Ion channel thermodynamics studied with temperature jumps measured at the cell membrane

  1. Carlos A.Z. Bassetto
  2. Bernardo I. Pinto
  3. Ramon Latorre
  4. Francisco Bezanilla

  • Curated by Biophysics Colab

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    Evaluation statement (17 January 2024)

    The study by Bassetto Jr. et al. presents an elegant and pioneering technique to rapidly manipulate membrane temperature by up to 10 ºC in less than 1.5 ms, thereby enabling high temporal resolution of the temperature dependence of ion channel currents. The approach combines the cut-open oocyte voltage clamp technique with laser illumination to heat the sub-membrane melanosome layer. Temperature is quantified from observed changes in membrane capacitance. Recordings of Kir1.1, TRPM8, and TRPV1 channels are used to validate the effectiveness of the technique. A limitation is that, in its current form, the technique can be used only on melanosome-containing Xenopus oocyte membranes.

    Biophysics Colab recommends this study to scientists working on the temperature dependence of ion channels and other membrane proteins.

    Biophysics Colab has evaluated this study as one that meets the following criteria:

    • Rigorous methodology
    • Transparent reporting
    • Appropriate interpretation

    (This evaluation refers to the version of record for this work, which is linked to and has been revised from the original preprint following peer review.)

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  1. Biophysics Colab

    Evaluation statement (17 January 2024)

    The study by Bassetto Jr. et al. presents an elegant and pioneering technique to rapidly manipulate membrane temperature by up to 10 ºC in less than 1.5 ms, thereby enabling high temporal resolution of the temperature dependence of ion channel currents. The approach combines the cut-open oocyte voltage clamp technique with laser illumination to heat the sub-membrane melanosome layer. Temperature is quantified from observed changes in membrane capacitance. Recordings of Kir1.1, TRPM8, and TRPV1 channels are used to validate the effectiveness of the technique. A limitation is that, in its current form, the technique can be used only on melanosome-containing Xenopus oocyte membranes.

    Biophysics Colab recommends this study to scientists working on the temperature dependence of ion channels and other membrane proteins.

    Biophysics Colab has evaluated this study as one that meets the following criteria:

    • Rigorous methodology
    • Transparent reporting
    • Appropriate interpretation

    (This evaluation refers to the version of record for this work, which is linked to and has been revised from the original preprint following peer review.)

  2. Biophysics Colab

    Authors' Response (15 August 2023)

    GENERAL ASSESSMENT

    The preprint by Bassetto Jr et al. presents an elegant and pioneering technique to rapidly manipulate membrane temperature and study the temperature dependence of ion channel currents. The work is a tour de force that combines the cut-open oocyte voltage clamp technique with laser illumination to achieve heating. Upon exposure to a laser pulse, the sub-membrane melanosome layer heats up, and the ensuing changes in membrane temperature are calculated from the observed changes in membrane capacitance (Cm) using a quasi-linear relationship between membrane capacitance and membrane temperature in the 15 to 45 ºC range. The approach enables the authors to achieve ~10 ºC temperature jumps within 1.5 ms and maintain the achieved test temperature for up to 150 ms. Recordings of Kir1.1, TRPM8, and TRPV1 channels are used to validate the technique. The rationale and the in situ detection of membrane temperature changes with ms time resolution is novel, and represents an important technical advance to study kinetic behavior of thermally-sensitive ion channels. Its great advantages notwithstanding, there are some limitations of the new technique in comparison with steady-state bath heating in that 1) In its current form it can be used only on Xenopus oocytes membranes (which contain melanosomes), and 2) In the cut-open setup it can be used only for the study of macroscopic, but not single-channel, currents. Also, the implementation details would benefit from some extra explanations as discussed in the recommendations below.

