Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Tuesday Speaker Abstracts

EMPLOYING THE DOUBLE-KNOT TOXIN-TRPV1 ION CHANNEL COMPLEX AS A MODEL SYSTEM TO INTERROGATE THE ROLES OF PROTEIN-MEMBRANE INTERACTIONS IN PROTEIN FUNCTION Jeet Kalia ; 1 Indian Institute of Science Education and Research (IISER) Bhopal, Biological Sciences, Bhopal, India Elucidating the roles of membrane lipids in the function of ion channel proteins is a long-cherished goal of ion channel biologists. These efforts have been tremendously facilitated by the recent success in obtaining high-resolution structures of ion channels complexed with membrane lipids. In this regard, the structure of the transient receptor potential vanilloid-1 (TRPV1) ion channel bound to its potent bi-lobed peptide agonist, the double-knot spider toxin (DkTx), is particularly insightful as it depicts the toxin, the channel, and membrane lipids forming an intimate tripartite complex. In my talk, I will describe how using this structure as a blueprint, we have investigated the role of the membrane in DkTx-activation of TRPV1. In one class of experiments, we generated a series of site-directed variants of DkTx at its membrane and channel-interacting sites and characterized their TRPV1-activation and membrane partitioning properties. These experiments demonstrated that the lipid-interacting residues of DkTx play a profound role in endowing it with both its high TRPV1-activating potency and its extremely slow wash-off rates, and that different toxin-membrane interfaces contribute in different ways to the mechanism of toxin-mediated channel activation. Additionally, experiments on a series of DkTx variants possessing altered lengths and rigidity of the peptide linker between the two lobes of the toxin revealed that the wild-type linker ensures optimal partitioning of the two lobes of the toxin into the membrane. I will also describe our studies that involved the creation of diverse DkTx-based toxin constructs possessing a range of valences and membrane affinities, that enables us to tease apart the contributions of the toxin’s bivalency and its high membrane affinity to its TRPV1-activation properties. Taken together, these studies establish that the membrane plays a crucial role in the activation of TRPV1 by DkTx.

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