Supplementary Figure 4: Single-channel properties of PC2^P185–D723.

Representative single-channel currents of PC2^P185-D723 in (a) symmetrical 210 mM KCl at +60 mV and (b) in asymmetrical 810 mM (cis) and 210 mM (trans) KCl solutions at +30 mV and (c) -50 mV are shown. An expanded portion of each trace (highlighted in blue) illustrates the rapid gating between multiple sub-conductance levels and the sudden shifting of gating into different preferred sub-conductance states (shown by **). In each trace, the most frequently visited sub-conductance levels are highlighted with green and purple dotted lines and with arrows. The fully open (O) and closed (C) channel levels are indicated with black dashed lines. The all-points histograms shown on the right were obtained from 2 min of consecutive recording and the colored arrows correspond to the predominant sub-conductance levels identified in those traces. (d) Linear regression of the current-voltage relationships for PC2^P185-D723 in the same symmetrical KCl (closed circles, n=5 independent experiments) and gradient KCl (open circles, n=6 independent experiments) ionic conditions as in (a-c). Each data point represents the mean ± s.e.m. (e-f) Bar charts compare the probability of PC2^P185-D723 gating to the full open state (Po_FULL) (e) and to sub-conductance open states (Po_SUB) (f) in symmetrical 210 mM KCl solutions (blue) and in KCl gradient (purple). Po_FULL is significantly higher at negative holding potential irrespective of whether the channels were in symmetrical (n=5 independent experiments,*P <0.05) or gradient conditions (n=6 independent experiments,*P<0.05). Under all the conditions examined, PC2^P185-D723 channel exhibits high levels of activity preferentially dwelling in sub-conductance states (note the different scales on the y-axis in (e) and (f)). Data shown are the mean values ± s.e.m. (g) Example of bilayer recording where control ‘empty’ liposomes were used. (h) Effect of Ca²⁺: Typical control single-channel recordings in 210 mM KCl at -60 mV are shown (top trace). A reduction in current amplitude is observed after adding 10 mM trans Ca²⁺ (middle trace) which is reversed after washout of the Ca²⁺ (bottom trace). (i) Effect of amiloride: A representative control recording in symmetrical 210 mM KCl at -60 mV is shown in the top trace and the effects of adding symmetrical 1 mM amiloride to both cis and trans chambers is shown in the bottom trace. The majority of recordings were of multiple channels and therefore (j) shows bar charts illustrating the reduction in mean current (mean I) that was observed after addition of 10 mM trans Ca²⁺ (black bar; n=6 independent experiments;*** P < 0.001) or 1 mM symmetrical amiloride (grey bar; n=5 independent experiments;*** P < 0.001) to channels gating in symmetrical 210 mM KCl at +40 mV (controls, white bars). Data shown are the mean values ± s.e.m. (Student’s t-test, *P<0.05; *** P < 0.001).