    RECOMMENDATIONS

    Essential revisions:

    1. Based on the experimental arrangement, it is unclear whether the temperature indeed increases homogeneously over the entire dome-shaped membrane area from which the current is recorded. The hemispherical membrane surface is exposed to a homogeneous laser that illuminates a major portion of the top dome in the top chamber, not the complete area of the clamped membrane. Thus, at the periphery, the absorbed power per unit membrane area is expected to be smaller than around the center of the dome. In this case, the membrane capacitance would be a mixture of capacitances for areas of membrane under varied temperatures: some time-variable and others constant. The channels in all these areas are mixed. Is there a way to estimate these fractions? How quickly does the heat transfer from the shined area to the dark area, and what is the temperature gradient at the boundary area? Given the shape of the beam and curvature of the domed membrane, it is not straightforward that the curved membranes are heated evenly. Can this be tested? There are three immediate heat sinks in the setup – the external solution, the cytosol, and dark membrane (not shined). It would be helpful to estimate or discuss how these affects the temperature clamping, local temperature gradients, and the speed of Tjump.

    To address this, we performed new experiments using a thermal camera to assess the homogeneity of heating during Tjumps. We observed near identical heating of the center pixels, while pixels at the edge of the illuminated area were on average < 1 C cooler. We now show these data in a new supplemental figure (Fig. S8) and refer to it in the results and methods section. We believe these new data support our claim that the oocyte dome experiences a homogeneous increase in temperature during Tjumps with our illumination conditions.

    1. Because of varying Rm, it is not clear how Tsteps can be accurately determined and be clamped at the target temperatures in real-time, without feedback as shown in Figs. 3 and 4. The empirical adjustment of h and the frequency of laser pulses is mentioned, but it remains unclear how this can be generally applied to different oocytes on a regular basis.

    We agree with the reviewers that the Tstep method does not provide precise temperature control, as could be achieved using feedback control. Our approach empirically determines a laser modulation that achieves the desired Tstep, which works for oocytes expressing the same construct from the same frog. We better explain this empirically determined laser modulation for Tsteps in the revised manuscript.

    1. The laser pulse method is used to elicit brief temperature jumps and "sustained" temperature steps lasting up to 150 ms. However, the gating of many temperature-sensitive ion channels is slow, with burst and interburst durations in the seconds (or tens of seconds) range. For such ion channels, much longer time periods at a given temperature are required to allow the channel population to relax to an equilibrium. Would the method presented here be suitable for generating test temperatures that are stable for seconds or tens of seconds?

    Our current Tstep method is limited to durations on the order of hundreds of milliseconds due to the empirical determination of the laser modulation pulses and the fact that for long durations, short laser pulses are required every ms. This produces fluctuations on the membrane temperature. In principle these issues could be addressed using continued illumination and controlling the light intensity directly, which would allow for longer stimulation at a constant temperature.

    1. To express membrane capacitance from the imaginary part of the membrane impedance, an approximation (Eq. 3) is used. The approximation is shown to hold under conditions of a resting membrane, i.e., when no ion channels are activated and thus the membrane resistance is large (~1 MΩ). Does the approximation hold also under conditions when large ionic currents are activated and Rm falls to <0.01 MΩ?

    This is an excellent point. To address it, we tested the method using only the imaginary part of the impedance to determine capacitance over a wide range of frequencies (Fig. S4). Considering values of Cm of 18.2 nF and Rm 1.17 MΩ W. We find that for frequencies larger than 300 Hz the term (𝜔𝐶𝑚𝑅𝑚)2 >> 1, which allows us to approximate the capacitance from the imaginary part of the impedance. Using Equation 7 thus follows that if Rm decreases 10 times the frequencies should increase 10 times for the approximation to hold. If Rm falls to <0.01 MΩ it might be better to fit the total impedance (real + imaginary) as in supplementary figure 7. We must point out that we adjusted and measured the capacitance in voltage ranges where the expressed channel is not conductive.

    1. For a temperature jump from 13 to 18.7 ºC (e.g. in Fig. 2), the membranes and channels might be expected to undergo an endothermic process, and thus dH > 0, not < 0, would be expected. In this case, dE / dS = T > 0 ==> dS > 0. Relief of inactivation was proposed and interpreted as increased dislodging of the internal blocking cations with higher entropy. On the other hand, the origin of these thermodynamic parameters may be complex. Can the authors comment on the derived thermodynamic values for Fig. 2d? Also, given that the system is unlikely to be at equilibrium, it may be simpler to just report phenomenological descriptors such as Q10.

