ABSTRACT
Canonical 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 (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors 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. CaM 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 CaM-induced regulatory mechanism of canonical TRP channels.
Subject(s)
Calmodulin/metabolism , Membrane Transport Modulators/pharmacology , Pyridazines/pharmacology , TRPC Cation Channels/drug effects , Zebrafish Proteins/drug effects , Animals , Binding Sites , Calmodulin/chemistry , Calmodulin/genetics , HEK293 Cells , Humans , Ligands , Membrane Potentials , Membrane Transport Modulators/chemistry , Membrane Transport Modulators/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Pyridazines/chemistry , Pyridazines/metabolism , Sf9 Cells , Structure-Activity Relationship , TRPC Cation Channels/chemistry , TRPC Cation Channels/genetics , TRPC Cation Channels/metabolism , Xenopus , Zebrafish Proteins/chemistry , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
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 Å. 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.