With a frequency of 1 Hz discrete voltage steps of 300 ms duration to potentials ranging from -120 mV to +80 mV in steps of 10 mV were applied. Currents were digitized at 5 kHz with MacLab/200 (AD Instruments, Spechbach, Germany). Oocytes were injected with 50 nl of solution containing cRNA coding for one of the genes (0.4 μg/μl) or water and then incubated in modified Barth’s solution (10 mM HEPES, pH 7.5, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO 3, 0.82 mM MgSO 4, 0.34 mM Ca(NO 3) 2, 0.41 mM CaCl 2, 100 units/ml penicillin, 100 μg/ml streptomycin) at 18☌ for 3 days before measurements.įunctional characterization in Xenopus oocytesĮlectrophysiological experiments were performed using an Oocyte Clamp OC-725 (Warner Instrument Corp., Hamden, USA) two-electrode voltage clamp amplifier. Polyadenylated cRNAs coding for Tb9 (TbVCL1), Tb90 (TbVCL2) and Tb90 (TbVC元) were prepared in vitro with the mMESSAGE mMACHINE kit (Ambion, Austin, TX, USA). Xenopus laevis oocytes were prepared, injected and defollicated as described previously. Surgery of Xenopus laevis to obtain the oocytes was done under anesthesia with 0.2% tricaine solution (ethyl aminobenzoate, MS222) and analgesic treatment with 25mg/kg Flunixin-Meglumin. brucei procyclic forms all three proteins localize intracellularly.Īnimal experiments were carried out in strict accordance to the Swiss ethical guidelines, and have been approved by the Kantonstierarzt of the Canton Bern, Kantonaler Veterinärdienst Bern (BE85/15). All three proteins are reversibly inhibited by 4,4-diisothiocyanostilbene-2,2-disulphonate (DIDS) and transport different anions such as chloride, bromide, iodide and nitrate. After expression Xenopus oocytes the resulting currents were functionally characterized. Here, we show that three proteins conveying this function are present in T. In trypanosomes, no chloride transporters have yet been described. CLC-3 to CLC-7 have intracellular functions in synaptic vesicles and/or endosomes and lysosomes (reviewed in ). CLC-2, CLC-Ka and CLC-Kb are implied in transepithelial transport. Human CLC-1 is located in the skeletal muscle plasma membrane and involved in the stabilization of the membrane potential. In the human genome there are nine isoforms, termed CLC-1 to CLC-7, CLC-Ka and CLC-Kb. Some mammalian CLC proteins also incorporate the β-subunits Barttin, Ostm1 or GlialCam. Formation of homodimers as well as heterodimers has been observed in heterologous expression systems. CLCs are dimers and have two ion translocation pathways, one in each subunit. Later it became apparent that some CLCs function as electrogenic Cl -/H +-exchangers. The first CLC member was identified in Torpedo marmorata and described as voltage-gated chloride channel. Īnion transport proteins that are members of the CLC gene family are found in all three domains of life. brucei transport systems has so far mainly concentrated on transporters of organic nutrients such as amino acids, sugars, nucleosides, nucleobases and metabolites. Until recently, transporters or channels selective for inorganic ions have attracted little attention. During their life cycle, the parasites must tightly regulate their internal ion composition to survive and proliferate in different environments such as the tsetse fly gut, salivary glands, or the human blood. The parasites are transmitted by the tsetse fly ( Glossina spp.). are the causative agents of human African trypanosomiasis (HAT), also called sleeping sickness, and of a related disease in livestock, called nagana. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was supported by Sinergia grant CRSII3_141913/1 from the Swiss National Science Foundation ( ) (PB, DR, PM, ES). Received: AugAccepted: NovemPublished: December 15, 2017Ĭopyright: © 2017 Steinmann et al. Mongin, Albany Medical College, UNITED STATES (2017) Identification and characterization of the three members of the CLC family of anion transport proteins in Trypanosoma brucei. Citation: Steinmann ME, Schmidt RS, Macêdo JP, Kunz Renggli C, Bütikofer P, Rentsch D, et al.
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