Steinmann ME et al. (2017), TbIRK is a signature sequence free potassium ch...

XB-ART-53603

Sci Rep 2017 Apr 06;71:656. doi: 10.1038/s41598-017-00752-1.

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TbIRK is a signature sequence free potassium channel from Trypanosoma brucei locating to acidocalcisomes.

Steinmann ME , Schmidt RS , Bütikofer P , Mäser P , Sigel E .

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Potassium channels from prokaryotes and eukaryotes are usually recognized by a typical amino acid sequence TXTGY(F)G representing the ionic selectivity filter. Using a screening approach with ion channel family profiles but without the above motif, we identified a gene in Trypanosoma brucei that exhibits homology to inward rectifying potassium channels. We report here cloning of this ion channel named TbIRK. The protein is localized to acidocalcisomes in procyclic and in bloodstream form parasites. Functional properties of this channel were established after expression in Xenopus oocytes. Currents recorded in potassium medium show inward rectification and little time dependence. Surprisingly, this channel retains selectivity for potassium ions over sodium ions >7, in spite of the lack of the classical selectivity filter. The sequence GGYVG was predicted in silico to replace this filter motif. Point mutations of the corresponding glycine residues confirmed this at the functional level. The channel is inhibited by caesium ions but remains unaffected by barium ions up to 10 mM. TbIRK is to our knowledge the first potassium channel in T. brucei that localizes to the acidocalcisomes, organelles involved in the storage of phosphates and the response to osmotic stress that occurs during the life cycle of trypanosomes.

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Species referenced: Xenopus laevis
Genes referenced: dnai1 hspa5

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	Figure 1. The unusual filter motif of TbIRK. (A) The protein sequence of TbIRK was aligned with representatives from six groups of similar proteins, as identified by HHBlits (Homology detection by iterative HMM-HMM comparison) within SWISS-MODEL30. Transmembrane domains unequivocally identified by different algorithms in all of the proteins are indicated in blue. The region between transmembrane domains is indicated by a red box. (B) Section indicated by the red box in (A), containing pore loop and filter motif, indicated in blue. Residues mutated within this study are indicated with a red asterisk.
	Figure 2. Electrophysiological characterization of TbIRK expressed in X. laevis oocytes. The 2-electrode voltage-clamp was used to study currents in oocytes injected with cRNA coding for TbIRK or with water. All recordings were done by applying the voltage-step protocol depicted in (E). The holding potential was −40 mV. With a frequency of 1 Hz voltage-steps of 300 ms duration were applied from −110 mV to 50 mV in 10 mV intervals. Currents recorded from a water-injected oocyte in potassium medium (KME) and sodium medium (NaME) are shown in (A) and (B). The corresponding representative current traces of a TbIRK-expressing oocyte in potassium medium and sodium medium (NaME) are shown in (C) and (D). (F) Recording of the same oocyte in potassium medium in presence of 10 mM CsCl.
	Figure 3. Current-voltage relationship. (A) Current-voltage relationship of TbIRK expressing oocytes in sodium medium (squares), potassium medium (circles) and N-methyl-D-glucamine medium (open triangels) (mean ± S.D.; n = 31 for sodium and potassium medium and n = 23 for N-methyl-D-glucamine medium). (B) The corresponding I/V relationship of water-injected oocytes in sodium medium (squares), potassium medium (circles) and N-methyl-D-glucamine medium (open triangels) (mean ± S.D.; n = 25 for sodium and potassium medium and n = 19 for N-methyl-D-glucamine medium). (C) Comparison of the I/V relationship of TbIRK expressing oocytes in potassium medium (circles) or sodium medium (squares) in presence (open symbols) or absence (closed symbols) of 10 mM CsCl (mean ± S.D.; n = 31 and 16, respectively for TbIRK-expressing oocytes and n = 25 an 14, respectively for water-injected control oocytes). (D) Inhibition by caesium was determined by changing from sodium medium to potassium medium containing different CsCl concentrations at −80 mV. The observed currents normalized to the response elicited by potassium medium in the absence of caesium. The inhibition curve was fitted with an IC50 of 0.74 ± 0.11 mM (mean ± S.E.M.; n = 7).
	Figure 4. Loss of selectivity upon mutation of G132A located in the suspected selectivity filter. All recordings were done by applying the voltage-step protocol depicted in Fig. 2E. Representative current traces obtained from TbIRK-expressing oocytes in sodium medium (NaME), potassium medium (KME) and potassium medium supplemented with 10 mM CsCl are shown in the upper row. Recordings obtained with a oocyte expressing the G132A-mutant are shown in the lower row.
	Figure 5. TbIRK co-localizes with a marker for acidocalcisomes. Co-localization of hemagglutinin (HA)-tagged TbIRK (green) with the acidocalcisomal marker TbVP1 (red) for C-terminally tagged TbIRK in procyclic form parasites (PCF, A) and N-terminally tagged TbIRK in bloodstream form parasites (BSF, B). (C) With the HA-tag at the C-terminus of TbIRK, its signal (green) partially co-localizes with TbVP1 (red, top) and partially with the ER marker BiP (red, bottom). Cells were counterstained with DAPI, shown in blue, visualizing the nuclear and kinetoplast DNA. DIC, differential interference contrast. Scale bars indicate 10 µm.
	Figure 6. RNAi-mediated downregulation of TbIRK in procyclic form parasites. (A) Growth of non-induced (open circles) and induced (triangles) was monitored over 10 days (mean ± S.D., n = 3; error bars are smaller than the symbols). Induction of RNAi against TbIRK did not result in a growth phenotype. Changes in target mRNA levels were confirmed by qPCR (B). The black bar represents the mRNA level of non-induced cells and the white bar represents the mRNA level of TbIRK in induced cells. TbIRK mRNA was downregulated by 80 ± 9% (mean ± S.D., n = 3).

References [+] :

Alexander, Guide to Receptors and Channels (GRAC), 3rd edition. 2008, Pubmed