RESUMO
Since its discovery, the light-gated cation channel Channelrhodopsin-2 (ChR2) has proven to be a long-sought tool for the noninvasive, light-activated control of neural cells in culture and in living animals. Although ChR2 is widely used in neurobiological applications, little is known about its molecular mechanism. In this work, the unitary conductance of ChR2 was determined for different cations, for example 40 fS at 200 mM NaCl and -60 mV, using noise analysis. The kinetics of the ion channel obtained by noise analysis is in excellent agreement with the photocurrent kinetics obtained by voltage-clamp and time-resolved spectroscopy. The inward rectification of the channel could be explained by the single channel parameters. ChR2 represents an ion channel with a 7 transmembrane helix motif, even though the sequence homology of its essential amino acids to those of the light-driven H(+) pump bacteriorhodopsin (bR) is high. Here, we also show that when ChR2 is expressed in electrofused giant HEK293 cells or reconstituted on planar lipid membranes, it can indeed act as an outwardly driven H(+) pump, demonstrating that ChR2 is bifunctional, and in-line with other microbial rhodopsins, a H(+) pump but with a leak that shows ion channel properties.
Assuntos
Proteínas de Transporte/fisiologia , Canais Iônicos/fisiologia , Bombas de Próton/fisiologia , Proteínas de Transporte/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Guanidina/farmacologia , Humanos , Canais Iônicos/genética , Cinética , Luz , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Técnicas de Patch-Clamp , Bombas de Próton/genética , Cloreto de Sódio/farmacologia , Temperatura , TransfecçãoRESUMO
The 2 nanomotors of rotary ATP synthase, ionmotive F(O) and chemically active F(1), are mechanically coupled by a central rotor and an eccentric bearing. Both motors rotate, with 3 steps in F(1) and 10-15 in F(O). Simulation by statistical mechanics has revealed that an elastic power transmission is required for a high rate of coupled turnover. Here, we investigate the distribution in the F(O)F(1) structure of compliant and stiff domains. The compliance of certain domains was restricted by engineered disulfide bridges between rotor and stator, and the torsional stiffness (kappa) of unrestricted domains was determined by analyzing their thermal rotary fluctuations. A fluorescent magnetic bead was attached to single molecules of F(1) and a fluorescent actin filament to F(O)F(1), respectively. They served to probe first the functional rotation and, after formation of the given disulfide bridge, the stochastic rotational motion. Most parts of the enzyme, in particular the central shaft in F(1), and the long eccentric bearing were rather stiff (torsional stiffness kappa > 750 pNnm). One domain of the rotor, namely where the globular portions of subunits gamma and epsilon of F(1) contact the c-ring of F(O), was more compliant (kappa congruent with 68 pNnm). This elastic buffer smoothes the cooperation of the 2 stepping motors. It is located were needed, between the 2 sites where the power strokes in F(O) and F(1) are generated and consumed.
Assuntos
Escherichia coli/enzimologia , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Rotação , Citoesqueleto de Actina/metabolismo , Complacência (Medida de Distensibilidade) , Ativação Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Hidrólise , Magnetismo , Microesferas , Modelos Moleculares , Oxirredução , Estrutura Terciária de Proteína , Pontos QuânticosRESUMO
Optogenetics revolutionizes basic research in neuroscience and cell biology and bears potential for medical applications. We develop mutants leading to a unifying concept for the construction of various channelrhodopsins with fast closing kinetics. Due to different absorption maxima these channelrhodopsins allow fast neural photoactivation over the whole range of the visible spectrum. We focus our functional analysis on the fast-switching, red light-activated Chrimson variants, because red light has lower light scattering and marginal phototoxicity in tissues. We show paradigmatically for neurons of the cerebral cortex and the auditory nerve that the fast Chrimson mutants enable neural stimulation with firing frequencies of several hundred Hz. They drive spiking at high rates and temporal fidelity with low thresholds for stimulus intensity and duration. Optical cochlear implants restore auditory nerve activity in deaf mice. This demonstrates that the mutants facilitate neuroscience research and future medical applications such as hearing restoration.
Assuntos
Potenciais de Ação , Vias Auditivas/fisiologia , Neurônios/fisiologia , Optogenética/métodos , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Audição/fisiologia , Humanos , Camundongos , Mutação , Técnicas de Patch-Clamp , Permeabilidade , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Xenopus laevisRESUMO
An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.
Assuntos
Cálcio/metabolismo , Quimiocinas/metabolismo , Luz , Animais , Linhagem Celular Tumoral , Clatrina/metabolismo , Citosol/metabolismo , Endocitose , Células HEK293 , Humanos , Camundongos , Técnicas de Patch-Clamp , Ratos , Receptores CXCR4/metabolismoRESUMO
The light-gated cation channel channelrhodopsin-2 (ChR2) has rapidly become an important tool in neuroscience, and its use is being considered in therapeutic interventions. Although wild-type and known variant ChR2s are able to drive light-activated spike trains, their use in potential clinical applications is limited by either low light sensitivity or slow channel kinetics. We present a new variant, calcium translocating channelrhodopsin (CatCh), which mediates an accelerated response time and a voltage response that is ~70-fold more light sensitive than that of wild-type ChR2. CatCh's superior properties stem from its enhanced Ca²(+) permeability. An increase in [Ca²(+)](i) elevates the internal surface potential, facilitating activation of voltage-gated Na(+) channels and indirectly increasing light sensitivity. Repolarization following light-stimulation is markedly accelerated by Ca²(+)-dependent BK channel activation. Our results demonstrate a previously unknown principle: shifting permeability from monovalent to divalent cations to increase sensitivity without compromising fast kinetics of neuronal activation. This paves the way for clinical use of light-gated channels.