Pinto-Anwandter BI et al. (2025), Energy landscape of a Kv channel revealed by te...

XB-ART-61340

Nat Commun 2025 Apr 09;161:3379. doi: 10.1038/s41467-025-58443-9.

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

Pinto-Anwandter BI , Bassetto CAZ , Latorre R , Bezanilla F .

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Voltage-dependent potassium channels (Kv) play a crucial role in membrane repolarization during action potentials. They undergo voltage-dependent structural conformational transitions according to their distribution across their energy landscape. Understanding these transitions helps us comprehend their molecular function. Here, we used sudden and sustained temperature changes (Tstep) combined with different voltage protocols and mutations to dissect the energy landscape of the Shaker K+ channel. We used two mutations, ILT (V369I, I372L, and S376T) and I384N, that affect the coupling between the voltage sensor (VSD) and the pore domain (PD), to obtain the temperature dependence of VSD last transition and the intrinsic temperature dependence of the pore, respectively. Our findings support a loose or tight conformation of the electromechanical coupling. In the loose conformation, the movement of the VSD is necessary but not sufficient to efficiently propagate the electromechanical energy to open the pore. In contrast, this movement is effectively translated into pore opening in the tight conformation. Our results describe the energy landscape of the Shaker channel and how its temperature dependence can be modulated by affecting its electromechanical coupling.

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Species referenced: Xenopus laevis
GO keywords: potassium channel activity

