Mapping Conformational Dynamics of the 5-HT3 Serotonin-Gated Ion Channel Using Voltage-Clamp Fluorometry

Core Concepts

Voltage-clamp fluorometry reveals distinct conformational states of the 5-HT3 serotonin receptor, including resting, inhibited, pre-active, and active states, which are differentially stabilized by agonists, partial agonists, and antagonists.

Abstract

The study used voltage-clamp fluorometry (VCF) to simultaneously monitor conformational changes and channel opening of the 5-HT3 serotonin-gated ion channel expressed in Xenopus oocytes. Four fluorescent sensors were engineered at different locations within the extracellular domain (ECD) to report local and global conformational rearrangements.
Key findings:

Competitive antagonists elicit agonist-like reorganizations at the orthosteric site, but these conformational effects do not spread to the vestibular and ECD-TMD interface regions.
Strong agonists promote a concerted motion of the whole protein during activation, with correlated changes in fluorescence and current.
Partial agonists and loss-of-function mutations stabilize intermediate "pre-active" conformations characterized by robust fluorescence changes but no channel opening.
The fluorescence signatures of the different sensors match well with known high-resolution cryo-EM structures, allowing functional annotation of these static structures.
The data provide insights into the allosteric mechanisms governing 5-HT3 receptor activation and reveal a common gating mechanism among pentameric ligand-gated ion channels involving structurally dynamic intermediate states.

Stats

The EC50 values for current (EC50c) and fluorescence (EC50f) responses to agonists (5-HT, mCPBG, varenicline) at labeled and unlabeled m5-HT3A mutants are provided in Table 2.

Quotes

"VCF data highlight a progressive propagation of the signal following ligand binding. Setrons elicit local reorganizations shown by the sensors located around the orthosteric site, partial agonists elicit local reorganizations at the four sensors indicating a motion of the whole ECD with partial pore opening, and strong agonists elicit reorganizations detected by all four sensors together with channel opening."
"VCF data thus identify four families of conformations endowed with distinct ΔI/ΔF signatures contributing to signal transduction: resting-like apo, setron-inhibited, partial agonist-elicited pre-active and active states."

Key Insights Distilled From

Mapping the molecular motions of 5-HT3 serotonin-gated channel by Voltage-Clamp Fluorometry

by Peverini,L.,... at www.biorxiv.org 09-28-2023

https://www.biorxiv.org/content/10.1101/2023.09.28.559960v2

Deeper Inquiries

How do the conformational dynamics of the 5-HT3 receptor revealed by VCF relate to its physiological functions in the gut-brain axis and potential involvement in neurological disorders

The conformational dynamics of the 5-HT3 receptor revealed by Voltage-Clamp Fluorometry (VCF) provide valuable insights into its physiological functions in the gut-brain axis and potential involvement in neurological disorders. The 5-HT3 receptor is a cation-selective excitatory channel expressed in both the central and peripheral nervous systems, playing a crucial role in neuronal communication. The receptor is involved in various neurological disorders such as schizophrenia, Parkinson's disease, and depression. By monitoring the receptor's conformational changes simultaneously with channel opening, VCF allows for the identification of different families of conformations that contribute to signal transduction. These conformations include "resting-like" without ligand, "inhibited-like" with setrons, "pre-active-like" with partial agonists, and "active-like" with strong agonists. Understanding these conformational changes is essential for elucidating the allosteric mechanisms that contribute to signal transduction in the gut-brain axis and in neurological disorders. The data from VCF can help in annotating high-resolution structures to physiologically relevant states, providing a better understanding of the receptor's function in health and disease.

What are the structural determinants that allow partial agonists to selectively stabilize the pre-active conformations of the 5-HT3 receptor

Partial agonists selectively stabilize the pre-active conformations of the 5-HT3 receptor through specific structural determinants. In the study, it was observed that partial agonists promote distinct intermediate conformations characterized by fluorescence variations but no current, especially when loss-of-function mutations are engineered. These intermediate conformations are partially populated for partial agonists, indicating a stabilization of both active and pre-active conformations. The structural determinants that allow partial agonists to selectively stabilize these pre-active conformations involve specific interactions with the receptor at the orthosteric site and other regions within the receptor's structure. By binding to the receptor, partial agonists induce conformational changes that lead to the activation of the receptor while maintaining it in a pre-active state. This selective stabilization of pre-active conformations by partial agonists highlights the complex allosteric mechanisms involved in receptor activation and modulation.

Could the fluorescent sensors developed in this study be used to screen for novel 5-HT3 receptor modulators with therapeutic potential

The fluorescent sensors developed in this study could be valuable tools for screening novel 5-HT3 receptor modulators with therapeutic potential. These sensors provide a way to monitor conformational changes in the receptor in real-time, allowing for the identification of compounds that can induce specific structural rearrangements associated with receptor activation or inhibition. By using these sensors in screening assays, researchers can assess the effects of potential modulators on the receptor's conformational dynamics and functional activity. This approach can help in identifying novel compounds that target the 5-HT3 receptor with high specificity and efficacy, paving the way for the development of new therapeutic agents for conditions related to the gut-brain axis and neurological disorders.