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The metabotropic glutamate receptor 7a (mGluR7a), a heptahelical Gαi/o-coupled protein, has been shown to be important for presynaptic feedback inhibition at central synapses and certain forms of long term potentiation and long term depression. The intracellular C terminus of mGluR7a interacts with calmodulin in a Ca2+-dependent manner, and calmodulin antagonists have been found to abolish presynaptic inhibition of glutamate release in neurons and mGluR7a-induced activation of G-protein-activated inwardly rectifying K+ channel (GIRK) channels in HEK293 cells. Here, we characterized the Ca2+ dependence of mGluR7a signaling in Xenopus oocytes by using channelrhodopsin-2 (ChR2), a Ca2+-permeable, light-activated ion channel for triggering Ca2+ influx, and a GIRK3.1/3.2 concatemer to monitor mGluR7a responses. Application of the agonist (S)-2-amino-4-phosphonobutanoic acid (l-AP4) (1–100 μm) caused a dose-dependent inward current in high K+ solutions due to activation of GIRK channels by G-protein βγ subunits released from mGluR7a. Elevation of intracellular free Ca2+ by light stimulation of ChR2 markedly increased the amplitude of l-AP4 responses, and this effect was attenuated by the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester). l-AP4 responses were potentiated by submembranous [Ca2+] levels within physiological ranges and with a threshold close to resting [Ca2+]i values, as determined by recording the endogenous Xenopus Ca2+-activated chloride conductance. Together, these results show that l-AP4-dependent mGluR7a signaling is potentiated by physiological levels of [Ca2+]i, consistent with a model in which presynaptic mGluR7a acts as a coincidence detector of Ca2+ influx and glutamate release.

Uveal melanoma is associated with a high mortality rate once metastases occur, with over >90% of metastatic patients dying within less than 1 year from metastases to the liver. The intraarterial hepatic (iah) administration of the alkylating agent fotemustine holds some promise with response rates of 36% and median survival of 15 months. Here, we investigated whether the DNA-repair-protein MGMT may be involved in the variability of response to fotemustine and temozolomide in uveal melanoma. Epigenetic inactivation of MGMT has been demonstrated to be a predictive marker for benefit from alkylating agent therapy in glioblastoma. We found a methylated MGMT promoter in 6% of liver metastases from 34 uveal melanoma patients. The mean MGMT activity measured in liver metastases with negligible liver tissue content was significantly lower than in liver tissue (146 versus 523 fmol/mg protein, p = 0.002). Expression of the MGMT protein was detectable in 50% of 88 metastases by immunohistochemistry on a tissue microarray. Expression was heterogeneous, and in accordance with MGMT activity data, usually lower than in the surrounding liver. Differential MGMT activity/expression between metastasis and liver tissue and more efficient depletion of MGMT with higher doses of alkylating agent therapy using iah delivery may provide the pharmacologic window for the higher response rate. However, these results do not support MGMT methylation status or protein expression as predictive markers for treatment outcome to iah chemotherapy with alkylating agents. © 2008 Wiley-Liss, Inc.

Activation of c-Jun, a major component of the AP-1 transcription factor, represents a paradigm for transcriptional response to stress. Transactivation of c-Jun is regulated by Jun-N-terminal kinases (JNKs) through phosphorylation at serine 63 and 73 (S63/S73), as well as at threonine 91 and 93 (T91/T93). How these two groups of phosphoacceptor sites respond to different grades of genotoxic stress and whether DNA-damage pathways influence the extent of their JNK-dependent phosphorylations remain to be elucidated. Here, we show that following a short exposure to the DNA-damaging compound etoposide, c-Jun phosphorylation is restricted to S63/S73. In contrast, JNK-dependent phosphorylation of T91/T93 requires continuous exposure to the drug and is impaired by caffeine treatment or alanine substitution of the adjacent threonine 95 (T95). Conversely, c-Jun mutations switching the T95/Q96 site into a canonical site for mitogen activated protein kinase (MAPK) phosphorylation (T95/P96) rescues T91/T93 phosphorylation in presence of caffeine, suggesting that a preceding phosphorylation at T95 exposes T91/T93 to JNK-dependent phosphorylation. Moreover, we show that alanine substitution at T95 impairs c-Jun transactivation and c-Jun-mediated cell death, indicating that negatively charged T95 is a general constraint for c-Jun activation. Hence, our study suggests that c-Jun may sense the strength of genotoxic stress through DNA-damage dependent phosphorylation of T95, which in turn augments c-Jun transactivation by JNKs.

