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

Figure 1. Maxi‐Cl is constitutively active in membrane blebs, is functional after membrane protein fractionation of blebs and reconstitution into giant liposomes, and is sensitive to Gd3+ .

Figure 1

  1. The activity of Maxi‐Cl (right panel) recorded in a patch membrane on a single‐membrane bleb (left image).
  2. I–V relationships of Maxi‐Cl currents recorded on membrane blebs bathed in normal Ringer solution (circles) and in Ringer solution in which all NaCl was replaced with equimolar Na‐glutamate (squares). Patch pipettes were filled with normal Ringer solution (circles and squares) or NMDG‐Cl Ringer solution (triangles). Leftward shift of the reversal potential (by −44 mV) observed in Na‐glutamate conditions is indicative of anion selectivity. The slope conductance is 411 ± 6 pS in normal Ringer solution; n = 7–23 (circles), n = 6–48 (squares), and n = 11–18 (triangles) were obtained from four to five different patches.
  3. Representative record of the macro‐patch current inactivation at −50 mV and +50 mV in an excised membrane bleb patch; the dashed line corresponds to the zero‐current level.
  4. Voltage dependence of open probability for Maxi‐Cl in excised membrane bleb patches. Half‐maximal inactivation was observed at −35.1 ± 2.2 mV (n = 11) and +18.7 ± 2.8 mV (n = 11) in the negative and positive voltage ranges, respectively.
  5. Mean patch currents recorded at +25 mV from excised membrane blebs in the absence (Control: white column) and presence of Gd3+ (50 μM: red column) in the pipette solution. *P < 0.05 (Student's t‐test). n = 27 for Control (from 23 different bleb preparations) and n = 7 for Gd3+ (from three different bleb preparations).
  6. Maxi‐Cl activity (right panel) recorded on a single giant proteoliposome (left image) after reconstitution with the fraction‐7 (F7) derived from total bleb‐membrane proteins.
  7. I–V relationships of Maxi‐Cl activity for F7. Proteoliposomes were bathed in a reconstitution buffer supplemented with 1 mM CaCl2 and 1 mM MgCl2 (circles) and in buffer in which all KCl was replaced with equimolar K‐glutamate (squares). Leftward shift of the reversal potential by −37 mV is indicative of anion selectivity; n = 5–12 from five to six different patches. The slope conductance is 410 ± 7 pS under symmetrical conditions.
Data information: (B, D, E, G) Error bars, SEM.