International Journal of Molecular Sciences Article Cytoplasmic Inter-Subunit Interface Controls Use-Dependence of Thermal Activation of TRPV3 Channel Lucie Macikova 1,2 , Lenka Vyklicka 1, Ivan Barvik 3, Alexander I. Sobolevsky 4 and Viktorie Vlachova 1,* 1 Department of Cellular Neurophysiology, Institute of Physiology Czech Academy of Sciences, 142 20 Prague, Czech Republic 2 Department of Physiology, Faculty of Science, Charles University, 128 00 Prague, Czech Republic 3 Division of Biomolecular Physics, Institute of Physics, Faculty of Mathematics and Physics, Charles University, 121 16 Prague, Czech Republic 4 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA * Correspondence: [email protected]; Tel.: +420-296-442-711 Received: 23 July 2019; Accepted: 13 August 2019; Published: 16 August 2019 Abstract: The vanilloid transient receptor potential channel TRPV3 is a putative molecular thermosensor widely considered to be involved in cutaneous sensation, skin homeostasis, nociception, and pruritus. Repeated stimulation of TRPV3 by high temperatures above 50 ◦C progressively increases its responses and shifts the activation threshold to physiological temperatures. This use-dependence does not occur in the related heat-sensitive TRPV1 channel in which responses decrease, and the activation threshold is retained above 40 ◦C during activations. By combining structure-based mutagenesis, electrophysiology, and molecular modeling, we showed that chimeric replacement of the residues from the TRPV3 cytoplasmic inter-subunit interface (N251–E257) with the homologous residues of TRPV1 resulted in channels that, similarly to TRPV1, exhibited a lowered thermal threshold, were sensitized, and failed to close completely after intense stimulation. Crosslinking of this interface by the engineered disulfide bridge between substituted cysteines F259C and V385C (or, to a lesser extent, Y382C) locked the channel in an open state. On the other hand, mutation of a single residue within this region (E736) resulted in heat resistant channels. We propose that alterations in the cytoplasmic inter-subunit interface produce shifts in the channel gating equilibrium and that this domain is critical for the use-dependence of the heat sensitivity of TRPV3. Keywords: transient receptor potential; transient receptor potential vanilloid 1 (TRPV1); noxious heat; ankyrin repeat; nociception 1. Introduction Transient receptor potential vanilloid 3 (TRPV3) is an ion channel implicated in the regulation of skin homeostasis, thermo-sensing, and nociception [1–5]. Its temperature sensitivity is strongly use-dependent which means that the activation threshold and the steepness of temperature responses progressively and irreversibly decrease as the channel is repeatedly activated [4,6–8]. This enables TRPV3 to change from a transducer of extremely noxious heat (>50 ◦C) to a non-noxious mild warmth sensing detector (<33 ◦C). Previous studies have identified several sites in temperature-sensitive vanilloid TRP channels from which the thermal threshold and the steepness of temperature dependence can be affected [9–17]. Two transferable mutually exclusive regions have been proposed, each as a bona fide protein domain conferring the characteristic steep temperature dependence. The first is the pore domain identified in TRPV1 that is capable of conferring specific heat sensitivity when transplanted into the Shaker Kv channel [16]. The second region responsible for the strong temperature dependence Int. J. Mol. Sci. 2019, 20, 3990; doi:10.3390/ijms20163990 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 3990 2 of 18 of all temperature-sensitive vanilloid family homologues is the membrane proximal domain (MPD) connecting the N-terminal ankyrin repeat domain (ARD) to the first transmembrane segment S1 [18]. An insertion of a single specific amino acid residue into the MPD linker region (V408–L422) results in a less temperature stable conformation of TRPV3 [8]. While these previous studies have seemingly narrowed down the discrete domains mediating temperature sensitivity, heat responses can also be dramatically affected by as little as a single residue mutation in the N-terminal ARD remotely located Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 18 from the two aforementioned regions [17]. Clearly, there is still no definitive answer as to whether thermally activatedfor the strong channels temperature use dependence a local, domain-based,of all temperature-sensitive mechanism vanilloid forfamily sensing homologues the is temperature, or whether athe global membrane response, proximal distributed domain (MPD) over connecting the channel the N-terminal structure, ankyrin is underlyingrepeat domain their(ARD) strongto thermal the first transmembrane segment S1 [18]. An insertion of a single specific amino acid residue into the sensitivity [19MPD–22 ].linker region (V408–L422) results in a less temperature stable conformation of TRPV3 [8]. The crystalWhile structure these previous of the studies cytoplasmic have seemingly N-terminal narrowed ARDdown oftheTRPV3 discrete isolateddomains mediating from the rest of the protein exhibitstemperature a stable sensitivity, conformation heat responses of a loopcan also between be dramatically ankyrin affected repeats by as 3 andlittle as 4, calleda single finger 3 [23] residue mutation in the N-terminal ARD remotely located from the two aforementioned regions (Figure1A–C).