Fig. 6: eM-associated transport domain movement detected by voltage-clamp fluorometry.
From: Elevator-like movements of prestin mediate outer hair cell electromotility
a Representative fluorescence recordings from Xenopus oocytes expressing either wild-type rPres or R150C mutant, pretreated with MTS-TAMRA. Voltage-clamped oocytes were subjected to voltage steps ranging from −250 mV (red) to +200 mV (purple). b F-V curves derived from the recordings shown in (a). Continuous line indicates Boltzmann fit to the data (V1/2 = −134.4 mV, α = 54.5 mV). c Predicted reorientation of R150 (side chain highlighted) between ES (green) and CS (orange). Side view; ESQg, and CSQg are aligned by the relatively immobile scaffold domain (shown as surface rendering). d Zoom-views of R150 in the CSQg (top) and ESQg (bottom). Residues within 5 Å of R150’s guanidinium group are shown as sticks. Hydrogen bonds/electrostatic interactions indicated by yellow dotted lines. e Average of F-V curves recorded from 10 oocytes (± SEM) as in (d, e). Data were normalized to saturating fluorescence change as obtained from Boltzmann fits to each individual measurement. For comparison, average F-V relation of zPres-R151C is replotted from Fig. 5f (gray). f Derivative of F-V curve from (d) shows close agreement with eM-associated charge movement (NLC; blue). NLC was recorded from CHO cells (±SEM, n = 6 independent cells) expressing rPres-R150C prelabeled with MTS-TAMRA. g Representative fluorescence recordings measured as in (a), in the absence or presence of 10 mM extracellular salicylate. h Average F-V curves from 10 oocytes (±SEM) as in (b) before (black) and during application of salicylate (red). i Salicylate-induced shift of voltage dependence from measurements shown in (g). j Displacement charge (Qd) for voltages −200, 0, and +200 mV. Traces visualized as windowed averages (1 ns window) over the mean of 3 replicates (dark) with SD (light). k Average charge displacement vs. voltage for CS and ES. Mean (± SD) of Qd calculated from pooled 25 ns simulation blocks (n = 3 independent replicate simulations each), with SD propagated from the individual replicates. Qg is calculated as the offset constant between the linear fits of prestin in the CS and ES state. Mean and standard deviation for Qg derived from linear fits of the 3 × 25 ns simulation blocks. Source data are provided as a Source data file.
