Functional characterization of a ClC transporter by solid-supported membrane electrophysiology

EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H⁺/Cl⁻ exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.     

UDP-sugar pyrophosphorylase controls the activity of proceeding sugar-1-kinases enzymes

Plant cell wall synthesis requires a number of different nucleotide sugars which provide the building blocks of the different polymers. These nucleotide sugars are mainly provided by de novo synthesis but recycling pathways also contribute to the pools. The last enzyme of the recycling pathway is UDP-sugar pyrophosphorylase (USP), a single copy gene in Arabidopsis, of which a knockout is lethal for pollen development. Here we analyze the dependency between USP enzyme activity and the upstream glucuronokinase. Gene silencing of USP by miRNA cause a concomitant reduction of USP and of glucuronokinase activity presumably to prevent the accumulation of sugar-1-phosphates interfering with normal metabolism and depleting the phosphate pool of the cell.     

Das Vordringen der Hochgebirgsvegetation in den Tiroler Alpen

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