Biomedical subjects

Ernst Bamberg

Publications and source records attributed to Ernst Bamberg.

At least 19 recordsLinked to original sources

PubMed2006

Fluorometric measurements of intermolecular distances between the alpha- and beta-subunits of the Na+/K+-ATPase.

The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the alpha-subunit, which mediates ATP hydrolysis and ion translocation, as well as the beta-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the alpha- and beta-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the alpha- and beta-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the alpha-subunit and the beta-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the beta-subunit in relation to the alpha-subunit and suggest that the alpha- and beta-subunits move toward each other during the E2 to E1 conformational transition.

Animals↗

PubMed2006

Photo-induced covalent attachment of agonists as a tool to study allosteric mechanisms of nicotinic acetylcholine receptors.

Muscular and neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels and contain either two or five binding sites for acetylcholine (ACh). Binding of ACh molecules on the nAChR will trigger the fast opening of the channel and subsequent slow desensitization process. Neuronal alpha7 nicotinic receptors are made up of five identical subunits and possess five binding sites for ACh; this raises the question of how many sites must be occupied before channel opening. However, the effect of each ligand binding on gating is difficult to assess because of the reversible aspect of ligand binding at each site. One solution is to photochemically tether agonists to their binding sites. Such methodology has been applied elegantly and successfully on the homotetrameric cyclic-nucleotide-gated channels to evaluate the functional effects of each ligand binding on gating (Ruiz and Karpen, 1997). We therefore decided to develop a similar approach on Torpedo and neuronal alpha7 nAChRs with the photoactivatable agonist AC5 to investigate the effect of binding site occupancy on allosteric transitions of the receptor. In the dark, AC5 (see structure below) evokes robust currents on oocytes expressing Torpedo nAChR, displaying maximal amplitude comparable to ACh, with EC50 = 1.2 microM (Mourot et al., 2005). When the voltage-clamp oocyte was exposed to UV light in the presence of 30 microM AC5 for 50 s, there was a prolonged activation of the Torpedo nAChR, not reversible by washing, but inhibited by the noncompetitive blockers tetracaine and proadifen (see structure below). Both UV light and AC5 are required for this effect. However, further studies are required to determine whether the gradual decrease of the inward current reflects a slow desensitization process. AC5 is thus a potent photoactivatable agonist of the nAChR, which is able, upon UV irradiation, to incorporate covalently into the ACh-binding sites and to prolong activation of the nAChR. By extending this methodology to patch-clamp experiments, we will be able to incorporate one or several AC5s covalently into the muscular and neuronal nAChR at the single-channel level. Such study will help us understand the observed cooperative effect of gating and will contribute decisively to the controversial concerted vs sequential models for nAChR allosteric transitions.

Acetylcholine↗

PubMed2005

Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.

For studying the function of specific neurons in their native circuitry, it is desired to precisely control their activity. This often requires dissection to allow accurate electrical stimulation or neurotransmitter application , and it is thus inherently difficult in live animals, especially in small model organisms. Here, we employed channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii, in excitable cells of the nematode Caenorhabditis elegans, to trigger specific behaviors, simply by illumination. Channelrhodopsins are 7-transmembrane-helix proteins that resemble the light-driven proton pump bacteriorhodopsin , and they also utilize the chromophore all-trans retinal, but to open an intrinsic cation pore. In muscle cells, light-activated ChR2 evoked strong, simultaneous contractions, which were reduced in the background of mutated L-type, voltage-gated Ca2+-channels (VGCCs) and ryanodine receptors (RyRs). Electrophysiological analysis demonstrated rapid inward currents that persisted as long as the illumination. When ChR2 was expressed in mechanosensory neurons, light evoked withdrawal behaviors that are normally elicited by mechanical stimulation. Furthermore, ChR2 enabled activity of these neurons in mutants lacking the MEC-4/MEC-10 mechanosensory ion channel . Thus, specific neurons or muscles expressing ChR2 can be quickly and reversibly activated by light in live and behaving, as well as dissected, animals.

Animals↗

PubMed2005

Millisecond-timescale, genetically targeted optical control of neural activity.

