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. 2017 Oct 18;36(22):3309–3324. doi: 10.15252/embj.201796685

Figure 7. Recombinant SLCO2A1 proteins exhibit Maxi‐Cl channel activity in reconstituted giant proteoliposomes.

Figure 7

  1. SDS–PAGE electrophoresis at the different purification stages for FLAG‐tagged SLCO2A1 proteins prepared from transfected HEK293T cells: wash 1, 2, 3, and 4 correspond to consecutive elution with wash buffer; three lanes for FLAG eluates correspond to consecutive elution with the FLAG peptide. Arrowheads point at faint protein bands.
  2. Western blotting with anti‐FLAG antibodies: The protein band corresponding to ˜70 kDa (which is close to 70,147 Da calculated from the amino acid sequence) is present in the total cell lysate of Slco2a1‐transfected cells, highly concentrated in the pooled FLAG eluate and absent in that of mock‐transfected cells.
  3. Maxi‐Cl activity recorded on giant proteoliposomes after reconstitution of the recombinant SLCO2A1 protein.
  4. I–V relationships of Maxi‐Cl activity recorded after reconstitution of the WT SLCO2A1 (white, blue, and green circles) or the K613G mutant (red circles). Recordings were obtained using normal Ringer solution in the bath and pipettes (white symbols), with normal Ringer solution in the bath and Ringer solution in which all Na+ ions were replaced with equimolar NMDG+ in pipettes (blue and red symbols), or under a KCl gradient (green symbols: 150/30 mM pipette/bath supplemented with 1 mM CaCl2 and 11 mM HEPES–KOH, pH 7.4). Error bars, SEM. For green symbols: The reversal potential for WT SLCO2A1 of −27.3 mV corresponds to PCl/PK = 9.3. The slope conductance for K613G (258 ± 5 pS) is significantly smaller than that for WT (354 ± 4 pS) at P < 0.05, and its reversal potential slightly shifted in a negative direction (red symbols). n = 3–15 from five different patches (see Appendix Table S2 for expression vectors used here.)
  5. Reversible blocking of WT SLCO2A1‐associated Maxi‐Cl activity in the giant proteoliposomes by 50 μM Gd3+. The data represent the results of five experiments.

Source data are available online for this figure.