RESUMEN
Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/µm2 . Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.
Asunto(s)
Optogenética , Retina , Channelrhodopsins/genética , Membrana Celular/metabolismo , Retina/metabolismo , Mutación , Microscopía FluorescenteRESUMEN
Viral rhodopsins are light-gated cation channels representing a novel class of microbial rhodopsins. For viral rhodopsin 1 subfamily members VirChR1 and OLPVR1, channel activity is abolished above a certain calcium concentration. Here we present a calcium-dependent spectroscopic analysis of VirChR1 on the femtosecond to second time scale. Unlike channelrhodopsin-2, VirChR1 possesses two intermediate states P1 and P2 on the ultrafast time scale, similar to J and K in ion-pumping rhodopsins. Subsequently, we observe multifaceted photocycle kinetics with up to seven intermediate states. Calcium predominantly affects the last photocycle steps, including the appearance of additional intermediates P6Ca and P7 representing the blocked channel. Furthermore, the photocycle of the counterion variant D80N is drastically altered, yielding intermediates with different spectra and kinetics compared to those of the wt. These findings demonstrate the central role of the counterion within the defined reaction sequence of microbial rhodopsins that ultimately defines the protein function.
Asunto(s)
Calcio , Rodopsinas Microbianas , Calcio/química , Calcio/metabolismo , Cinética , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismoRESUMEN
The technique of time-resolved macromolecular crystallography (TR-MX) has recently been rejuvenated at synchrotrons, resulting in the design of dedicated beamlines. Using pump-probe schemes, this should make the mechanistic study of photoactive proteins and other suitable systems possible with time resolutions down to microseconds. In order to identify relevant time delays, time-resolved spectroscopic experiments directly performed on protein crystals are often desirable. To this end, an instrument has been built at the icOS Lab (in crystallo Optical Spectroscopy Laboratory) at the European Synchrotron Radiation Facility using reflective focusing objectives with a tuneable nanosecond laser as a pump and a microsecond xenon flash lamp as a probe, called the TR-icOS (time-resolved icOS) setup. Using this instrument, pump-probe spectra can rapidly be recorded from single crystals with time delays ranging from a few microseconds to seconds and beyond. This can be repeated at various laser pulse energies to track the potential presence of artefacts arising from two-photon absorption, which amounts to a power titration of a photoreaction. This approach has been applied to monitor the rise and decay of the M state in the photocycle of crystallized bacteriorhodopsin and showed that the photocycle is increasingly altered with laser pulses of peak fluence greater than 100â mJâ cm-2, providing experimental laser and delay parameters for a successful TR-MX experiment.
Asunto(s)
Proteínas , Sincrotrones , Análisis Espectral , Proteínas/química , Cristalografía , LuzRESUMEN
Microbial rhodopsins have become an indispensable tool for neurobiology. Of thousands of identified microbial rhodopsins, a minute fraction has been studied so far and they have shown remarkable functional diversity suggesting more great promises that this large family holds. Effective production of recombinant microbial and viral rhodopsins is a prerequisite for the success of functional and structural studies of these proteins. Escherichia coli (E. coli) are suitable for high yield expression of many of microbial and viral rhodopsins and they facilitate rapid exploration of this large protein family.
Asunto(s)
Escherichia coli , Rodopsina , Escherichia coli/genética , Escherichia coli/metabolismo , Rodopsina/química , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genéticaRESUMEN
In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins (MPs) at a molecular level. High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs (two different proton pumps and a sodium light-driven ion pump). The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs - a surface usually accommodating hydrophobic chains of annular lipids (which are known structural and functional determinants for MPs). In conformity with these results, supplementary in silico molecular dynamics (MD) analysis predicted even greater numbers of argon and krypton binding positions on MP surface within the bilayer. These results indicate a potential importance of such interactions, particularly as related to the phenomenon of noble gas-induced anaesthesia.
Asunto(s)
Anestésicos , Criptón , Argón/química , Argón/farmacología , Cristalografía por Rayos X , Criptón/química , Criptón/metabolismo , LípidosRESUMEN
Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited. The challenge is that their 'visualization' requires ultrahigh-resolution structures of the ground and functionally important intermediate states to identify proton translocation events and perform their structural assignment. Our true-atomic-resolution structures of the light-driven proton pump bacteriorhodopsin, a model in studies of proton transport, show that CHBs and LBHBs not only serve as proton pathways, but also are indispensable for long-range communications, signaling and proton storage in proteins. The complete picture of CHBs and LBHBs discloses their multifunctional roles in providing protein functions and presents a consistent picture of proton transport and storage resolving long-standing debates and controversies.
