Fig. 2.

pH-dependent conductance response to APOL1 and its prevention by SRA. The voltage was held at −20 mV, except for a brief excursion to 0 mV (denoted by an asterisk; the current did not return to zero due to a small electrode offset). The solutions were 0.5 M KCl, 5 mM CaCl₂, 0.5 mM EDTA, supplemented with either 5 mM K-succinate, pH 5.3 (cis), or 5 mM K-Hepes, pH 7.2 (trans). When indicated, the cis side was adjusted to pH 7.3 with 20 μL 1 M Hepes, pH 7.5. Horizontal dashed bars indicate perfusion of the cis side with cis solution. A downward deflection in the current record represents an increase in the magnitude of the current. (A) Cis addition of 40 ng APOL1 caused a minor increase in current magnitude at pH 5.3 (Inset: current and time-scale expansion), and then soluble protein was removed by perfusion. Upon cis pH adjustment to 7.3, there was a large (∼200 pA) increase in current magnitude, which was reversed upon readjustment to pH 5.3. A stirring defect explains the delayed current response to the second Hepes addition. (B) When 5 μg SRA were added to the cis side, the subsequent addition of 40 ng APOL1 still caused a minor increase in current magnitude at pH 5.3 that was resistant to cis perfusion; however, the current actually decreased in magnitude upon adjustment of the cis side to pH 7.3 and remained low when the cis pH was returned to 5.3 (Inset: current scale expansion on the same time line). This procedure was then repeated on the same membrane with 5 μg SRA and 40 ng APOL1-G2 (which does not bind SRA), resulting in a normal current response (minimal increase in the current magnitude at low cis pH, followed by a large increase upon cis neutralization).