[17]. Whereas Clearly, there the is still finger no definitive 3 is a answer flexible as toregion whether thermally in a majority activated ofchannels vanilloid use a local, receptor ARD structures, indomain-based, TRPV3-ARD mechanism (Protein for Datasensing Bank the temperature, Code, PDB or whether ID, 4N5Q) a global it response, bends over distributed the inner helices of the ankyrinover repeats the channel 3 andstructure, 4 and is underlying is stabilized their strong by hydrogen thermal sensitivity bonds [19–22]. and hydrophobic packing [23], The crystal structure of the cytoplasmic N-terminal ARD of TRPV3 isolated from the rest of the indicating itsprotein unique exhibits structural a stable roleconformation in TRPV3. of a loop An between increasing ankyrin number repeats 3 of and structures 4, called finger of the3 [23] vanilloid TRP channels show(Figure that 1A–C). the region Whereas around the finger the 3 fingeris a flexible 3 forms region a in key a majority interface of vanilloid between receptor the ARD ARD and MPD of the adjacentstructures, subunit in (Figure TRPV3-ARD1C). (Protein The newest Data Bank cryo-electron Code, PDB ID, microscopic4N5Q) it bends over structures the inner helices of the full length of the ankyrin repeats 3 and 4 and is stabilized by hydrogen bonds and hydrophobic packing [23], TRPV3 capturedindicating in various its unique states structural along role the in TRPV3. activation An increasing cycle reveal number pronounced of structures ofinter-subunit the vanilloid interfaces formed by theTRP 3-stranded channels showβ thatsheet the region and thearound C-terminal the finger 3 loopforms ofa key one interface subunit between and the the ARD ARD and repeats 2–5, including fingerMPD 3,of the of adjacent the adjacent subunit subunit(Figure 1C). [ 24The,25 newest]. Since cryo-electron the interface-forming microscopic structures domains of the full are critically length TRPV3 captured in various states along the activation cycle reveal pronounced inter-subunit involved in temperatureinterfaces formed sensitivity by the 3-stranded of TRPV3 β sheet and and itsthe closeC-terminal relatives loop of[ one10, 17subunit,18,26 and,27 the], weARD reasoned that the unique propertiesrepeats 2–5, including of finger finger 3 might3, of the beadjacent important subunit [24,25]. for setting Since the the interface-forming thermal threshold domains for TRPV3 activation. Wearewere critically curious involved whether in temperature substitutions sensitivity of in TRPV3 finger and 3 its with close the relatives homologous [10,17,18,26,27], residues we of TRPV1 reasoned that the unique properties of finger 3 might be important for setting the thermal threshold can change thefor thermalTRPV3 activation. threshold We were of TRPV3 curious towhether resemble substitutions the one in offinger TRPV1. 3 with the homologous residues of TRPV1 can change the thermal threshold of TRPV3 to resemble the one of TRPV1. Figure 1. SchematicFigure 1. Schematic representation representation of the of the finger finger3 3 loop region region of transient of transient receptor receptor potential vanilloid potential vanilloid 3-ankyrin repeat3-ankyrin domain repeat (TRPV3-ARD).domain (TRPV3-ARD). (A ()A Sequence) Sequence logo logo along along thermally thermally activated activated TRPV channels TRPV channels (fish, frog, snake, chicken, mouse, rat, and human TRPV1-4) and amino acid alignment of human (fish, frog, snake,TRPV3, chicken,rat TRPV2, mouse,and rat TRPV1 rat, with and elements human of TRPV1-4)secondary structure and amino indicated acid above. alignment Positions of human TRPV3, rat TRPV2,are numbered and according rat TRPV1 to human with TRPV3 elements (above) ofand secondary rat TRPV1 (below). structure The residues indicated of the tip above. of Positions are numberedthe according finger 3 are indicated to human in colors. TRPV3 (B) Residues (above) within and the rat finger TRPV1 3 mutated (below). in this Thestudy residues shown in of the tip of the context of ankyrin repeats 3 and 4 (AR3 and AR4) of the