Biology subjects

Vlachova, V.

Publications and source records attributed to Vlachova, V..

2 recordsLinked to original sources

bioRxiv2025

(-)-Englerin A binds a conserved lipid site of TRPC5 and exposes a Met-aromatic motif in channel activation

TRPC4/5 cation channels are polymodal cellular sensors that play key roles in signal transduction/integration and have been implicated in various human pathologies, including anxiety, pain and cardiometabolic disease1-3. The plant natural product (-)-englerin A (EA)4 is a potent, selective TRPC4/5 agonist5,6 that has transformed fundamental and translational research on TRPC4/5 channels. However, the structural basis of interactions between EA and TRPC4/5 proteins has remained elusive, limiting our ability to fully understand and exploit mechanisms of TRPC4/5 channel activation by this intriguing natural product. Here, we present nine high-resolution cryo-EM structures (2.4-3.2 [A]) of human TRPC5 - representing different states and ligand occupancies - which show that EA binds to a conserved lipid binding site between transmembrane domains of adjacent TRPC5 subunits. Our structural models are consistent with the effects of mutagenesis of nearby residues on EAs potency, efficacy and activation kinetics, and allow us to rationalise competitive inhibition by other TRPC4/5 modulators as well as EAs selectivity profile within the TRPC family. Comparison of structures containing various TRPC5:EA stoichiometries revealed key structural and molecular determinants of EA-mediated TRPC5 activation - most notably the aromatic interaction network around Phe520 - underscoring the critical function of Met-aromatic motifs in ion channel structure and function. Binding of EA causes conformational changes of nearby amino acid residues, resulting in rearrangement of the pore helices into a pre-open state. Collectively, we provide structural insight into the mode-of-action of the most widely used TRPC4/5 agonist, which will underpin fundamental TRPC4/5 channel research as well as ongoing drug discovery programmes.

molecular biology↗

bioRxiv2021

The human TRPA1 intrinsic cold and heat sensitivity involves separate channel structures beyond the N-ARD domain

The human TRPA1 (hTRPA1) is an intrinsic thermosensitive ion channel responding to both cold and heat, depending on the redox environment. Here, we have studied purified hTRPA1 truncated proteins to gain further insight into the temperature gating of hTRPA1. We found in patch-clamp bilayer recordings that {Delta}1-688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas {Delta}1-854 hTRPA1 that is also lacking the S1-S4 voltage sensing-like domain (VSLD) gained sensitivity to cold but lost its heat sensitivity. The thiol reducing agent TCEP abolished the temperature sensitivity of both {Delta}1-688 hTRPA1 and {Delta}1-854 hTRPA1. Cold and heat activity of {Delta}1-688 hTRPA1 and {Delta}1-854 hTRPA1 were associated with different structural conformational changes as revealed by intrinsic tryptophan fluorescence measurements. Heat evoked major structural rearrangement of the VSLD as well as the C-terminus domain distal to the transmembrane pore domain S5-S6 (CTD), whereas cold only caused minor conformational changes. As shown for {Delta}1-854 hTRPA1, a sudden drop in tryptophan fluorescence occurred within 25-20{degrees}C indicating a transition between heat and cold conformations of the CTD, and thus it is proposed that the CTD contains a bidirectional temperature switch priming hTRPA1 for either cold or heat. In whole-cell patch clamp electrophysiology experiments, replacement of the cysteines 865, 1021 and 1025 with alanine modulated the cold sensitivity of hTRPA1 when heterologously expressed in HEK293T cells. It is proposed that the hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5-S6 pore region and the VSLD, respectively.

physiology↗