Giant HEK293 cells of 30-65 {mu}m in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 {+-} 0.1 {mu}F/cm{sup 2} for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 {mu}m) was found to be between 12.8 and 16.1 {mu}S/cm{sup 2}, which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludesmore » internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system.« less

Summary During classical conditioning, a positive or negative value is assigned to a previously neutral stimulus, thereby changing its significance for behavior. If an odor is associated with a negative stimulus, it can become repulsive. Conversely, an odor associated with a reward can become attractive. By using Drosophila larvae as a model system with minimal brain complexity, we address the question of which neurons attribute these values to odor stimuli. In insects, dopaminergic neurons are required for aversive learning, whereas octopaminergic neurons are necessary and sufficient for appetitive learning [1–4]. However, it remains unclear whether two independent neuronal populations are sufficient to mediate such antagonistic values. We report the use of transgenically expressed channelrhodopsin-2 [5], a light-activated cation channel, as a tool for optophysiological stimulation of genetically defined neuronal populations in Drosophila larvae. We demonstrate that distinct neuronal populations can be activated simply by illuminating the animals with blue light. Light-induced activation of dopaminergic neurons paired with an odor stimulus induces aversive memory formation, whereas activation of octopaminergic/tyraminergic neurons induces appetitive memory formation. These findings demonstrate that antagonistic modulatory subsystems are sufficient to substitute for aversive and appetitive reinforcement during classical conditioning.

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 α 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.

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 =240 nM ; Δ H=−17.6 kJ mol −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.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed in many epithelia and in the heart. Phosphorylation of CFTR by protein kinases is thought to be an absolute prerequisite for the opening of CFTR channels. In addition, nucleoside triphosphates were shown to regulate the opening of phosphorylated CFTR. Here, we report that phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates human CFTR, resulting in ATP responsiveness of PIP(2)-treated CFTR. PIP(2) alone is not sufficient to open CFTR, but ATP opens nonphosphorylated CFTR after application of PIP(2). The effect of PIP(2) is independent of protein kinases, as PIP(2) activates CFTR in the complete absence of Mg. Phosphatidylinositol and phosphatidylinositol monophosphate activate CFTR less efficiently than PIP(2). PIP(2) application to phosphorylated CFTR may inhibit the CFTR chloride current. We suggest that regulation of CFTR by PIP(2) is a previously unrecognized, alternative mechanism to control chloride conductance.

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.

The rat organic cation transporter (rOCT)-2 was characterized by electrical and tracer flux measurements compared with rOCT1. By applying choline gradients to voltage-clamped Xenopus oocytes expressing rOCT2, potential-dependent currents could be induced in both directions. Tracer flux measurements with seven organic cations revealed similar Michaelis-Menten constant values for both transporters, with the exception of guanidine. In parallel experiments with rOCT2 and rOCT1, inhibition of tetraethylammonium transport by 12 cations, 2 weak bases, corticosterone, and the anions para-amminohippurate, α-ketoglutarate, and probenecid was characterized. The IC50values of many inhibitors were similar for both transporters, whereas others were significantly different. Mepiperphenidol and O-methylisoprenaline showed an ∼70-fold lower and corticosterone a 38-fold higher affinity for rOCT2. With the use of these inhibitors together with previous information on cation transporters, experimental protocols are proposed to dissect out the individual contributions of rOCT2 and rOCT1 in intact proximal tubule preparations. Inhibition experiments at different pH levels strongly suggest that the weak base quinine passively permeates the plasma membrane at physiological pH and inhibits rOCT2 from the intracellular side.

