Thermodynamic Analysis of Ligand-Gated Ion Channels

Creative Bioarray is committed to providing clients with thermodynamic analysis services of ligand-gated ion channels (LGICs), which involve measuring the energy associated with the gating of LGICs through techniques such as single-channel electrophysiology to advance the study of the isomerization mechanism of LGICs.

Introduction

Allosteric proteins regulate the flow of substances and information through signaling and metabolism pathways. These macromolecular systems isomerize between sets of alternative conformations with distinct functional outputs. And, the probability of adopting each conformation is affected by multiple environmental factors, including electrical, chemical, mechanical, or thermal energy deposited at discrete "sensor" sites in the protein, leading to the establishment of a new conformational equilibrium.

LGICs are allosteric proteins that isomerize between the O(pen) and C(losed) conformations. Differences in ligand affinity in different shapes can have an impact on the O↔C "gating" equilibrium constant. The enthalpy, free energy, and entropy of gating change upon ligand binding by LGICs. In addition, electrophysiological experiments suggested the existence of one or several intermediate gating states between O and C, but their structural relevance and the mechanisms that stimulate energy flow between binding sites and gates are unclear. Therefore, it is necessary to explore the thermodynamic cycle of ligand binding and channel gating to provide important information for elucidating the isomerization mechanism of LGICs.

Fig. 1 The ligand binding site of AChBP with carbamylcholine (CCh). (Auerbach, 2013)

Our Services

We have successfully helped our clients analyze the thermodynamics of the allosteric process in mouse neuromuscular acetylcholine receptors (AChRs) using a variety of methods such as advanced electrophysiological techniques. Building on our previous success, our services can be extended to the energetic analysis of a variety of LGICs. Our services include but not limited to:

• Determination of the free energy differences between the ground states of the isomerization using single-channel electrophysiology, including ΔG₀ (G_O-G_C; unliganded gating) and ΔG₂ (G_A2O-G_A2C; diliganded gating).
• Measurement of the change of ΔG₂, ΔG₀, ΔG_B (mean free energy resulting from the change in affinity for the agonist at each binding site) and equilibrium dissociation constant (K_d) to analyze the influence of mutations on gating.
• Measurement of the intrinsic gating free energy and free energy from the ligand.
• Analysis of temperature dependence of LGICs gating.
• Analysis of intermediate gating states by single-channel currents detection, open current analysis, and rate-equilibrium analysis.
• Site-gate energy transfer analysis.

Applications

We are committed to providing our clients with thermodynamic analysis of LGICs, which can be applied to the following research:

• Study on the mechanism of the actual work of the gating isomerization
• Research on the properties of the gated transition state energy landscapes

Advantages

• Ph.D. level experienced technicians
• Patient, open and respectful communication
• Powerful and advanced data analysis tools

In the increasingly competitive global market, Creative Bioarray takes the pursuit of excellence as our winning philosophy. We are committed to providing our clients with high-quality and reliable scientific services to achieve sound growth. If you are interested in our services of thermodynamic analysis, please contact us for more details.

Reference

1. Auerbach, A. The energy and work of a ligand-gated ion channel. Journal of molecular biology, 2013, 425(9): 1461-1475.