Please use this identifier to cite or link to this item: http://dr.iiserpune.ac.in:8080/xmlui/handle/123456789/4388
Title: The mechanism of activation of the TRPV1 ion channel by the double-knot spider toxin
Authors: KALIA, JEET
SARKAR, DEBAYAN
Dept. of Biology
20143323
Keywords: TRPV1 ion channel
Double-knot toxin
protein-lipid interface
membrane partitioning
2020
Issue Date: Jan-2020
Abstract: The transient receptor potential vanilloid 1 (TRPV1) ion channel is activated by a diverse range of stimuli including noxious heat and pain-inducing agents. The molecular mechanisms underlying this polymodal channel activation are poorly understood. The recently solved cryo-electron microscopy structure of TRPV1 bound to its potent agonist, the double-knot toxin (DkTx), in a native membrane bilayer environment provides an ideal blueprint for investigating these mechanisms. To interrogate the functional roles of toxin-membrane and toxin-channel interfaces uncovered by this structure, I generated a series of site-directed toxin variants and studied their TRPV1-activation properties by employing two-electrode voltage clamp electrophysiology. Together with membrane partitioning experiments, these studies revealed that toxin-membrane interfaces play profound roles in channel activation. Additionally, I explored the functional role of the seven amino acids-long linker of DkTx that connects its two channel-binding motifs by characterizing the TRPV1-activating and membrane partitioning properties of toxin variants possessing altered linker lengths and rigidity. These studies revealed that in addition to imparting bivalency, the linker plays an important role in fine-tuning the membrane-partitioning propensity of the toxin. Over the course of performing these studies, a requirement for a robust fluorescence-based quantitative assay for characterizing the binding of the toxin to cellular membranes was perceived. Towards this goal, I attempted several approaches for fluorescently labelling DkTx without disrupting its function and was successful in doing so by utilizing the sortase enzyme-based bioconjugation technology. Taken together, my studies on the DkTx-TRPV1 system reveal that the membrane can play a dominant role in the function of membrane-embedded protein-protein complexes and set the stage for utilizing DkTx as a powerful tool for studying protein-lipid interactions.
URI: http://dr.iiserpune.ac.in:8080/xmlui/handle/123456789/4388
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