Fig. 4: Conformational dynamics of FLYC1 protomer.

Side (a) and top (b) views of superposed up and down classes. Movement of helices denoted by red arrows. F572 shown as sticks. In top view, TM6a of all subunits is shown. c Expanded view of window in a, showing the presence of a salt bridge between R334 and D598 in the up (left) but not in the down class (right). d Left, representative stretch-activated currents from WT (black) or R334E mutant (red) FLYC1 in the cell-attached patch clamp mode in response to 1 s pulse of −70 mmHg pressure at −80 mV. For comparison, currents are normalized and overlaid. Top, single channel current; bottom, macroscopic current. Inset depicts enlarged section of the macroscopic trace to highlight that channel activation and inactivation are largely similar between WT and R334E. Right, quantification of maximal current response from cells transfected with FLYC1 plasmid (N = 6), R334E mutant (N = 6), or D598K mutant (N = 9). ****p = 0.000036; * p = 0.0104, one-way ANOVA. e Mean deactivation kinetics of WT (N = 6) or mutant (N = 6) FLYC1 channel; ** p = 0.0043, Mann–Whitney test, two-tailed. In panel d and e, individual cells are illustrated as scatter and mean is represented by grey bars. N = number of cells tested and pooled from different experimental days. f Superposition of hypothetical C7 ‘all-up’ and ‘all-down’ classes. Two top left and two top right subunits show only down and up protomers, respectively. F572 ring is shown for all subunits in both conformations. The position and orientation of K558 are shown in pink for only two subunits. g First frame of first component of 3D variability analysis. Subunits are labeled up (U) or down (D) depending on their conformation. Lipid-like density is colored purple.