Temporally precise, noninvasive control of activity in well-defined neuronal populations is a long-sought goal of systems neuroscience. We adapted for this purpose the naturally occurring algal protein Channelrhodopsin-2, a rapidly gated light-sensitive cation channel, by using lentiviral gene delivery in combination with high-speed optical switching to photostimulate mammalian neurons. We demonstrate reliable, millisecond-timescale control of neuronal spiking, as well as control of excitatory and inhibitory synaptic transmission. This technology allows the use of light to alter neural processing at the level of single spikes and synaptic events, yielding a widely applicable tool for neuroscientists and biomedical engineers.

Action Potentials↗

PubMed2005

Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates sucrose efflux under the control of the sucrose gradient and the proton motive force.

The phloem network is as essential for plants as the vascular system is for humans. This network, assembled by nucleus- and vacuole-free interconnected living cells, represents a long distance transport pathway for nutrients and information. According to the Münch hypothesis, osmolytes such as sucrose generate the hydrostatic pressure that drives nutrient and water flow between the source and the sink phloem (Münch, E. (1930) Die Stoffbewegungen in der Pflanze, Gustav Fischer, Jena, Germany). Although proton-coupled sucrose carriers have been localized to the sieve tube and the companion cell plasma membrane of both source and sink tissues, knowledge of the molecular representatives and the mechanism of the sucrose phloem efflux is still scant. We expressed ZmSUT1, a maize sucrose/proton symporter, in Xenopus oocytes and studied the transport characteristics of the carrier by electrophysiological methods. Using the patch clamp techniques in the giant inside-out patch mode, we altered the chemical and electrochemical gradient across the sucrose carrier and analyzed the currents generated by the proton flux. Thereby we could show that ZmSUT1 is capable of mediating both the sucrose uptake into the phloem in mature leaves (source) as well as the desorption of sugar from the phloem vessels into heterotrophic tissues (sink). As predicted from a perfect molecular machine, the ZmSUT1-mediated sucrose-coupled proton current was reversible and depended on the direction of the sucrose and pH gradient as well as the membrane potential across the transporter.

Animals↗

PubMed2005

pH-dependent photoisomerization of retinal in proteorhodopsin.

The early steps in the photocycle of the bacterial proton pump proteorhodopsin (PR) were analyzed by ultrafast pump/probe spectroscopy to compare the rate of retinal isomerization at alkaline and acidic pH values. At pH 9, the functionally important primary proton acceptor (Asp97, pK(a) = 7.7) is negatively charged; consequently, a reaction cycle analogous to the archaeal bacteriorhodopsin (BR) is observed. The excited electronic state of PR displays a pronounced biphasic decay with time constants of 400 fs and 8 ps. At pH 6 where Asp97 is protonated a similar biphasic decay is observed, although it is significantly slower (700 fs and 15 ps). The results indicate, in agreement to similar findings in other retinal proteins, that also in PR the charge distribution within the chromophore binding pocket is a major determinant for the rate and the efficiency of the primary reaction.

Archaeal Proteins↗

PubMed2005

Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation.

The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na+ and K+ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic alpha-subunit (four isoforms) and an ancillary beta-subunit (three isoforms). Mutations in the alpha2-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase alpha2-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [3H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. 86Rb+-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase alpha2-isoform.

Humans↗

PubMed2005

The beta subunit of the Na+/K+-ATPase follows the conformational state of the holoenzyme.

The Na+/K+-ATPase is a ubiquitous plasma membrane ion pump that utilizes ATP hydrolysis to regulate the intracellular concentration of Na+ and K+. It is comprised of at least two subunits, a large catalytic alpha subunit that mediates ATP hydrolysis and ion transport, and an ancillary beta subunit that is required for proper trafficking of the holoenzyme. Although processes mediated by the alpha subunit have been extensively studied, little is known about the participation of the beta subunit in conformational changes of the enzyme. To elucidate the role of the beta subunit during ion transport, extracellular amino acids proximal to the transmembrane region of the sheep beta1 subunit were individually replaced for cysteines. This enabled sulfhydryl-specific labeling with the environmentally sensitive fluorescent dye tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes. Investigation by voltage-clamp fluorometry identified three reporter positions on the beta1 subunit that responded with fluorescence changes to alterations in ionic conditions and/or membrane potential. These experiments for the first time show real-time detection of conformational rearrangements of the Na+/K+-ATPase through a fluorophore-labeled beta subunit. Simultaneous recording of presteady-state or stationary currents together with fluorescence signals enabled correlation of the observed environmental changes of the beta subunit to certain reaction steps of the Na+/K+-ATPase, which involve changes in the occupancy of the two principle conformational states, E1P and E2P. From these experiments, evidence is provided that the beta1-S62C mutant can be directly used to monitor the conformational state of the enzyme, while the F64C mutant reveals a relaxation process that is triggered by sodium transport but evolves on a much slower time scale. Finally, shifts in voltage dependence and kinetics observed for mutant K65C show that this charged lysine residue, which is conserved in beta1 isoforms, directly influences the effective potential that determines voltage dependence of extracellular cation binding and release.

