recent visible absorption and current measurements of CaChR1 [Sineshchekov, O. A., et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys. J. 104, 807-817; Li, H., et al. (2014) Role of a helix B lysine residue in the photoactive site in channelrhodopsins. Biophys. J. 106, 1607-1617].

Archaerhodopsin-3 (AR3) is a light-driven proton pump from Halorubrum sodomense, but little is known about its photocycle. Recent interest has focused on AR3 because of its ability to serve both as a high-performance, genetically-targetable optical silencer of neuronal activity and as a membrane voltage sensor. We examined light-activated structural changes of the protein, retinal chromophore, and internal water molecules during the photocycle of AR3. Low-temperature and rapid-scan time-resolved FTIR-difference spectroscopy revealed that conformational changes during formation of the K, M, and N photocycle intermediates are similar, although not identical, to bacteriorhodopsin (BR). Positive/negative bands in the region above 3,600 cm( - 1), which have previously been assigned to structural changes of weakly hydrogen bonded internal water molecules, were substantially different between AR3 and BR. This included the absence of positive bands recently associated with a chain of proton transporting water molecules in the cytoplasmic channel and a weakly hydrogen bonded water (W401), which is part of a hydrogen-bonded pentagonal cluster located near the retinal Schiff base. However, many of the broad IR continuum absorption changes below 3,000 cm( - 1) assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR were very similar in AR3. This work and subsequent studies comparing BR and AR3 structural changes will help identify conserved elements in BR-like proton pumps as well as bioengineer AR3 to optimize neural silencing and voltage sensing.

Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.

Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3 to 11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (O(all-trans)) is found. At higher pH a second 13-cis retinal N-like species (N(13-cis)) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N(13-cis) detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N(13-cis) to M(13-cis) levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential.

Channelrhodopsins (ChRs) from green algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs in order to bioengineer their properties for specific optogenetic applications. Most studies have focused so far on channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Here, low-temperature and time-resolved rapid scan FTIR-difference spectroscopy, along with kinetic UV-Visible transient absorbance spectroscopy, are applied to study protein conformational changes occurring during the photocycle of the red-shifted channelrhodopsin-1 from Chlamydomonas augustae (CaChR1). Substitutions with isotope-labeled retinals or the retinal analog A2, site-directed mutagenesis, hydrogen-deuterium exchange and H218O substitution are used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to CrChR2, almost exclusively all-trans to 13-cis isomerization of the retinal chromophore, similar to the primary phototransition of bacteriorhodopsin (BR). A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1 which, along with amide I bands, reflects alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2 indicating a more heterogeneous environment. Based on the data from mutations involving the residues Glu169 and Asp299 (homologs of the Asp85 and Asp212 Schiff base counterions in BR), as well as Phe139 (Lys132 in the C1C2 chimera located within hydrogen bonding distance of the homologs of Asp85 and Asp212), we propose a model for protonation changes occurring near the Schiff base during the P1 and P2 steps of the photocycle.

Channelrhodopsins (ChRs) control phototaxis in green algae and function as light-gated cation channels when expressed in animal cells. Because ChRs can be functionally expressed in neuronal membranes, this distinct family of microbial rhodopsins have rapidly become an important tool in neuroscience. While the light-activated molecular changes occurring in channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) have been extensively studied, little is known about such changes in the diverse groups of other ChRs including the major class of channelrhodopsin-1 (ChR1). Here, we have characterized the structure and molecular changes in ChR1 from Chlamydomonas augustae (CaChR1). This ChR has properties advantageous for light modulated neuronal control including a red-shifted λmax and slow light inactivation compared to CrChR2 (Hou, S. et al. (2012) Photochem Photobiol 88, 119-128). Near-infrared confocal resonance Raman spectroscopy (RRS) reveals that in contrast to CrChR2, which contains a mixture of retinal isomers, the retinal chromophore structure of CaChR1 is almost completely all-trans in the light-adapted state similar to many microbial rhodopsins including bacteriorhodopsin. In addition, unlike other ChR1s, such as CrChR1 (ChR1 from Chlamydomonas reinhardtii), which exhibit significant shifts in λmax at different pHs, the RRS of CaChR1 including the ethylenic frequency which reflects λmax remains largely unaltered over a wide pH range. This insensitivity to pH is despite the presence of the residue Glu87 (CrChR1 sequence numbering) which has been previously associated with pH sensitivity. Low-temperature FITR difference-spectroscopy reveals that the primary phototransition of CaChR1 to the K photointermediate involves an all-trans to 13-cis retinal isomerization and significant changes in the protein structure including structural changes in the peptide backbone, Asp/Glu residues, Cys residues and internal water molecules. These results are discussed in terms of possible differences in the molecular mechanism of various ChRs.