    The thermodynamic parameters we derived for the relief of rectification in Kir1.1b may originate from complex processes that are difficult to interpret mechanistically. The ΔH values reported are obtained by fitting the V1⁄2 values using equation 4. Due to the fast blocking of Kir channels by internal polyamines (faster than the speed of our voltage clamp), we can consider the system at equilibrium during the temperature jumps. We explain these results mechanistically in the following paragraph:

    "The rectification of Kir channels arises from the block of the pore by cytoplasmic polyamines and magnesium. The block of the channel would produce a decrease in the entropy of the blocker molecule. This entropy change needs to be offset by an enthalpy change for the blockage to be reversible. The interaction between the positive charges of the blocker with negatively charged amino acids and substantial hydrophobic interactions within the internal vestibule may contribute to these large enthalpic changes and thus explain the temperature dependence observed.

    1. For the sinusoidal wave measurements and the determination of Cm in real time in a quasi-stationary circuit with a short period (say 1 ms), it is helpful to estimate the accuracy of phase-shift measurement with a limited number of data points in such a short period, and the size of errors in Cm measurements.

    Since we sample the current and voltage at 1MHz we don't expect any problems due to sampling. To test our approach using the Hilbert transform to extract the capacitance at times shorter than the period of the voltage wave, we simulated an RC system in which we simulated a rapid change in capacitance, modeled after the change in capacitance from a 1 ms temperature jump (Figure S5.). We can observe that the Hilbert transform method can follow the change in capacitance accurately in these cases.

    Optional suggestions:

    1. The data in Fig. 1f demonstrate a substantial difference between the temperatures measured using a calibrated pipette positioned at ~1 um from the membrane and that reported for the membrane itself by the capacitance-based temperature measurement (CTM) method. That difference suggests a steep spatial temperature gradient along the membrane normal. Might such a microscopic temperature gradient affect the function of ion channels or membrane proteins in general?

    The temperature gradient shown in Fig. 1f does suggests a microscopic gradient along the membrane normal during heating. From our simulations, it is not plausible that a significant temperature drop across a nanometer-length scale would occur. It is important to note that we expect that the temperature does not change at the _ membrane _. Of course, we acknowledge that small variations are possible, but we don't expect that to be significant. The difference between the measurement by the pipette and the capacitance is that the pipette is placed near the oocyte (μm), which is different from the capacitance that measures at the membrane. This is main reason for the different values for the temperature when measured with these two different methods.

    1. In Extended Data Fig. 3a, the recording was done at a holding potential of -80 mV (Vm = constant). As the capacitance change over time is nearly linear during the period of "Laser On", the current I(t) = d(Cm Vm)/dt = Vm * d(Cm)/dt is expected to follow the Cm(t) change, and be at a nearly constant level. During this period, the Iop shows a sharp ON phase and a decay. It would be helpful to readers if these two phases are explained, are compared side-by-side with the data in Fig. 1e, and used to highlight the advantages of Cm measurements using sinusoidal waves.

    The sharp ON phase occurs because the melanin layer quickly absorbs the laser pulse energy, creating a rapid temperature jump at the membrane. This fast heating causes a transient capacitance change that manifests as a large ON current (I=V dC/dt). After the initial spike, the temperature and, consequently, the capacitance reaches constant value state where the incoming laser energy balances the heat dissipation into the surrounding solution. During this phase, the capacitance doesn't change. Thus there is no optocapacitive current. We included a paragraph to explain this:

    "This pulse protocol produces a large optocapacitive current during the temperature rising phase. Since the subsequent laser pulses maintain the temperature constant, they do not produce large optocapacitive currents."

    1. It would help if the authors clarified how the speed of T steps (which the authors report is 1.5 ms for a 9 ºC jump) is expected to change with the magnitude change in temperature.

    For a given laser power density, the speed of the temperature jump will depend on how much the temperature needs to rise and how quickly the heat dissipates (which depends on factors like heated membrane area, the distance of the melanin to the membrane, etc.).

    In general, we empirically adjust the laser power for different magnitude temperature jumps to achieve a rapid jump in ~1-2 ms. The exact relationship between temperature jump speed and magnitude is complex and would require extensive modeling and calibration and can be seen in detail in Shapiro et al. 2012 (https://doi.org/10.1038/ncomms1742).

    However, in practice, we can achieve rapid jumps over a wide temperature range by empirically tuning the laser power as needed.

    1. For Tjumps in channel-expressing oocytes in Fig. 2, the T jump is measured separately, not in real-time with the voltage-series. It would be helpful as a control to show the residual currents in the presence of Ba2+ at say -100 mV. Is there any irreversible pore blocker that can be used so that Tjumps can be done in the middle of the voltage pulses? Additionally, do membranes with high densities of channels themselves alter the capacitive measurements?

    We have added a new supplemental figure (Fig. S2E) showing Kir currents before and after the Ba2+ block, with and without a Tjump. This demonstrates the isolation of the optocapacitive current for subsequent subtraction. We did not test any irreversible blocker.

    We have unpublished results with Kv channels that show measuring the effect of temperature on gating currents, which can be considered as a type of capacitive current. In those cases, we are careful to do the temperature measurements at a voltage where these "extra" capacitive currents are absent.

    1. The data points in Fig. 4c/d are clustered together. It will be helpful to have data points at T = 13.5 and 21.5 ºC in Fig. 4c and at T = 24 and 34 ºC in Fig. 4d to show a broad range of temperatures, and compare the data with the steady-state data from the same types of oocytes clamped at these temperatures by bath perfusion.

    We did not test bath temperature changes on TRP channels.

    1. In Fig. 4, a plot of log(I/Imax) vs. 1/T2 at different voltages would be helpful to illustrate its connection to the enthalpic changes (Equation 6).

    Since we have deemphasized the van't Hoff analysis in the revised manuscript, we opted not to include these specific plots. However, it could provide helpful background, especially for readers interested in the thermodynamic framework.

    1. Can the change in single channel conductance () for TRPM8 in the early phase of Tjump (Fig. 5c) be compared with conductance immediately before in order to estimate both Q10 at different voltages and H? This would be a test of the voltage-independence of channel conductance. The same for TRPV1.

    Comparing the single channel conductance before and during the Tjump would allow an estimation of the Q10 and enthalpy change associated with conductance.

    However, accurately isolating the conductance component is difficult, given the time resolution. For TRPs the conductance increase onset overlaps with the subsequent change in open probability during the Tjump.

    While we could not reliably quantify Q10 for the conductance, we agree that this is an important question.

    1. The assumption that channel gating is a 2-state process might complicate estimation of van't Hoff enthalpy/entropy parameters for Kir channels.

    The two-state model we employed is a simplification for interpreting the thermodynamic parameters for Kir channel gating. These channels likely have multiple closed and open states. However, given the experimental results, this provides a valuable model to explain the temperature dependence of Kir.

    1. A question that might be pertinent for the study of TRP channels, is how easily these methods can be used to probe the cross-modulation of temperature and TRP channel agonists. Capsaicin and hydrophobic ligands are likely to partition in the membrane – would that alter the membrane capacitance or capacitance vs temperature calibrations?

    You raise a valid point we had not previously considered - hydrophobic agonists partitioning into the membrane could alter the capacitance relationship we rely on for calibration. Another calibration curve with those ligands at the membrane may be necessary to check whether this would affect the previously calibrated relationship However, if we performed the temperature measurement by capacitance at the beginning of the experiment before the application of any agonists this should fine.

    1. Some supportive evidence that melanin addition to cell or vesicle membranes would allow this method to be used in other membranes would greatly enhance the applicability of this approach.

    We agree that this is an important next step. Gold nanoparticles have been previously used to heat the membrane of neurons; similar approaches might render this technique in new preparations.

    1. A side-by-side comparison of this approach with other approaches for studying temperature jumps in ion channels would be incredibly useful for many readers to visualize the relative abilities of various methods to deliver and measure temperature jumps.

    (This is a response to peer review conducted by Biophysics Colab on version 2 of this preprint.)

  3. Biophysics Colab

    Consolidated peer review report (12 September 2022)

    GENERAL ASSESSMENT

    The preprint by Bassetto Jr et al. presents an elegant and pioneering technique to rapidly manipulate membrane temperature and study the temperature dependence of ion channel currents. The work is a tour de force that combines the cut-open oocyte voltage clamp technique with laser illumination to achieve heating. Upon exposure to a laser pulse, the sub-membrane melanosome layer heats up, and the ensuing changes in membrane temperature are calculated from the observed changes in membrane capacitance (Cm) using a quasi-linear relationship between membrane capacitance and membrane temperature in the 15 to 45 ºC range. The approach enables the authors to achieve ~10 ºC temperature jumps within 1.5 ms and maintain the achieved test temperature for up to 150 ms. Recordings of Kir1.1, TRPM8, and TRPV1 channels are used to validate the technique. The rationale and the in situ detection of membrane temperature changes with ms time resolution is novel, and represents an important technical advance to study kinetic behavior of thermally-sensitive ion channels. Its great advantages notwithstanding, there are some limitations of the new technique in comparison with steady-state bath heating in that 1) In its current form it can be used only on Xenopus oocytes membranes (which contain melanosomes), and 2) In the cut-open setup it can be used only for the study of macroscopic, but not single-channel, currents. Also, the implementation details would benefit from some extra explanations as discussed in the recommendations below.

    RECOMMENDATIONS

    Essential revisions:

    1. Based on the experimental arrangement, it is unclear whether the temperature indeed increases homogeneously over the entire dome-shaped membrane area from which the current is recorded. The hemispherical membrane surface is exposed to a homogeneous laser that illuminates a major portion of the top dome in the top chamber, not the complete area of the clamped membrane. Thus, at the periphery, the absorbed power per unit membrane area is expected to be smaller than around the center of the dome. In this case, the membrane capacitance would be a mixture of capacitances for areas of membrane under varied temperatures: some time-variable and others constant. The channels in all these areas are mixed. Is there a way to estimate these fractions? How quickly does the heat transfer from the shined area to the dark area, and what is the temperature gradient at the boundary area? Given the shape of the beam and curvature of the domed membrane, it is not straightforward that the curved membranes are heated evenly. Can this be tested? There are three immediate heat sinks in the setup – the external solution, the cytosol, and dark membrane (not shined). It would be helpful to estimate or discuss how these affects the temperature clamping, local temperature gradients, and the speed of Tjump.
    2. Because of varying Rm, it is not clear how Tsteps can be accurately determined and be clamped at the target temperatures in real-time, without feedback as shown in Figs. 3 and 4. The empirical adjustment of h and the frequency of laser pulses is mentioned, but it remains unclear how this can be generally applied to different oocytes on a regular basis.
    3. The laser pulse method is used to elicit brief temperature jumps and "sustained" temperature steps lasting up to 150 ms. However, the gating of many temperature-sensitive ion channels is slow, with burst and interburst durations in the seconds (or tens of seconds) range. For such ion channels, much longer time periods at a given temperature are required to allow the channel population to relax to an equilibrium. Would the method presented here be suitable for generating test temperatures that are stable for seconds or tens of seconds?
    4. To express membrane capacitance from the imaginary part of the membrane impedance, an approximation (Eq. 3) is used. The approximation is shown to hold under conditions of a resting membrane, i.e., when no ion channels are activated and thus the membrane resistance is large (~1 MΩ). Does the approximation hold also under conditions when large ionic currents are activated and Rm falls to <0.01 MΩ?
    5. For a temperature jump from 13 to 18.7 ºC (e.g. in Fig. 2), the membranes and channels might be expected to undergo an endothermic process, and thus dH > 0, not < 0, would be expected. In this case, dE / dS = T > 0 ==> dS > 0. Relief of inactivation was proposed and interpreted as increased dislodging of the internal blocking cations with higher entropy. On the other hand, the origin of these thermodynamic parameters may be complex. Can the authors comment on the derived thermodynamic values for Fig. 2d? Also, given that the system is unlikely to be at equilibrium, it may be simpler to just report phenomenological descriptors such as Q10.
    6. For the sinusoidal wave measurements and the determination of Cm in real time in a quasi-stationary circuit with a short period (say 1 ms), it is helpful to estimate the accuracy of phase-shift measurement with a limited number of data points in such a short period, and the size of errors in Cm measurements.

    Optional suggestions:

    1. The data in Fig. 1f demonstrate a substantial difference between the temperatures measured using a calibrated pipette positioned at ~1 um from the membrane and that reported for the membrane itself by the capacitance-based temperature measurement (CTM) method. That difference suggests a steep spatial temperature gradient along the membrane normal. Might such a microscopic temperature gradient affect the function of ion channels or membrane proteins in general?
    2. In Extended Data Fig. 3a, the recording was done at a holding potential of -80 mV (Vm = constant). As the capacitance change over time is nearly linear during the period of "Laser On", the current I(t) = d(Cm Vm)/dt = Vm * d(Cm)/dt is expected to follow the Cm(t) change, and be at a nearly constant level. During this period, the Iop shows a sharp ON phase and a decay. It would be helpful to readers if these two phases are explained, are compared side-by-side with the data in Fig. 1e, and used to highlight the advantages of Cm measurements using sinusoidal waves.
    3. It would help if the authors clarified how the speed of T steps (which the authors report is 1.5 ms for a 9 ºC jump) is expected to change with the magnitude change in temperature.
    4. For Tjumps in channel-expressing oocytes in Fig. 2, the T jump is measured separately, not in real-time with the voltage-series. It would be helpful as a control to show the residual currents in the presence of Ba2+ at say -100 mV. Is there any irreversible pore blocker that can be used so that Tjumps can be done in the middle of the voltage pulses? Additionally, do membranes with high densities of channels themselves alter the capacitive measurements?
    5. The data points in Fig. 4c/d are clustered together. It will be helpful to have data points at T = 13.5 and 21.5 ºC in Fig. 4c and at T = 24 and 34 ºC in Fig. 4d to show a broad range of temperatures, and compare the data with the steady-state data from the same types of oocytes clamped at these temperatures by bath perfusion.
    6. In Fig. 4, a plot of log(I/Imax) vs. 1/T2 at different voltages would be helpful to illustrate its connection to the enthalpic changes (Equation 6).
    7. Can the change in single channel conductance () for TRPM8 in the early phase of Tjump (Fig. 5c) be compared with conductance immediately before in order to estimate both Q10 at different voltages and H? This would be a test of the voltage-independence of channel conductance. The same for TRPV1.
    8. The assumption that channel gating is a 2-state process might complicate estimation of van't Hoff enthalpy/entropy parameters for Kir channels.
    9. A question that might be pertinent for the study of TRP channels, is how easily these methods can be used to probe the cross-modulation of temperature and TRP channel agonists. Capsaicin and hydrophobic ligands are likely to partition in the membrane – would that alter the membrane capacitance or capacitance vs temperature calibrations?
    10. Some supportive evidence that melanin addition to cell or vesicle membranes would allow this method to be used in other membranes would greatly enhance the applicability of this approach.
    11. A side-by-side comparison of this approach with other approaches for studying temperature jumps in ion channels would be incredibly useful for many readers to visualize the relative abilities of various methods to deliver and measure temperature jumps.

    REVIEWING TEAM

    Reviewed by:

    Sandipan Chowdhury, Assistant Professor, University of Iowa, USA: ion channel structure-function and temperature gating

    László Csanády, Director of the Institute of Biochemistry and Molecular Biology, Semmelweis University, Hungary: structure-function of TRP channels

    Marcel P. Goldschen-Ohm, Assistant Professor, The University of Texas at Austin, USA: ion channel structure-function and kinetics

    Qiu-Xing Jiang, Associate Professor, University of Buffalo, USA: high resolution ion channel structure and functional thermodynamics

    Curated by:

    Marcel P. Goldschen-Ohm, Assistant Professor, The University of Texas at Austin, USA

    (This consolidated report is a result of peer review conducted by Biophysics Colab on version 2 of this preprint. Comments concerning minor and presentational issues have been omitted for brevity.)

  4. Version published to 10.1016/j.bpj.2023.01.015
  5. Version published to 10.1101/2022.07.11.499616v2 on bioRxiv
  6. Version published to 10.1101/2022.07.11.499616v1 on bioRxiv