Amino Acid Sequence↗

PubMed2004

"Vision" in single-celled algae.

Photosynthetic unicellular algae have a unique visual system. In Chlamydomonas reinhardtii, the pigmented eye comprises the optical system and at least five different rhodopsin photoreceptors. Two of them, the channelrhodopsins, are rhodopsin-ion channel hybrids switched between closed and open states by photoisomerization of the attached retinal chromophore. They promise to become a useful tool for noninvasive control of membrane potential and intracellular ion concentrations.

Animals↗

PubMed2003

Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.

Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seven-transmembrane alpha helices of ChR2 are characteristic for G protein-coupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination.

Algal Proteins↗

PubMed2003

Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket.

Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp, Gln, Ala), Asp804 (Glu, Asn, Ala), and Asp808 (Glu, Asn, Ala). Electrogenic cation transport properties of these 12 mutants were analyzed in two-electrode voltage-clamp experiments on Xenopus laevis oocytes by measuring the voltage dependence of K+-stimulated stationary currents and pre-steady-state currents under electrogenic Na+/Na+ exchange conditions. Whereas mutants D804N, D804A, and D808A hardly showed any Na+/K+ pump currents, the other constructs could be classified according to the [K+] and voltage dependence of their stationary currents; mutants N776A and E779Q behaved similarly to the wild-type enzyme. Mutants E779D, E779A, D808E, and D808N had in common a decreased apparent affinity for extracellular K+. Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the current-voltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na+/Na+ exchange currents are rather voltage-independent and at potentials above -60 mV faster than the wild type. Thus, the characteristic voltage-dependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn776 and Asp804, respectively, decreases the extracellular Na+ affinity.

Amino Acid Substitution↗

PubMed2003

Probing the sensory rhodopsin II binding domain of its cognate transducer by calorimetry and electrophysiology.

Sensory rhodopsin II, a repellent phototaxis receptor from Natronobacterium pharaonis (NpSRII) forms a tight complex with its cognate transducer (NpHtrII). Light excitation of the receptor triggers conformational changes in both proteins, thereby activating the cellular two-component signalling cascade. In membranes, the two proteins form a 2:2 complex, which dissociates to a 1:1 heterodimer in micelles. Complexed to the transducer sensory rhodopsin II is no longer capable of light-driven proton pumping. In order to elucidate the dimerisation and the size of the receptor-binding domain of the transducer, isothermal titration calorimetry and electrophysiological experiments have been carried out. It is shown, that an N-terminal sequence of 114 amino acid residues is sufficient for tight binding (K(d)=240nM; DeltaH=-17.6kJmol(-1)) and for inhibiting the proton transfer. These data and results obtained from selected site-directed mutants indicate a synergistic interplay of transducer transmembrane domain (1-82) and cytoplasmic peptide (83-114) leading to an optimal and specific interaction between receptor and transducer.

Animals↗

PubMed2003

Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry.

The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active Na(+)K(+)-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5-M6 loop of the sheep alpha(1)-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the catalytic cycle. After expression of the single cysteine mutants in Xenopus oocytes and covalent attachment of tetramethylrhodamine-6-maleimide, only mutant N790C reported molecular rearrangements of the M5-M6 loop by showing large, ouabain-sensitive fluorescence changes ( approximately 5%) on addition of extracellular K(+). When the enzyme was subjected to voltage jumps under Na(+)Na(+)-exchange conditions, we observed fluorescence changes that directly correlated to transient charge movements originating from the E(1)P-E(2)P transition of the transport cycle. The voltage jump-induced fluorescence changes and transient currents were abolished after replacement of Na(+) by tetraethylammonium or on addition of ouabain, showing that conformational flexibility is impaired under these conditions. Voltage-dependent fluorescence changes could also be observed in the presence of subsaturating K(+) concentrations. This allowed to monitor the time course of voltage-dependent relaxations into a new stationary distribution of states under turnover conditions, showing the acceleration of relaxation kinetics with increasing K(+) concentrations. As a result, the stationary distribution between E(1) and E(2) states and voltage-dependent relaxation times can be determined at any time and membrane potential under Na(+)Na(+) exchange as well as Na(+)K(+) turnover conditions.