The genetic disease cystic fibrosis is caused by mutation of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Controversial studies reported regulation of the epithelial sodium channel (ENaC) by CFTR. We found that uptake of (22)Na(+) through ENaC is modulated by activation of CFTR in oocytes, coexpressing CFTR and ENaC, depending on extracellular chloride concentration. Furthermore we found that the effect of CFTR activation could be mimicked by other chloride channels. Voltage- and patch-clamp measurements, however, showed neither stimulation nor inhibition of ENaC-mediated conductance by activated CFTR. We conclude that the observed modulation of (22)Na(+) uptake by activated CFTR is due to the effect of CFTR-mediated chloride conductance on the membrane potential. These findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR.

Properties of the cytoplasmic binding sites of the rabbit Na(+)/glucose cotransporter, SGLT1, expressed in Xenopus oocytes were investigated using the giant excised patch clamp technique. Voltage and substrate dependence of the outward cotransport were studied using alpha-methyl D-glucopyranoside (alphaMDG) as a substrate. The apparent affinity for alphaMDG depends on the cytoplasmic Na(+) concentration and voltage. At 0 mV the K(M) for alphaMDG is 7 mM at 110 mM Na(+) and 31 mM at 10 mM Na(+). The apparent affinity for alphaMDG and Na(+) is voltage dependent and increases at positive potentials. At 0 mV holding potential the outward current is half-maximal at about 70 mM. The results show that SGLT1 can mediate sugar transport out of the cell under appropriate concentration and voltage conditions, but under physiological conditions this transport is highly improbable due to the low affinity for sugar.

The lipid bilayer and the giant patch technique were used to study the Na+,K(+)-ATPase. In excised patches from ventricular myocytes, Na+,K(+)-pump currents show a saturable ATP dependence with a Km of approximately 150 microM at 24 degrees C. Partial reactions in the transport cycle were investigated by generating ATP concentration jumps through photolytic release of ATP from caged ATP at pH 7.4 and 6.3. Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slow decay to a stationary current. Experiments with purified pig kidney Na+,K(+)-ATPase attached to a planar lipid bilayer resulted in similar pump current signals and the same outcome. It was concluded that the fast rate constant of approximately 200 s-1 at 24 degrees C reflects a step rate limiting the electrogenic Na+ release.

Purified ( Na + + K + )-ATPase from pig kidney was attached to black lipid membranes and ATP-induced electric currents were measured as described previously by Fendler et al. ((1985) EMBO J. 4, 3079–3085). An ATP concentration jump was produced by an ultraviolet-light flash converting non-hydrolysable caged ATP to ATP. In the presence of Na+ and Mg2+ this resulted in a transient current signal. The pump current was not only ATP dependent, but also was influenced by the ATP/caged ATP ratio. It was concluded that caged ATP binds to the enzyme (and hence inhibits the signal) with a K i of approx. 30 μM, which was confirmed by enzymatic activity studies. An ATP affinity of approx. 2 μM was determined. The addition of the protonophore 1799 and the Me + / H + exchanger monensin made the bilayer conductive leading to a stationary pump current. The stationary current was strongly increased by the addition of K+ with a K 0.5 of 700 μM. Even in the absence of K+ a stationary current could be measured, which showed two Na+-affinities: a high-affinity ( K 0.5 ⩽ 1 mM ) and a low-affinity ( K 0.5 ⩾ 0.2 M ). In order to explain the sustained electrogenic Na+ transport during the Na+-ATPase activity, it is proposed, that Na+ can replace K+ in dephophorylating the enzyme, but binds about 1000-times weaker than K+. The ATP requirement of the Na+-ATPase was the same ( K 0.5 = 2 μ M ) with regard to the peak currents and the stationary currents. However, for the (Na + + K + )-ATPase the stationary currents required more ATP. The results are discussed on the basis of the Albers-Post scheme.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel which is activated by protein phosphorylation and nucleoside triphosphates. We demonstrate here that fusion of the soluble catalytic subunit of cAMP-dependent protein kinase to the membrane protein bacteriorhodopsin yields a constitutively active protein kinase which activates CFTR effectively. As it is membrane-bound it is particularly useful for continuous perfusion of excised inside-out patches. We also tested the effect of a naturally membrane-bound protein kinase, cGMP-dependent protein kinase II, on CFTR. Both kinases, when continuously active, increase apparent affinity of CFTR to ATP about two-fold emphasizing the role of phosphorylation in modulating the interaction of ATP with the nucleotide binding domains.