Melanopsin, found in intrinsically photosensitive retinal ganglion cells, drives circadian rhythm and pupillary response. A variety of studies indicate that melanopsin shares many properties with invertebrate rhodopsin such as found in the eyes of squid including activation by an initial photon absorption and regeneration to the original state with a second red-shifted photon. We report the expression and initial spectroscopic characterization of recombinant expressed melanopsin. Mouse melanopsin, carrying a C-terminal deca-his-tag, was expressed in Sf9 insect cells, using recombinant baculovrius. Good expression levels of intact melanopsin protein were achieved (2-4 mg/l). To regenerate the active photopigment 11-cis retinal was added. However, solubilization efficiency was quite low for most detergents. Best results were obtained with dodecylphosphocholine (DPC). Efficient single-step purification was achieved with affinity chromatography over immobilized Ni-2+ ions. So far only a small percentage of the purified melanopsin was photoactive, showing a maximum absorbance at 480-500 nm. Preliminary low-temperature FTIR difference and resonance Raman spectroscopic measurements are reported for melanopsin and squid rhodopsin in order to compare structural features and conformational changes upon light activation including retinal isomerization and protein backbone changes. Supported by a grants from the NIH-NEI to KJR and from NWO-CW to WJdG.

Archaerhodopsin 3 (AR3) and Archaerhodopsin T (ArchT) are light-driven proton pumps that are used as genetically targetable neuronal silencers and fluorescent sensors of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 and ArchT incorporate into the plasma membranes of E. coli and mammalian cells. We used near-IR resonance Raman confocal microscopy and time resolved UV-Vis pump-probe absorption spectroscopy to study the effects of pH and membrane potential on the retinal chromophore structure and photocycle kinetics of these proteins. Measurements were performed on AR3 and ArchT reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3. The retinal chromophore structure of AR3 is an all-trans configuration almost identical to BR over pH ranging from 3-11. Small changes detected in the retinal ethylenic stretching frequency and Schiff base (SB) hydrogen-bonding strength relative to BR may be related to an alternate water structure near the SB. In the case of the mutant D95N, an all-trans retinal O-like species (Oall-trans) is found at neutral pH. At higher pH, a 13-cis retinal N-like species (N13-cis) is detected which is attributed to a slowly decaying intermediate in the D95N red-light photocycle. However, the amount of N13-cis detected is reduced in E. coli but restored upon dissipation of the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N13-cis to M13-cis equilibrium in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism offers a possible explanation for the observed dependence on transmembrane potential of the fluorescence of AR3, ArchT, and other microbial rhodopsins.

We examined structural changes of the protein, retinal chromophore and internal water molecules during the photocycle of Archaerhodopsin-3 (AR3), a light-driven proton pump found in Halobrubrum sodemense. This protein, which belongs to the archael rhodopsin family, has a ∼75% sequence homology with the more extensively studied bacteriorhodopsin (BR) proton pump from Halobacterium salinarum. Recent interest has focused on AR3 because of its ability to serve both as a high-performance genetically targetable optical silencer of neuronal activity and as a membrane voltage sensor. Low-temperature and rapid-scan time-resolved FTIR-difference spectroscopy revealed that the conformational changes during formation of the K, M and N photocycle intermediates are similar, although not identical, to BR. Positive/negative bands in the region above 3600 cm-1 which have previously been assigned to structural changes of weakly hydrogen bonded internal water molecules were substantially different between AR3 and BR. This included the absence of positive bands recently associated with a chain of proton transporting water molecules in the cytoplasmic channel and a weakly hydrogen bonded water (W401) which is part of a hydrogen-bonded pentagonal cluster located near the retinal Schiff base. However, many of the broad IR continuum changes below 3000 cm-1 assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions of the protein were very similar in AR3 and BR.