Vinayagam, D. | Search bioRxiv

bioRxiv2024

Ideal efficacy photoswitches for TRPC4/5 channels harness high potency for spatiotemporally-resolved control of TRPC function in live tissues

Directly probing the endogenous biological roles of target proteins with high spatial and temporal resolution, as non-invasively and reproducibly as possible, is a shared conceptual goal for research across many fields, as well as for targeted therapies. Here we describe the rational conceptual design and test-case practical implementation of a photopharmacological paradigm to empower high-performance photomodulation studies in vivo. TRPC4/5 ion channels are involved in many spatiotemporally resolved circuits, from pain and anxiety, to reproductive signaling, digestion, and obesity. To unpick their biology requires spatiotemporally precise tools, which were lacking. We developed "ideal efficacy photoswitch" ligands to control their diverse functions in situ. These E{leftrightarrows}Z-photoswitchable ligands bias TRPC[4]/5 channel activity with exquisite photocontrol, from strong agonism under 360 nm, to low agonism at 385 nm, to strong antagonism at 410-460 nm. Cryo-EM structures of both TRPC4 and TRPC5 with both Z-agonists and E-antagonists support the rationale for efficacy switching through competitive E/Z isomer binding. Crucially, since the E/Z ratio is exclusively determined by the light wavelength applied, their channel photocontrol is exclusively wavelength-dependent, yet drug-concentration-independent: so is reproducible from cell culture to >millimetre-depth tissues. Indeed, we were able to photocontrol both direct and downstream TRPC4/5 biology in cell lines or primary cells in culture, from calcium flux, to primary neuron excitability and adrenaline release; and even in tissues, photoswitching small intestine motility and peristalsis. The TRPC4/5 ligands we develop will thus unlock a range of high-precision investigations in TRP biology. More broadly, we propose that the success of this efficacy photoswitch program, from concept to tissue level translation, is mainly a consequence of how biology has evolved proteins for efficacy control. We therefore foresee that a variety of functionally responsive protein targets, not only sensory and signaling ion channels and receptors, will be amenable to similarly high-performance photocontrol even in vivo, if a new generation of reagent development adopts this paradigm of ideal efficacy photoswitching. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/602451v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1f8e527org.highwire.dtl.DTLVardef@18c72d0org.highwire.dtl.DTLVardef@1c5d648org.highwire.dtl.DTLVardef@177203d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Structural basis of TRPC4 regulation by calmodulin and pharmacological agents

ABSTRACTCanonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin. Molecular details of both calmodulin and drug binding have remained elusive so far. Here we report structures of TRPC4 in complex with a pyridazinone-based inhibitor and a pyridazinone-based activator and calmodulin. The structures reveal that both activator and inhibitor bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand binding site can be transmitted to the central ion-conducting pore of TRPC4. Calmodulin binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel calmodulin-induced regulatory mechanism of canonical TRP channels.Competing Interest StatementThe authors declare no competing interests. M.Y., M.W.L, and G.M. are or were shareholders of Goldfinch Bio.View Full Text

biochemistry↗

bioRxiv2018

Electron cryo-microscopy structure of the canonical TRPC4 ion channel

Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 [A]. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.

biochemistry↗