Adenosine Triphosphate↗

PubMed2003

Charge displacements during ATP-hydrolysis and synthesis of the Na+-transporting FoF1-ATPase of Ilyobacter tartaricus.

Transient electrical currents generated by the Na(+)-transporting F(o)F(1)-ATPase of Ilyobacter tartaricus were observed in the hydrolytic and synthetic mode of the enzyme. Two techniques were applied: a photochemical ATP concentration jump on a planar lipid membrane and a rapid solution exchange on a solid supported membrane. We have identified an electrogenic reaction in the reaction cycle of the F(o)F(1)-ATPase that is related to the translocation of the cation through the membrane bound F(o) subcomplex of the ATPase. In addition, we have determined rate constants for the process: For ATP hydrolysis this reaction has a rate constant of 15-30 s(-1) if H(+) is transported and 30-60 s(-1) if Na(+) is transported. For ATP synthesis the rate constant is 50-70 s(-1).

Adenosine Diphosphate↗

PubMed2003

Conformational dynamics of Na+/K+- and H+/K+-ATPase probed by voltage clamp fluorometry.

We used the method of site-directed fluorescence labeling in combination with voltage-clamp fluorometry for time-resolved recording of localized conformational transitions of the Na(+)/K(+)- and H(+)/K(+)-ATPase. Therefore, single cysteine mutations were introduced into the extracellular TM5-TM6 loop of the sheep Na(+)/K(+)-ATPase alpha(1)-subunit devoid of other extracellular cysteines. Upon expression in Xenopus oocytes and covalent attachment of tetramethylrhodamine-maleimide (TMRM) as a reporter fluorophore, Cys-mutant N790C showed large fluorescence changes of up to 5% in response to extracellular K(+) that were completely abolished by ouabain. When voltage jumps were applied under Na(+)/Na(+)-exchange conditions, we observed fluorescence changes that paralleled the transient currents originating from the E(1)P<-->E(2)P transition. These fluorescence changes were also completely inhibited by ouabain, as were the voltage jump-induced transient currents. Transient fluorescence changes could also be measured as a function of increasing K(+) concentrations, that is, under turnover conditions. As a result, the distribution between E(1) and E(2) states can be determined at any time and membrane potential. Very similar fluorescence signals were obtained for rat gastric H(+)/K(+)-ATPase upon expression in oocytes, when a single cysteine was introduced at a position homologous to N790 in Na(+)/K(+)-ATPase for attachment of the fluorophore. As to the high sequence similarity among P-type ATPases within the TM5 helix and the TM5-TM6 loop region, our results enable new means of kinetic investigation for these pumps under physiological conditions in living cells.

Amino Acid Sequence↗

PubMed2003

Two-electrode voltage-clamp analysis of Na,K-ATPase asparagine 776 mutants.

Steady-state and pre-steady-state currents of Asn(776) mutants of Na,K-ATPase are presented. The stationary current generated by N776Q strongly depends on the membrane potential, but has a negative slope, opposite to that of the wild-type enzyme. The apparent rate constant of the reaction sequence E(1)P(Na(+)) <--> E(2)P + Na(+) of this mutant is rather independent of the membrane potential and is at resting and depolarizing membrane potential higher than that of the wild-type enzyme. Thus, the voltage-dependent increase of the rate coefficient of the wild type that is associated with extracellular Na(+) rebinding is almost absent in the N776Q mutant. These findings indicate that dislocating the carboxamide group of Asn(776) decreases the affinity of sodium at its extracellular binding site.

Alanine↗

PubMed2002

Proteorhodopsin is a light-driven proton pump with variable vectoriality.

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions. The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.

Animals↗

PubMed2002

Channelrhodopsin-1: a light-gated proton channel in green algae.

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.

Amino Acid Sequence↗