Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Poster Abstracts

26-POS Board 7 DISTINCT OPEN STATES OF TRPV1 AND ENERGETICS OF RTX ACTIVATION Shisheng Li 1 ; Bo Hyun Lee 1 ; Jie Zheng 1 ; 1 UC Davis, Physiology and Membrane Biology, Davis, CA, USA TRPV1 is a major nociceptor for plant toxins such as capsaicin and resiniferatoxin (RTX), protons and heat. Activation of the multi-subunit TRPV1 channel is known to be allosteric. However, most ligands including capsaicin activate TRPV1 rapidly, making it difficult to capture transition states between the initial closed state and the fully liganded open state by functional or structural methods. RTX activates TRPV1 slowly but irreversibly, offering an opportunity to obtain a glimpse of the transition states from patch-clamp recordings. We found the Y512A mutation makes RTX binding reversible. Incorporating Y512A mutation into concatemers allowed fixing RTX binding in wildtype subunits and removing RTX from mutant subunits, hence gaining control of the exact number of bound RTX to investigate transition states from steady current recordings. Using this system, we showed that binding of RTX to each of the four subunits yields an equal stabilization energy, which we estimated to be 1.78 kcal/mol. We further confirmed that a different number of RTX binding changes only the open probability but not the single-channel conductance, as predicted by a classic allosteric model. However, using different ligands (capsaicin, 2-APB) or activation methods (heat or proton), we did observe distinct single- channel conductance levels as well as ion-ion interactions, suggesting the existence of distinct open states.

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