Energy landscape of a Kv channel revealed by temperature steps while perturbing its electromechanical coupling

Fig. 1. Temperature dependence of ionic conduction in WT and mutant channels. a Schematic representation of the experimental set-up used to apply fast-temperature steps to the oocyte membrane. A current-regulated homogenized laser light is used to illuminate the upper dome of the oocyte in the cut-open set-up, where the current recording occurs. The absorption of the visible light by melanin under the membrane of the oocyte generates a temperature change that is modulated by using the PWM of the laser light. b Structural location of the ILT mutations highlighting the location in the C-terminus of S4. c Structural location of the I384N mutation at the N-terminus of the S4-S5 linker, indicating its interaction with the S6 segment in the pore domain (PD), interacting pore subunit shown in blue, PDB: 7SIP15. d–f Current response to temperature steps application (red arrow) during a voltage pulse protocol (inset) for d Wild-type (WT) Shaker IR e ILT, and f I384N channels. The Tstep profile is shown on an expanded scale below the current traces. Scales for the current and temperature are shown for each trace family. The bath temperature is shown in blue, and the temperature reached during the…

Fig. 2. VSD displacement in response to temperature produces a time-dependent delay in activation. a Gating current measurements during a voltage and Tstep protocol. Tsteps were applied in the middle of a voltage step after the gating currents had subsided (indicated by the red arrow). The Tstep profile (red) is shown in an expanded scale above the current traces and voltage protocol. b Detail of temperature-dependent charge movement at different voltages. c Temperature-dependent charge movement vs. voltage (Q–V) curves for 4 different cells. d Ionic currents in response to a voltage step protocol, shown without a temperature step (No Tstep, gray) and with Tstep applied at 100 ms (yellow), 25 ms (green), and 2 ms (red) before the voltage pulse, arrows indicate the corresponding current traces. The inset depicts the voltage protocol; arrows in the voltage protocol indicate the time at which the Tstep is applied. The expanded Tstep trace is presented above the voltage protocol. e Delay in ionic current onset as a function of the time between Tstep and voltage step. Inset shows the time course of the charge movement calculated by the integral of the gating current in response to a similar…

Fig. 3. Temperature-dependent VSD displacement in ILT and I384N. Gating current measurements during a voltage and Tstep protocol for a ILT-V478W. Tstep were applied in the middle of a voltage step after gating currents had subsided (indicated by the red arrow) to analyze the effect of temperature on gating charge movement. The Tstep profile (red) is shown in an expanded scale above the current traces. b Detail of temperature-dependent charge movement on a at different voltages. c Gating current measurement during a voltage and Tstep protocol for a I384N-W434F. d Detail of temperature-dependent charge movement on c at different voltages. e, f Temperature-dependent charge movement vs. voltage curves for e ILT-V478W (n = 6 cells) and f I384N-W434F (n = 5 cells). g Fraction of the total gating charge moved at different voltages for WT = from 15.6 ± 0.3 °C to 22.8 ± 0.7 °C, ILT = from 22.2 ± 0.3 °C to 30.5 ± 0.9 °C, and I384N = from 15.4 ± 0.2 °C to 22.8 ± 1.1 °C (number of cells N for WT = 4, ILT = 6, I384N = 5). Data are shown as mean ± SEM.

Fig. 4. Pore opening and VSD movement thermodynamics in WT, ILT, and I384N channels. Normalized G–V plots for WT (a), ILT (b), and I384N (c) channels under various temperatures indicated by different colors. The continuous line corresponds to a two-state model fitting. d Free energy (zFV—see “Methods”) vs. temperature for the G–V relations. The WT (gray) and I384N (blue) constructs show a negative ΔS (-slope), while ILT (red) exhibits a positive ΔS. e Q–V relationship for the V478W channel. f Free energy vs. temperature plot for the V478W Q–V. g Q–V relationship for the ILT-V478W channel. h Free energy vs. temperature plot for the ILT-V478W Q–V. i Q–V relationship for the I384N-W434F mutant. j Free energy vs. temperature plot for the I384N-W434F mutant. The free energy vs. temperature plot shows the energy for the first (ΔG1, red) and second (ΔG2, blue) component of the Q–V, the sum for the tetramer (4(ΔG1 + ΔG2), green) and using the V-median (gray). I384N only has one distinct component (ΔG1, red). The continuous lines in the Q–V curves are illustrative to help show the shifts. Parameters used for fitting are found in Supplementary Table 1, Tables 1 and 2. For (a–c), (e), (g) and (i) N…

Fig. 5. Simulating the effects of temperature steps on the gating transitions of Shaker WT, I384N, and ILT. a Schematic representation of different conformational states of the channel, from left to right: Resting (VSD down, pore closed), Intermediate (VSD intermediate, pore closed), Active (VSD up, pore closed), and Open (VSD up and pore open). The color of the transmembrane helix denotes the movement of the S4 segment in the resting (purple), intermediate (yellow) and active state (red). Created in BioRender. Pinto, B. (2025) https://BioRender.com/p64k114. b, c, d are the ionic and gating current simulations when Tstep is applied in the middle of a voltage protocol. The model used and its equations are shown in Supplementary Fig. 6. The parameters used for simulation are shown in the Supplementary Tables 2 and 3. (e-g) Simulated Q–V (red), G–V (blue) and the fraction of the charge moved (gray) for the WT (e), ILT (f), and I384N (g) mutants at 290 (filled symbols) and 300 K (empty symbols). The voltage protocols used are the same as presented in Fig. 1.