Asunto(s)
Proteínas , Protones , Enlace de HidrógenoRESUMEN
Ribonucleases (RNases) are a non-mutagenic alternative to harmful DNA-damaging anticancer drugs. Targeting of RNases with antibodies to surface antigens that are selectively expressed on tumor cells endows specificity to the cytotoxic actions of RNases. Barnase, a ribonuclease from Bacillus amyloliquefaciens, is a promising candidate for targeted delivery to cancer cells because of its insusceptibility to the ubiquitous cytoplasmic ribonuclease inhibitor, and its high stability and catalytic activity. Here, we characterized in vitro and in vivo an immunoRNase, scFv 4D5-dibarnase, which consists of two barnase molecules that are fused serially to the single-chain variable fragment (scFv) of humanized 4D5 antibody. The latter is directed against the extracellular domain of human epidermal growth factor receptor 2 (HER2), a cancer marker that is overexpressed in many human carcinomas. The scFv 4D5-dibarnase exerted a specific cytotoxic effect on HER2-overexpressing SKBR-3 and BT-474 human breast carcinoma cells (IC(50) = 4.1 and 2.4 nM, respectively) via induction of apoptosis. Ten doses of 0.7 mg/kg scFv 4D5-dibarnase to BALB/c nude mice that bore SKBR-3 human breast cancer xenografts resulted in a 76% reduction in tumor growth. A single injection of scFv 4D5-dibarnase at a total course dose of 7 mg/kg did not cause severe side effects in BALB/c nude or BDF1 mice. The cytotoxicity and selectivity of scFv 4D5-dibarnase merit consideration of this immunoRNase as a potent anticancer agent.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Receptor ErbB-2/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Ribonucleasas/metabolismo , Ribonucleasas/uso terapéutico , Anticuerpos de Cadena Única/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacología , Proteínas Bacterianas , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Inyecciones Intravenosas , Riñón/efectos de los fármacos , Riñón/patología , Ratones , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Ribonucleasas/química , Ribonucleasas/farmacología , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/químicaRESUMEN
Cluster crystals are periodic structures with lattice sites occupied by several, overlapping building blocks, featuring fluctuating site occupancy, whose expectation value depends on thermodynamic conditions. Their assembly from atomic or mesoscopic units is long-sought-after, but its experimental realization still remains elusive. Here, we show the existence of well-controlled soft matter cluster crystals. We fabricate dendritic-linear-dendritic triblock composed of a thermosensitive water-soluble polymer and nanometer-scale all-DNA dendrons of the first and second generation. Conclusive small-angle X-ray scattering (SAXS) evidence reveals that solutions of these triblock at sufficiently high concentrations undergo a reversible phase transition from a cluster fluid to a body-centered cubic (BCC) cluster crystal with density-independent lattice spacing, through alteration of temperature. Moreover, a rich concentration-temperature phase diagram demonstrates the emergence of various ordered nanostructures, including BCC cluster crystals, birefringent cluster crystals, as well as hexagonal phases and cluster glass-like kinetically arrested states at high densities.
Asunto(s)
Dendritas/química , Nanoestructuras/química , Estructura Molecular , Transición de Fase , Dispersión del Ángulo Pequeño , TemperaturaRESUMEN
Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans. The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor.
Asunto(s)
Bombas de Protones/química , Rodopsina/química , Transporte Iónico , Luz , Filogenia , Dominios Proteicos , Rodopsina/fisiologíaRESUMEN
The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Flavobacteriaceae/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Cristalografía por Rayos X , Escherichia coli/metabolismo , Simulación de Dinámica Molecular , Pliegue de Proteína , Rodopsina/química , Rodopsina/metabolismo , Sodio/metabolismo , Difracción de Rayos XRESUMEN
Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na+/K+-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca2+ ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca2+ permeability.
Asunto(s)
Fitoplancton/virología , Rodopsina/química , Rodopsina/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Animales , Calcio/metabolismo , Cationes , Células Cultivadas , Channelrhodopsins/metabolismo , Células HEK293 , Humanos , Activación del Canal Iónico , Luz , Neuronas/metabolismo , Filogenia , Conformación Proteica , Ratas Wistar , Rodopsina/genética , Relación Estructura-Actividad , Proteínas Virales/genética , Difracción de Rayos XRESUMEN
Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.
Asunto(s)
Neoplasias de la Mama/patología , Medios de Contraste , Técnica del Anticuerpo Fluorescente/métodos , Aumento de la Imagen/métodos , Región Variable de Inmunoglobulina , Microscopía Fluorescente/métodos , Puntos Cuánticos , Neoplasias de la Mama/inmunología , Línea Celular Tumoral , Humanos , Región Variable de Inmunoglobulina/inmunologíaRESUMEN
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.
