Fig. 3: Effect of PPIases on the gating of SthK.
From: Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel
a Representative single-channel recordings of WT SthK at +100 mV in the presence of 100 µM cAMP, 0 µM cAMP + 1 µM CypD, and 100 µM cAMP + 1 µM CypD, as indicated. Dashed lines indicate the closed level. b Normalized all amplitude histogram from single-channel recordings of WT SthK in the presence of 100 µM cAMP, without PPIase (black, dashed line) and in the presence of 1 µM SlyD (orange) or 1 µM CypD (purple) at +100 mV. These experiments were performed three times with similar outcome. c Schematic state model (blue cartoon is the channel, orange triangle is the ligand) for the proline switch in SthK. Column labels indicate apo, cAMP-bound, and open states. Row labels indicate cis and trans Pro channel forms. Vertical transitions are modulated by prolyl isomerization. Gradient lines indicate the shift in the cis/trans equilibrium between the apo and the open state. d Initial rates of Tl+ flux obtained from the stopped-flow assay (Eqs. (2) and (3)) are plotted as functions of the cAMP concentration. Lines represent fits according to Eq. (4). Data with 2.5 s delay time are shown for SthK P300A in blue (EC50 = 24 ± 2 µM, nH = 2.9 ± 0.6, n = 3 independent experiments), WT SthK in the presence of 1 µM SlyD in orange (EC50 = 36 ± 2 µM, nH = 4 ± 0.9, n = 3 independent experiments). The fit for WT SthK in the absence of PPIase is shown as dashed line. WT SthK after about 45 min delay time is shown as open circles and the fit as continuous black line (EC50 = 59 ± 1 µM, nH = 3 ± 0.9, n = 4 independent experiments). Symbols represent mean ± S.D. Source data are provided as a Source Data file.
