Fig. 2: Probing the conformational landscape by accessibility scanning.

a Changes of peak NLC in response to application of MTS reagents (1 mM; MTSES, dark gray; MTSET, light gray; control, black) to the extracellular (left) or intracellular (right) side of cysteine substitution mutants expressed in CHO cells. Bars show residual NLC post-MTS reagent, normalized to signal before MTS application (±SEM; data from n ≥ 5 independent cells, see Source data file). Significance level ***p ≤ 0.005 (two-sided Dunnett’s test). Upper panel, mutants in TM3; Lower panel, mutants in TM10. Coloration highlights positions sensitive to MTS reagents at the extracellular (blue) or intracellular (red) side. In mutant L401C, MTS reagents did not change NLC amplitude, but intracellular MTSET shifted voltage-dependence (Supplementary Figure 5f). b Extra- and intracellular water accessibility (WA) determined from MD simulations. Bars indicate average number of water molecules visiting each residue (100 ps/frame n = 6 MD replicas; error bars show SD within each respective cluster) originating from the extracellular solution (left) or intracellular solution (right). Orange, water contacts in the CS conformational cluster; green, contacts in the ES cluster. Positions exhibiting substantial water contact frequency consistent with experimental cysteine accessibility are highlighted in blue (extracellular WA) and red (intracellular WA). c Structural interpretation of experimental data. The extended conformation (ES, green) but not the compact state (CS, orange) is compatible with intracellular access to TM10 positions through V139 (TM3), including the anion-binding site centered approximately around S396. Corresponding experimental MTS reactivity thus confirms occupancy of ES under physiological conditions. Extracellular access of V139 at the lower end of helical TM3 is predicted only for the compact state (CS, orange). Thus, sensitivity of V139C to extracellular MTS demonstrates occupancy of CS. d Representative NLC recordings rPres with all endogenous cysteines replaced by alanines show insensitivity to MTS reagents applied either from the extracellular (upper panels) or intracellular side (lower panels). Left panels: Time course of NLC normalized to amplitude before application of MTSES (dark gray), MTSET (light gray), or control solution (black). Middle panels: representative NLC traces before (black) and after application of MTSES (dark gray). Right panels: representative NLC traces before (black) and after application of MTSET (light gray). e–g Representative recordings obtained as in (d) show inhibition of NLC selectively by extracellular MTS for L142C and G145C, but two-sided accessibility for V139. Source data are provided as a Source data file.