Asunto(s)
Rodopsina/química , ATPasa Intercambiadora de Sodio-Potasio/química , Sodio/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Conformación Molecular , Mutación , Unión Proteica , Multimerización de Proteína , Rodopsina/genética , Rodopsina/metabolismo , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Relación Estructura-ActividadRESUMEN
Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists.
Asunto(s)
Phycodnaviridae/metabolismo , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/ultraestructura , Bacteriorodopsinas , Cristalografía por Rayos X , Halobacterium salinarum , Activación del Canal Iónico , Canales Iónicos , Luz , Simulación de Dinámica Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Rodopsinas Microbianas/fisiologíaRESUMEN
We successfully cloned and expressed a single-chain antibody (425scFv), that is directed to human epidermal growth factor receptor HER1 (EGFR) in transgenic tobacco plants as a fusion with bacterial barstar gene (425scFv-barstar). Plant-produced recombinant 425scFv-barstar was recovered using barstar-barnase system. Based on barstar-barnase affinity, during purification of the plant-produced 425scFv-barstar, we generated bispecific scFv-antibody heterodimers from individual single-chain fragments initially produced in different host systems with binding activity to both HER1 and HER2/neu tumor antigens. We demonstrated by flow cytometry and indirect immunofluorescent microscopy that both the components of heterodimer retain its specific cell-binding activity.
Asunto(s)
Receptores ErbB/inmunología , Fragmentos de Inmunoglobulinas/biosíntesis , Nicotiana/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Bacterianas/genética , Western Blotting , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Microscopía Fluorescente , Plantas Modificadas Genéticamente , Receptor ErbB-2 , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
The light-gated ion channel channelrhodopsin 2 (ChR2) from Chlamydomonas reinhardtii is a major optogenetic tool. Photon absorption starts a well-characterized photocycle, but the structural basis for the regulation of channel opening remains unclear. We present high-resolution structures of ChR2 and the C128T mutant, which has a markedly increased open-state lifetime. The structure reveals two cavities on the intracellular side and two cavities on the extracellular side. They are connected by extended hydrogen-bonding networks involving water molecules and side-chain residues. Central is the retinal Schiff base that controls and synchronizes three gates that separate the cavities. Separate from this network is the DC gate that comprises a water-mediated bond between C128 and D156 and interacts directly with the retinal Schiff base. Comparison with the C128T structure reveals a direct connection of the DC gate to the central gate and suggests how the gating mechanism is affected by subtle tuning of the Schiff base's interactions.
Asunto(s)
Channelrhodopsins/química , Secuencia de Aminoácidos , Channelrhodopsins/genética , Channelrhodopsins/ultraestructura , Chlamydomonas reinhardtii , Cristalografía por Rayos X , Transporte Iónico , Optogenética , Conformación Proteica , Alineación de SecuenciaRESUMEN
One of the major and essential classes of transmembrane (TM) receptors, present in all domains of life, is sensor histidine kinases, parts of two-component signaling systems (TCSs). The structural mechanisms of TM signaling by these sensors are poorly understood. We present crystal structures of the periplasmic sensor domain, the TM domain, and the cytoplasmic HAMP domain of the Escherichia coli nitrate/nitrite sensor histidine kinase NarQ in the ligand-bound and mutated ligand-free states. The structures reveal that the ligand binding induces rearrangements and pistonlike shifts of TM helices. The HAMP domain protomers undergo leverlike motions and convert these pistonlike motions into helical rotations. Our findings provide the structural framework for complete understanding of TM TCS signaling and for development of antimicrobial treatments targeting TCSs.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de la Membrana/química , Fosfoproteínas/química , Cristalización/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Dominios Proteicos , Transducción de SeñalRESUMEN
Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins.
Asunto(s)
Optogenética , Bombas de Protones/metabolismo , Rodopsina/metabolismo , Archaea/metabolismo , Sitios de Unión , Línea Celular , Cromatografía Líquida de Alta Presión , Escherichia coli/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Luz , Liposomas , Modelos Moleculares , Optogenética/métodos , Unión Proteica , Conformación Proteica , Protones , Retina/metabolismo , Rodopsina/química , Análisis EspectralRESUMEN
Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.
Asunto(s)
Bacteriorodopsinas/metabolismo , Secuencia de Aminoácidos , Bacteriorodopsinas/genética , Cristalografía por Rayos X , Escherichia coli/metabolismo , Halobacterium salinarum/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , ARN Mensajero/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de SecuenciaRESUMEN
Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.