Background and Purpose The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers regulating numerous biological processes. Malfunctional cNMP signalling is linked to diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in Caenorhabditis elegans is restricted to soluble adenylyl cyclases, the membrane‐bound Blastocladiella emersonii CyclOp and hyperpolarizing rhodopsins; yet missing are membrane‐bound photoactivatable adenylyl cyclases and hyperpolarizers based on K+ currents. Experimental Approach For the characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with cyclic nucleotide‐gated channels in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to depolarize or hyperpolarize cells, we performed behavioural analyses, measured cNMP content in vitro, and compared in vivo expression levels. Key Results We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine‐control of cGMP levels. We established photoactivatable membrane‐bound adenylyl cyclases, based on mutated versions (“A‐2x”) of Blastocladiella and Catenaria (“Be,” “Ca”) CyclOp, as N‐terminal YFP fusions, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two‐component optogenetics, we introduced the cAMP‐gated K+‐channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A‐2x), or YFP‐BeCyclOp(A‐2x). As an alternative, we implemented the B. emersonii cGMP‐gated K+‐channel BeCNG1 together with BeCyclOp. Conclusion and Implications We established a comprehensive suite of optogenetic tools for cNMP manipulation, applicable in many cell types, including sensory neurons, and for potent hyperpolarization. Deutsche Forschungsgemeinschaft (DFG) http://dx.doi.org/10.13039/501100001659

We report on the first successful expression of the light driven H+ pump, bacteriorhodopsin, into the plasma membrane of oocytes from Xenopus laevis. The light induced photocurrents which reflect the pumping of H+ by BR were analysed under voltage clamp conditions. At least 100 active BR molecules per μm2 were expressed in the plasma membrane so that both the voltage clamp and giant patch clamp method could be applied. We show that H+ pumping by BR is modulated by the membrane potential, i.e. the pump current shows strong voltage dependence in the range measured between −165 mV to +60 mV.

When the gene mutated in cystic fibrosis (CF) was identified and named the cystic fibrosis transmembrane conductance regulator (CFTR), it was not immediately clear what its function might be. Subsequent analysis showed that it is an anion channel, but also other functions were proposed. This protocol aims to shortly describe the two-electrode voltage-clamp (TEVC) technique and to highlight some precautions when studying CFTR with it. A short description of the series resistance (RS) and its influence on measurements of channel characteristics is given.

The Ca 2+ concentration and voltage dependence of the relaxation kinetics of the Na-Ca exchanger after a Ca 2+ concentration jump was measured in excised giant membrane patches from guinea pig heart. Ca 2+ concentration jumps on the cytoplasmic side were achieved by laser flash-induced photolysis of DM-nitrophen. In the Ca-Ca exchange mode a transient inward current is generated. The amplitude and the decay rate of the current saturate at concentrations >10 μ M. The integrated current signal, i.e., the charge moved is fairly independent of the amount of Ca 2+ released. The amount of charge translocated increases at negative membrane potentials, whereas the decay rate constant shows no voltage dependence. It is suggested that Ca 2+ translocation occurs in at least four steps: intra- and extracellular Ca 2+ binding and two intramolecular transport steps. Saturation of the amplitude and of the relaxation of the currrent can be explained if the charge translocating reaction step is preceded by two nonelectrogenic steps: Ca 2+ binding and one conformational transition. Charge translocation in this mode is assigned to one additional conformational change which determines the equilibrium distribution of states. In the Na-Ca exchange mode, the stationary inward current depends on the cytoplasmic Ca 2+ concentration and voltage. The K m for Ca 2+ is 4 μ M for guinea pig and 10 μ M for rat myocytes. The amplitude of the pre–steady-state current and its relaxation saturate with increasing Ca 2+ concentrations. In this mode the relaxation is voltage dependent.

N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O6-methylguanine (O6-MeG), which is repaired by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6-MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O6-MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) to monitor O6-MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O6-MeG in CRC cells. Using the highly sensitive and specific UPLC–MS/MS, TMZ-induced O6-MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O6-MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC–MS/MS measurements and dose–response modeling revealed a non-linear formation of hepatic and colonic O6-MeG adducts in WT, whereas linear O6-MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC–MS/MS analysis is highly sensitive and specific for O6-MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O6-methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures.