Panel A shows a molecular schematic for applied transmembrane potential simulations. The schematic includes ELIC5 colored by subunit with sodium ions in yellow, chloride ions in blue, and POPC as multicolored van der Waals surfaces. Propylamine is placed in all five agonist binding sites. Panel B presents a line graph of measured currents for each transmembrane potential of cryo-electron microscopy ELIC5. The x-axis represents applied potential in millivolts, ranging from minus 250 millivolts to 250 millivolts, and the y-axis represents current in picoamperes. Independent replicates are shown as dots, with the mean across replicates displayed as a box and the standard deviation as a crossbar. Conductance for positive and negative potentials is estimated as linear fits, shown as blue and red dashed lines, respectively, with the error of the linear fit as transparent areas. The experimental single-channel sodium ion conductance is shown as a solid black line. Panel C includes representative single-channel currents of WT ELIC and histograms of these currents. The top part shows current traces at positive 100 millivolts and minus 100 millivolts, while the bottom part shows probability density histograms for these currents. Panel D shows a line graph of current-voltage relationships for human alpha 7 nAChR, with the x-axis representing applied potential in millivolts and the y-axis representing current in picoamperes. The linear fit is forced to go through the origin. Panel E presents a similar line graph for zebrafish alpha 1 GlyR. Panel F shows a trace representing the activation of ELIC5 in response to 10 millimolar cysteamine at a holding potential of minus 80 millivolts, followed by deactivation events at positive 80 millivolts and minus 80 millivolts. Panel G displays a current-voltage relationship for WT ELIC activated by cysteamine, WT ELIC activated by propylamine, and ELIC5 from excised patch-clamp recordings of giant liposomes. The x-axis represents holding potential in millivolts, and the y-axis represents current in picoamperes. Replicates are shown as dots, the mean as a diamond, and the standard deviation as a crossbar. The dashed lines represent the line of best fit through the measured mean currents.
The cryo-EM structures of pLGICs display significant outward rectification. (A) A molecular schematic for the applied transmembrane potential simulations. The electric field is applied only in the direction of the membrane normal. ELIC5 is shown colored by subunit with sodium ions in yellow, chloride ions in blue, and POPC as multicolored van der Waals surfaces. Propylamine is placed in all five agonist-binding sites. (B) Measured currents for each transmembrane potential of cryo-EM ELIC5. Independent replicates are shown as dots. The mean across independent replicates at each potential is shown as a box with the standard deviation as a crossbar. Conductance for positive (0 to +250 mV) and negative (−250 to 0 mV) potentials is estimated as a linear fit of all data points and is displayed as blue and red dashed lines, respectively, with the error of the linear fit shown as the transparent area. The experimental single-channel Na+ conductance (γNa) is shown as a solid black line (standard deviation in gray). (C) Representative single-channel currents of WT ELIC (top) and histograms of the currents from these traces (bottom). (D and E) The results of computational electrophysiology measurements for (D) human α7 nAChR (PDB 7KOX) and (E) zebrafish α1 GlyR (PDB 3JAE) are shown with the same color scheme as in B. For D and E, the linear fit is forced to go through the origin. (F) A trace representing the activation of ELIC5 (black) in response to 10 mM cysteamine at a holding potential of −80 mV followed by the slow deactivation in response to withdrawal of the agonist. Representative deactivation events leading to distinct steps in current are shown for ELIC5 at holding potentials of +80 mV (blue) and −80 mV (red). (G) I–V relationship for WT ELIC activated by cysteamine (black, n = 5), WT ELIC activated by propylamine (green, n = 5), and ELIC5 (gray, n = 3) from excised patch-clamp recordings of giant liposomes. Replicates are shown as dots, the mean as a diamond, and the standard deviation as a crossbar. The dashed lines represent the line of best fit through the measured mean currents and are taken as γNa.