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The light-driven proton pump bacteriorhodopsin (bR) was functionally expressed in Xenopus laevis oocytes and in HEK-293 cells. The latter expression system allowed high time resolution of light-induced current signals. A detailed voltage clamp and patch clamp study was performed to investigate the DeltapH versus Deltapsi dependence of the pump current. The following results were obtained. The current voltage behavior of bR is linear in the measurable range between -160 mV and +60 mV. The pH dependence is less than expected from thermodynamic principles, i.e., one DeltapH unit produces a shift of the apparent reversal potential of 34 mV (and not 58 mV). The M(2)-BR decay shows a significant voltage dependence with time constants changing from 20 ms at +60 mV to 80 ms at -160 mV. The linear I-V curve can be reconstructed by this behavior. However, the slope of the decay rate shows a weaker voltage dependence than the stationary photocurrent, indicating that an additional process must be involved in the voltage dependence of the pump. A slowly decaying M intermediate (decay time > 100 ms) could already be detected at zero voltage by electrical and spectroscopic means. In effect, bR shows optoelectric behavior. The long-lived M can be transferred into the active photocycle by depolarizing voltage pulses. This is experimentally demonstrated by a distinct charge displacement. From the results we conclude that the transport cycle of bR branches via a long-lived M(1)* in a voltage-dependent manner into a nontransporting cycle, where the proton release and uptake occur on the extracellular side.

The method of voltage clamp fluorometry combined with sitedirected 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 ␣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 (Ϸ5%) 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 E1P-E2P 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 E1 and E2 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. P -type ATPases form a major class of primary active membrane transport proteins, so called because they become transiently phosphorylated on ATP hydrolysis. The most prominent member is the ubiquitously occurring Na ϩ ͞K ϩ -ATPase, which exports three Na ϩ ions and imports two K ϩ ions in each transport cycle and thereby maintains the electrochemical gradients of Na ϩ and K ϩ across the plasma membrane of most animal cells.The reaction cycle of the Na ϩ ͞K ϩ -ATPase is described in terms of the Albers-Post scheme (see Fig. 1A) (1, 2). The transduction of primary energy from ATP hydrolysis to active ion transport is brought about by conformational changes that occur for both the ␣ and ␤ subunit of the Na ϩ ͞K ϩ -ATPase (3-6). Several approaches were undertaken to reveal Na ϩ ͞K ϩ -ATPase conformational changes by using fluorescence labeling of the native enzyme with fluorescein-5Ј-isothiocyanate, N-(p-(2-benzimidazolyl)phenyl)-maleimide and styrylpyridinium dyes like RH421 (7-12), which were limited for several reasons. Purely biochemical assays to investigate conformationdependent proteolysis patterns cannot provide time-resolved data, in the case of fluorescence labeling with styryl dyes the site of...

A target-site mutation in Tu GluCl3 contributes to avermectin resistance in T. urticae. However, given the multitude of channel genes and the potential additive or synergistic effects of mutations, to what extent mutations determine the often extremely strong resistance phenotype in the field deserves further study. © 2017 Society of Chemical Industry.

؉ . 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 currentvoltage 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 voltagedependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn 776 and Asp 804 , respectively, decreases the extracellular Na ؉ affinity.

Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAi-based pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways.

Five new alkaloids, 6beta-hydroxystemofoline (1), 16-hydroxystemofoline (2), neostemofoline (3), protostemodiol (4), and 13-demethoxy-11(S*),12(R*)-dihydroprotostemonine (5), along with 10 known alkaloids, were isolated from stems and leaves of Stemona japonica. Their structures were elucidated by 1D and 2D NMR and other spectroscopic studies. The insecticidal activity of the agonist 16-hydroxystemofoline (2) and antagonist 13-demethoxy-11(S*),12(R*)-dihydroprotostemonine (5) was demonstrated by electrophysiological in vitro tests on the insect nicotinic acetylcholine receptor and by in vivo screenings against relevant agricultural insect pests.