43 research outputs found
Platymonas subcordiformis Channelrhodopsin-2 (PsChR2) Function II. RELATIONSHIP OF THE PHOTOCHEMICAL REACTION CYCLE TO CHANNEL CURRENTS*
Protein Sequence and Membrane Lipid Roles in the Activation Kinetics of Bovine and Human Rhodopsins.
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A Comparison between the Photoactivation Kinetics of Human and Bovine Rhodopsins.
Rhodopsin is a G-protein-coupled receptor important for vertebrate vision under dim light conditions. Many studies of the activation mechanism of bovine rhodopsin have been conducted, but there have been relatively few investigations of the human protein. A recent study of the late photointermediates of bovine rhodopsin studies at 15 掳C and pH 7.3, 8.0, and 8.7 revealed a rather complex activation mechanism involving two metarhodopsin I480 and metarhodopsin II intermediates. Human rhodopsin was studied under these same conditions using time-resolved optical absorption spectroscopy with measurements from 10 渭s to 200 ms after photolysis. The results show that the two proteins follow the same photoactivation mechanism, although their kinetics differ significantly. The comparison of bovine and human rhodopsins shows that the initial Schiff base deprotonation equilibrium is more forward shifted in human rhodopsin, and more of the reaction flows through the metarhodopsin I380 intermediate in human rhodopsin than in the bovine protein
Amplitude Analysis of Single-Wavelength Time-Dependent Absorption Data Does Not Support the Conventional Sequential Mechanism for the Reduction of Dioxygen to Water Catalyzed by Bovine Heart Cytochrome c
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Complexity of Bovine Rhodopsin Activation Revealed at Low Temperature and Alkaline pH.
The late intermediates involved in the activation mechanism of bovine rhodopsin are investigated by time-resolved optical absorption spectroscopy. Measurements from 10 渭s to 200 ms after photolysis were carried out on membrane suspensions of bovine rhodopsin at a temperature of 15 掳C and at pH of 7.3, 8.0, and 8.7. The time-resolved absorption spectra in the 330-650 nm range were analyzed by global exponential and kinetic scheme fitting methods. The results indicate an activation mechanism that is more complex than suggested previously. It involves interconnected branched pathways with two metarhodopsin I480 and two metarhodopsin II intermediates. The intermediates involved in this more complex mechanism need to be considered in spectroscopic studies that vary sample temperature and pH in order to enhance the presence of specific rhodopsin intermediates
Platymonas subcordiformis Channelrhodopsin-2 (PsChR2) Function: II. RELATIONSHIP OF THE PHOTOCHEMICAL REACTION CYCLE TO CHANNEL CURRENTS.
Channelrhodopsins, such as the algal phototaxis receptor Platymonas subcordiformis channelrhodopsin-2 (PsChR2), are light-gated cation channels used as optogenetic tools for photocontrol of membrane potential in living cells. Channelrhodopsin (ChR)-mediated photocurrent responses are complex and poorly understood, exhibiting alterations in peak current amplitude, extents and kinetics of inactivation, and kinetics of the recovery of the prestimulus dark current that are sensitive to duration and frequency of photostimuli. From the analysis of time-resolved optical absorption data, presented in the accompanying article, we derived a two-cycle model that describes the photocycles of PsChR2. Here, we applied the model to evaluate the transient currents produced by PsChR2 expressed in HEK293 cells under both fast laser excitation and step-like continuous illumination. Interpretation of the photocurrents in terms of the photocycle kinetics indicates that the O states in both cycles are responsible for the channel current and fit the current transients under the different illumination regimes. The peak and plateau currents in response to a single light step, a train of light pulses, and a light step superimposed on a continuous light background observed for ChR2 proteins are explained in terms of contributions from the two parallel photocycles. The analysis shows that the peak current desensitization and recovery phenomena are inherent properties of the photocycles. The light dependence of desensitization is reproduced and explained by the time evolution of the concentration transients in response to step-like illumination. Our data show that photocycle kinetic parameters are sufficient to explain the complex dependence of photocurrent responses to photostimuli
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Platymonas subcordiformis Channelrhodopsin-2 (PsChR2) Function: II. RELATIONSHIP OF THE PHOTOCHEMICAL REACTION CYCLE TO CHANNEL CURRENTS.
Channelrhodopsins, such as the algal phototaxis receptor Platymonas subcordiformis channelrhodopsin-2 (PsChR2), are light-gated cation channels used as optogenetic tools for photocontrol of membrane potential in living cells. Channelrhodopsin (ChR)-mediated photocurrent responses are complex and poorly understood, exhibiting alterations in peak current amplitude, extents and kinetics of inactivation, and kinetics of the recovery of the prestimulus dark current that are sensitive to duration and frequency of photostimuli. From the analysis of time-resolved optical absorption data, presented in the accompanying article, we derived a two-cycle model that describes the photocycles of PsChR2. Here, we applied the model to evaluate the transient currents produced by PsChR2 expressed in HEK293 cells under both fast laser excitation and step-like continuous illumination. Interpretation of the photocurrents in terms of the photocycle kinetics indicates that the O states in both cycles are responsible for the channel current and fit the current transients under the different illumination regimes. The peak and plateau currents in response to a single light step, a train of light pulses, and a light step superimposed on a continuous light background observed for ChR2 proteins are explained in terms of contributions from the two parallel photocycles. The analysis shows that the peak current desensitization and recovery phenomena are inherent properties of the photocycles. The light dependence of desensitization is reproduced and explained by the time evolution of the concentration transients in response to step-like illumination. Our data show that photocycle kinetic parameters are sufficient to explain the complex dependence of photocurrent responses to photostimuli