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	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 Tstep is shown in red. g–i Comparison of the time course of normalized currents (black, I/I0) and temperature (red) at different voltages for (g) WT, (h) ILT and (i) I384N channels. ∆γ and ∆P0 indicate the change in single-channel conductance and the change in the open probability, respectively. j–l Plot of the Q10 of ionic currents for the j WT, k ILT, and l I384N channels, colors indicate different initial bath temperatures. For (j), (k), and (l), N of at least 4 cells was measured for each bath temperature. (T: No Tstep, Tstep) indicates the value of bath temperature before the temperature step (No Tstep) and temperature reached during the temperature step (Tstep). Data are shown as mean ± SEM.
	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 Tstep in V478W at −60 mV (QTstep). f Relationship between delay in ionic current onset and voltage for different Tstep durations applied before the test 0 mV voltage step. Black squares indicate the delay in current onset in pulses without Tstep (No Tstep) (N = 4 independent measurements, Temperatures = 12 °C, before Tstep to 22 °C, after Tstep). Data are shown as mean ± SEM.
	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 = 4−9 cells measured for each temperature, in E 3 cells measured with a bath temperature of 10 °C with different Tstep magnitudes. Data are shown as mean ± SEM.
	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.
	Supplementary Figure 1: Effects of temperature on the W434F mutant. a Gating currents measurements during a voltage and laser step protocol for W434F. A continuous laser pulse applied in the middle of a voltage step produced a temperature change after the gating currents subsided. b Detail into representative current traces following the subtraction of using the trace without Tjump, a variation of Tstep, where a long pulse that generates a slow increase in temperature is applied rather than a step (16). c Current traces after the subtraction of non Tjump component the line indicates the isochronal used to measure the current to voltage (I-V) relationship. d I-V curve showed a positive current due to the appearance of ionic conduction when V> -40 mV, n=3 different cells. The optocapacitive component was not subtracted.
	Supplementary Figure 2: Representative current traces for G-V and Q-V measurements in WT channel. a, b Ionic currents in response to a voltage pulse protocol for Shaker-IR in the absence a and presence b of a Tstep applied before the voltage step. c, d Gating currents in response to a voltage pulse protocol for Shaker-IR-V478W in the absence c and presence d of a Tstep applied before the voltage step. The voltage step protocol and Tstep profile are shown above the recordings. The bath temperature is shown in blue, Tstep is shown in red, and arrows show the time when Tstep was applied.
	Supplementary Figure 3: Representative current traces for G-V and Q-V measurements in ILT mutant. a, b Ionic currents in response to a voltage pulse protocol for Shaker-IR-ILT in the absence a, and presence b of a Tstep applied before the voltage step. c, d Gating currents in response to a voltage pulse protocol for Shaker-IR-ILT-V478W in the absence c and presence d of a Tstep applied before the voltage step. The voltage step protocol and Tstep profile are shown above the currents. The bath temperature is shown in blue, Tstep temperature is shown in red, and the arrows show the time when Tstep was applied.
	Supplementary Figure 4: Representative current traces for G-V and Q-V measurements in I384N mutant. a, b Ionic currents in response to a voltage pulse protocol for Shaker-IR-I384N in the absence a, c and presence b, d of a Tstep applied before the voltage step for two different bath temperatures. e, f Gating currents in response to a voltage pulse protocol for Shaker-IRI384N-W434F in the absence e and presence f of a Tstep applied before the voltage step. The voltage step protocol and Tstep profile are shown above the recordings. The bath temperature is shown in blue, Tstep temperature is shown in red, and arrows show time when Tstep was applied.
	Supplementary Figure 5: Charge moved by temperature vs voltage. Fraction of the total gating charge moved at different voltages for a 10 ºC Tstep from a bath temperature of 10 ºC. Each point of the curve was calculated by either integrating the current induced by a Tstep (red – similar to what was calculated for the Figure 2 a-c) or using the gating currents at two different bath temperatures. For the green data point, we recorded the gating currents at two different bath temperatures for different voltages. Then, we calculated the gating charge. Next, we subtracted the gating charges at 20 ºC from the gating charge at 10 ºC. The gating currents were elicited like what was shown in Supplementary Figure 2 c, d. Note that the Tstep for the red and green were the same and both were calculated from the same cell.
	Supplementary Figure 6: Kinetic model for Shaker-IR channels used for simulating ionic and gating currents. a Kinetic model based on Zagotta, Hoshi and Aldrich model (17). The model consists of 16 states, where states C1-C15 are closed states and state O is the only open state. The pore only opens when all the voltage sensors are activated. The transition from state 15 to O represents the opening of the bundle crossing in the pore. b Rates constant used for the model shown in a. The values used for the enthalpic, entropic components and the charge associated with each transition are shown in the supplementary table 2. K is a constant related to the vibration frequency. This factor is linearly dependent on temperature and has the same value for all the transitions (500/ms at 290 K). c-e Energy landscape of the transitions from Resting to Active (upper) and from Active to Open (lower) state for WT (c), ILT (d) and I384N (e) at -60 (grey), 0 (red) and 60 mV (blue) for a temperature of 290 K (17 ºC) (solid lines) and 300 K (27 ºC) (dashed lines). The curves were calculated using the parameters shown in Supplementary Table 2, 3.
	Supplementary Figure 6: Kinetic model for Shaker-IR channels used for simulating ionic and gating currents. a Kinetic model based on Zagotta, Hoshi and Aldrich model (17). The model consists of 16 states, where states C1-C15 are closed states and state O is the only open state. The pore only opens when all the voltage sensors are activated. The transition from state 15 to O represents the opening of the bundle crossing in the pore. b Rates constant used for the model shown in a. The values used for the enthalpic, entropic components and the charge associated with each transition are shown in the supplementary table 2. K is a constant related to the vibration frequency. This factor is linearly dependent on temperature and has the same value for all the transitions (500/ms at 290 K). c-e Energy landscape of the transitions from Resting to Active (upper) and from Active to Open (lower) state for WT (c), ILT (d) and I384N (e) at -60 (grey), 0 (red) and 60 mV (blue) for a temperature of 290 K (17 ºC) (solid lines) and 300 K (27 ºC) (dashed lines). The curves were calculated using the parameters shown in Supplementary Table 2, 3.
	Supplementary Figure 7: Q-V for ILT using two different holding potentials. ILT does not enter in the relaxed state and the Q-V is the same for both 0 (red) and -80 mV (blue) holding potential. N of 9 and 3 cells for red and blue curve respectively, T=22 ºC. Data are shown as Mean ± SEM.
	Supplementary Figure 8: Cole-Moore shift delay determination. A representative ionic current trace (black trace) was elicited using the voltage protocol shown in Figure 2d. A Two-exponential function was fitted (red trace) and then extrapolated to the point where the current is 0 A (dashed horizontal line). The delay is then calculated by subtracting the extrapolated point from the time of onset voltage. This procedure was performed to currents in the presence and absence of Tstep. For representative purposes, we are displaying a representative ionic current trace without Tstep here.