Figure S5. Bilayer recordings show little leak and reveal subconductance states.
(A) The recordings from a multi-channel patch at ±140, ±130, and 0 mV (black) were showed to demonstrate that the recorded currents reached zero when all channels were closed, suggesting that the leaking currents were negligible and had very little effect in the recorded currents. The corresponding closing events were expanded in the bottom, which shows not only that the currents reached zero at multiple occasions, but also that there are subconductance states. (B) The recording at the −80 mV epoch in panel (A) was expanded to show the subconductance states (sub1 and sub2) besides the main state. (C) Average macroscopic currents from currents recoded in asymmetrical Cl− settings (42/166 mM) as in Fig 4A were plotted as a function of transmembrane voltage (Vm). Error bars: SD from three recordings. Fitting with a linear curve in the range of −40 to +80 mV was performed to deduce a reversal potential of ∼+30.5 mV, very close to the Nernst potential of Cl− (34.8 mV). The estimated chord conductance was ∼1.5 nS, suggesting 11 channels in the patch, which was used to calculate the estimated average open probability (<P0> = average current/(n × g × (Vm–ECl)); n is the number of channels and g the single-channel conductance). The red markers and red line represent <P0>. Because of a small amount of leak currents in bilayer membranes, the single-channel current versus Vm as shown in Fig 4B is more accurate in determining the reversal potential. (D) Single-channel recordings from CHGB under low, asymmetric Cl−, and high, symmetric K+. cis: 150 mM K-isethionate, 1.5 mM KCl, 10 mM MES, pH 5.5; trans: 135 mM K-isethionate, 15 mM KCl, 10 mM MES, pH 5.5. Vertical scale bar: 15 pA. (E) Single-channel currents from (D) versus Vm. Without sizeable currents for Vm varying from −50 to +100 mV under the solution conditions, a linear fitting (solid black line) was extrapolated to positive Vm to yield an estimated single-channel conductance of ∼58.4 pS and a reversal potential of ∼+65 mV. Error bars: SD of average single-channel currents obtained from 3 to 5 histograms of single-channel events in patch recordings.