Sucrose- and H+-dependent charge movements associated with the gating of sucrose transporter ZmSUT1

Background: In contrast to man the majority of higher plants use sucrose as mobile carbohydrate. Accordingly proton-driven sucrose transporters are crucial for cell-to-cell and long-distance distribution within the plant body. Generally very negative plant membrane potentials and the ability to accumulate sucrose quantities of more than 1 M document that plants must have evolved transporters with unique structural and functional features. Methodology/Principal Findings: To unravel the functional properties of one specific high capacity plasma membrane sucrose transporter in detail, we expressed the sucrose/H+ co-transporter from maize ZmSUT1 in Xenopus oocytes. Application of sucrose in an acidic pH environment elicited inward proton currents. Interestingly the sucrose-dependent H+ transport was associated with a decrease in membrane capacitance (Cm). In addition to sucrose Cm was modulated by the membrane potential and external protons. In order to explore the molecular mechanism underlying these Cm changes, presteady-state currents (Ipre) of ZmSUT1 transport were analyzed. Decay of Ipre could be best fitted by double exponentials. When plotted against the voltage the charge Q, associated to Ipre, was dependent on sucrose and protons. The mathematical derivative of the charge Q versus voltage was well in line with the observed Cm changes. Based on these parameters a turnover rate of 500 molecules sucrose/s was calculated. In contrast to gating currents of voltage dependent-potassium channels the analysis of ZmSUT1-derived presteady-state currents in the absence of sucrose (I = Q/τ) was sufficient to predict ZmSUT1 transport-associated currents. Conclusions: Taken together our results indicate that in the absence of sucrose, ‘trapped’ protons move back and forth between an outer and an inner site within the transmembrane domains of ZmSUT1. This movement of protons in the electric field of the membrane gives rise to the presteady-state currents and in turn to Cm changes. Upon application of external sucrose, protons can pass the membrane turning presteady-state into transport currents

Early Intermediates in the Transport Cycle of the Neuronal Excitatory Amino Acid Carrier Eaac1

Electrogenic glutamate transport by the excitatory amino acid carrier 1 (EAAC1) is associated with multiple charge movements across the membrane that take place on time scales ranging from microseconds to milliseconds. The molecular nature of these charge movements is poorly understood at present and, therefore, was studied in this report in detail by using the technique of laser-pulse photolysis of caged glutamate providing a 100-μs time resolution. In the inward transport mode, the deactivation of the transient component of the glutamate-induced coupled transport current exhibits two exponential components. Similar results were obtained when restricting EAAC1 to Na+ translocation steps by removing potassium, thus, demonstrating (1) that substrate translocation of EAAC1 is coupled to inward movement of positive charge and, therefore, electrogenic; and (2) the existence of at least two distinct intermediates in the Na+-binding and glutamate translocation limb of the EAAC1 transport cycle. Together with the determination of the sodium ion concentration and voltage dependence of the two-exponential charge movement and of the steady-state EAAC1 properties, we developed a kinetic model that is based on sequential binding of Na+ and glutamate to their extracellular binding sites on EAAC1 explaining our results. In this model, at least one Na+ ion and thereafter glutamate rapidly bind to the transporter initiating a slower, electroneutral structural change that makes EAAC1 competent for further, voltage-dependent binding of additional sodium ion(s). Once the fully loaded EAAC1 complex is formed, it can undergo a much slower, electrogenic translocation reaction to expose the substrate and ion binding sites to the cytoplasm

The β 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 α subunit that mediates ATP hydrolysis and ion transport, and an ancillary β subunit that is required for proper trafficking of the holoenzyme. Although processes mediated by the α subunit have been extensively studied, little is known about the participation of the β subunit in conformational changes of the enzyme. To elucidate the role of the β subunit during ion transport, extracellular amino acids proximal to the transmembrane region of the sheep β1 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 β1 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 β subunit. Simultaneous recording of presteady-state or stationary currents together with fluorescence signals enabled correlation of the observed environmental changes of the β 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 β1-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 β1 isoforms, directly influences the effective potential that determines voltage dependence of extracellular cation binding and release

On the Mechanism of Proton Transport by the Neuronal Excitatory Amino Acid Carrier 1

Uptake of glutamate from the synaptic cleft is mediated by high affinity transporters and is driven by Na+, K+, and H+ concentration gradients across the membrane. Here, we characterize the molecular mechanism of the intracellular pH change associated with glutamate transport by combining current recordings from excitatory amino acid carrier 1 (EAAC1)–expressing HEK293 cells with a rapid kinetic technique with a 100-μs time resolution. Under conditions of steady state transport, the affinity of EAAC1 for glutamate in both the forward and reverse modes is strongly dependent on the pH on the cis-side of the membrane, whereas the currents at saturating glutamate concentrations are hardly affected by the pH. Consistent with this, the kinetics of the pre–steady state currents, measured after saturating glutamate concentration jumps, are not a function of the pH. In addition, we determined the deuterium isotope effect on EAAC1 kinetics, which is in agreement with proton cotransport but not OH− countertransport. The results can be quantitatively explained with an ordered binding model that includes a rapid proton binding step to the empty transporter followed by glutamate binding and translocation of the proton-glutamate-transporter complex. The apparent pK of the extracellular proton binding site is ∼8. This value is shifted to ∼6.5 when the substrate binding site is exposed to the cytoplasm

Optogenetic Long-Term Manipulation of Behavior and Animal Development

Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used “slow” ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the “dauer” larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development

The parallel G-quadruplex structure of vertebrate telomeric repeat sequences is not the preferred folding topology under physiological conditions

G-quadruplex topologies of telomeric repeat sequences from vertebrates were investigated in the presence of molecular crowding (MC) mimetics, namely polyethylene glycol 200 (PEG), Ficoll 70 as well as Xenopus laevis egg extract by CD and NMR spectroscopy and native PAGE. Here, we show that the conformational behavior of the telomeric repeats in X. laevis egg extract or in Ficoll is notably different from that observed in the presence of PEG. While the behavior of the telomeric repeat in X. laevis egg extract or in Ficoll resembles results obtained under dilute conditions, PEG promotes the formation of high-order parallel topologies. Our data suggest that PEG should not be used as a MC mimetic

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

The discovery of channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time- resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(−) and 1,648(+) cm−1. These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance