Rapid Temperature Jumps and Steps Reveal Insights into Ion Channel Thermodynamics

核心概念

Newly developed techniques of rapid temperature jumps (Tjumps) and sustained temperature steps (Tsteps) provide a powerful tool to study the temperature dependence of ion channel function and gating mechanisms.

要約

The authors developed two complementary techniques to study ion channel thermodynamics:

Temperature Jumps (Tjumps):

Tjumps are generated by illuminating the Xenopus oocyte membrane with a homogenized laser beam, which heats the membrane through the absorption of light by melanin.
The authors used a capacitance-based temperature measurement (CTM) method to track the fast temperature changes at the membrane with sub-millisecond time resolution.

Temperature Steps (Tsteps):

Tsteps are generated by modulating the duration of a train of laser pulses, allowing for sustained temperature changes.
The CTM method is also used to monitor the temperature changes during Tsteps.

The authors demonstrated the applicability of these techniques by studying the effects of temperature on the inward rectifier potassium channel Kir1.1b, the cold-sensitive channel TRPM8, and the heat-sensitive channel TRPV1.
Key findings:

Tjumps on Kir1.1b revealed a high temperature dependence of the rectification process, with a large enthalpic component.
Tsteps on TRPM8 and TRPV1 showed a biphasic response, with an initial increase in single-channel conductance followed by changes in the open probability.
The Tstep technique allowed the authors to study the temperature dependence of channel gating kinetics at specific points during the activation process.
The authors conclude that the combination of Tjumps, Tsteps, and CTM provides a powerful tool to study the temperature dependence of ion channel function and gating mechanisms, offering new insights into membrane protein thermodynamics.

統計

The capacitance of the oocyte membrane changes approximately 1.070% ± 0.003 per 1°C increase in temperature.
The Kir1.1b channel exhibits a Q10 of 1.6 ± 0.2 for the single-channel conductance.
The Kir1.1b channel rectification has an enthalpy change (ΔH) of -71.7 ± 2.9 kcal/mol and an entropy change (ΔS) of -263 ± 10 cal/K·mol.

引用

"Temperature is an intensive physical property that results from the movement of atoms and molecules. As such, it affects from chemical reactions to biological processes."
"We expect that the combination of fast temperature changes and CTM can enrich our understanding of membrane protein thermodynamics."
"These newly developed approaches provide a general tool to study membrane proteins thermodynamics."

抽出されたキーインサイト

ION CHANNEL THERMODYNAMICS STUDIED WITH TEMPERATURE JUMPS MEASURED AT THE CELL MEMBRANE

by Bassetto,C. ... 場所 www.biorxiv.org 07-12-2022

https://www.biorxiv.org/content/10.1101/2022.07.11.499616v2

深掘り質問

How could the Tjump and Tstep techniques be adapted to study ion channel thermodynamics in other cell types, such as isolated neurons or cell lines?

The Tjump and Tstep techniques can be adapted to study ion channel thermodynamics in other cell types by modifying the experimental setup and methodology to suit the specific characteristics of the cells of interest. Here are some ways these techniques could be adapted:

Cell Type Consideration: Different cell types may have varying membrane properties and sensitivities to temperature changes. Therefore, it is essential to optimize the laser parameters, such as power, wavelength, and duration, to achieve precise and controlled temperature changes in the specific cell type under study.

Temperature Calibration: Just as in the Xenopus oocytes, temperature calibration using a calibrated pipette or other temperature probes can be employed to ensure accurate temperature measurements at the membrane level in isolated neurons or cell lines.

Optimization of Laser Setup: The laser setup for Tjump and Tstep techniques may need to be adjusted to accommodate the size and shape of different cell types. For example, in isolated neurons, the laser beam may need to be focused on specific regions of the cell to achieve localized temperature changes.

Integration with Patch-Clamp Technique: In the case of isolated neurons or cell lines, integrating the Tjump and Tstep techniques with the patch-clamp technique can provide a comprehensive understanding of ion channel thermodynamics at the single-cell level.

Validation and Calibration: Before conducting experiments in other cell types, it is crucial to validate the Tjump and Tstep techniques by comparing the results with established temperature-dependent mechanisms in ion channels and ensuring the accuracy and reliability of the temperature measurements.

What other types of membrane proteins, beyond ion channels, could benefit from the application of these temperature perturbation techniques?

Beyond ion channels, several other types of membrane proteins could benefit from the application of Tjump and Tstep techniques to study their temperature-dependent mechanisms. Some examples include:

Transporters: Membrane transporters, such as ATP-dependent transporters or secondary active transporters, often undergo conformational changes in response to temperature variations. Applying Tjump and Tstep techniques can help elucidate the temperature sensitivity of transporter kinetics and substrate binding.

Receptors: Temperature can modulate the binding affinity and activation of membrane receptors, including G protein-coupled receptors (GPCRs) and ligand-gated receptors. By using temperature perturbation techniques, researchers can investigate the temperature-dependent conformational changes and signaling pathways of these receptors.

Adhesion Proteins: Proteins involved in cell adhesion, such as integrins and cadherins, play crucial roles in cell-cell interactions and signaling. Studying the temperature dependence of their conformational changes and binding affinities can provide insights into cell adhesion dynamics.

Enzymes: Membrane-bound enzymes, such as ATPases or phospholipases, may exhibit temperature-dependent changes in catalytic activity and substrate specificity. Tjump and Tstep techniques can be used to investigate the thermodynamic properties of enzyme-substrate interactions at the membrane.

Structural Proteins: Proteins involved in membrane structure and organization, like scaffolding proteins or cytoskeletal components, may respond to temperature variations by altering membrane dynamics. Understanding the temperature sensitivity of these proteins can shed light on membrane architecture and function.

What insights could be gained by combining the Tjump and Tstep techniques with other biophysical methods, such as single-molecule studies or structural biology approaches, to further elucidate the temperature-dependent mechanisms of ion channel function?

Combining the Tjump and Tstep techniques with other biophysical methods, such as single-molecule studies or structural biology approaches, can provide a more comprehensive understanding of the temperature-dependent mechanisms of ion channel function. Here are some insights that could be gained from these combined approaches:

Single-Molecule Studies: By integrating Tjump and Tstep techniques with single-molecule studies, researchers can investigate the temperature-dependent conformational changes and dynamics of individual ion channels in real-time. This approach can reveal how temperature influences the stability, interactions, and functional states of ion channels at the single-molecule level.

Structural Biology Approaches: Combining Tjump and Tstep techniques with structural biology methods, such as X-ray crystallography or cryo-electron microscopy, can provide high-resolution structural insights into the temperature-induced conformational changes of ion channels. This can help in elucidating the structural basis of temperature sensitivity and gating mechanisms of ion channels.

Kinetic Analysis: Integrating Tjump and Tstep techniques with kinetic analysis methods can allow for the precise measurement of temperature-dependent rate constants, activation energies, and transition states involved in ion channel gating. This approach can provide a detailed kinetic model of temperature-dependent channel dynamics.

Thermodynamic Modeling: By combining temperature perturbation techniques with thermodynamic modeling, researchers can quantitatively analyze the energetics of ion channel function at different temperatures. This can lead to the determination of thermodynamic parameters, such as enthalpy and entropy changes, associated with temperature-induced conformational transitions in ion channels.

Pharmacological Studies: The integration of Tjump and Tstep techniques with pharmacological studies can help in exploring the temperature sensitivity of ion channel modulation by drugs or ligands. This approach can provide insights into how temperature influences the efficacy and binding kinetics of channel modulators.

Overall, the combination of Tjump and Tstep techniques with complementary biophysical methods can offer a multi-faceted approach to unraveling the complex temperature-dependent mechanisms underlying ion channel function.