Different ligands of the TRPV3 cation channel cause distinct conformational changes as revealed by intrinsic tryptophan fluorescence quenching

Abstract

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.

Keywords: camphor; fluorescence; human TRPV3; icilin; membrane protein; membrane reconstitution; protein purification; quenching; transient receptor potential channels (TRP channels).

FIGURE 1.

Expression and detergent screen of human TRPV3. A, Sf9 cells infected with recombinant baculovirus (top left), exposed to blue light to monitor GFP fluorescence (top right), and in the absence and presence of 10 mm camphor during a calcium imaging experiment (bottom left and right, respectively). B, calcium imaging experiment, showing the response of Sf9 cells expressing hTRPV3-GFP (red) and control (black) to 10 mm camphor. The dashed lines represent mean ± S.E. (n = 52). C, detergent screen of hTRPV3. The graph shows a superposition of FSEC profiles from detergent-solubilized hTRPV3-GFP. Comparison of peak amplitude and symmetry between different detergents reveals the superior extraction efficiency and stability of hTRPV3-GFP in DDM. OG, lauryl maltose neopentyl glycol; LMNG, n-octyl-β-d-glucopyranoside.

FIGURE 2.

Purification of human TRPV3. A, FSEC profile of solubilized Sf9 membranes expressing hTRPV3-GFP (analytical sample taken before incubation with GFP trap). mFU, millifluorescence units. B, FSEC profile of analytical sample from the flow-through from the same GFP trap. C, UV detection SEC profile of hTRPV3 after elution from the GFP trap. Inset, Coomassie-stained SDS gel and Western blot (WB) of the hTRPV3 peak fraction, carried out with goat polyclonal anti-TRPV3 IgG. mAU, milliabsorbance units.

FIGURE 3.

Thermostability-based FSEC screening for hTRPV3-stabilizing additives. A, melting curve of hTRPV3-GFP, yielding a melting temperature (i.e. the temperature at half-maximal fluorescence amplitude) of 36.2 ± 1.0 °C. Relative fluorescence is calculated from oligomeric peak amplitudes in consecutive FSEC runs with hTRPV3-GFP samples, preheated at different temperatures. Data represent mean ± S.E. (error bars) (n = 3). Inset, overlay of the oligomeric peak FSEC profiles. B, bar diagram shows relative fluorescence of oligomeric peak amplitudes from consecutive FSEC runs with a selection of additives tested for possible stabilizing effects on hTRPV3-GFP. Black and gray bars represent relative FSEC peak amplitudes of hTRPV3-GFP without additives, incubated at 4 and 40 °C, respectively (with control 4 °C set as 1). Blue bars, relative peak amplitudes of hTRPV3-GFP samples supplemented with various additives before incubation at 40 °C.

FIGURE 4.

Functional reconstitution of purified hTRPV3. A, representative current recording from a planar lipid bilayer containing a high number of hTRPV3 channels during a ramp voltage protocol from −100 to 100 mV. Arrows, traces before (control) and after the addition of 100 μm 2-APB. Dashed line, zero current level. B, representative current traces of single-channel activity recorded from a planar lipid bilayer clamped at 50 and 100 mV after the addition of 100 μm 2-APB (left). The closed channel current level is indicated by dashed lines. Shown are corresponding current amplitude histograms (right), yielding a single-channel conductance of 174 pS. C, representative current trace from a lipid bilayer containing multiple hTRPV3 channels recorded at 100 mV. The channels were activated by 100 μm 2-APB and subsequently inhibited by 10 μm ruthenium red. The arrow indicates where ruthenium red was added. D, representative current traces of single-channel activity recorded at 100 mV in the presence of 100 μm 2-APB, 200 μm menthol, 500 μm camphor, or 500 μm eucalyptol. The bottom trace shows inhibition of 2-APB-activated current by 10 μm icilin. The arrow indicates where icilin was added. E, scatter plot showing open probability (P_o) versus applied concentration of the tested agonists. Error bars, S.E.

FIGURE 5.

Quenching of intrinsic Trp fluorescence by TRPV3 ligands. The graphs show averaged fluorescence emission spectra (n = 3) of hTRPV3 in the presence of increasing concentrations 2-APB (A), menthol (B), camphor (C), and icilin (D). E, quenching plots of hTRPV3 and lysozyme in the presence of camphor, eucalyptol, and icilin show that the hTRPV3 quenching by these compounds is specific. F, bar diagram compares the quenching of wild type hTRPV3 with Trp mutants, recorded in the presence of a saturating concentration of camphor (10 mm) and icilin (1 mm). All data represent mean ± S.E. (error bars) (n = 3). None of the tested mutants exhibits a significant difference from wild type hTRPV3 in Q_max value (p > 0.05). mFU, millifluorescence units.

FIGURE 6.

Conservation and spatial location of Trp residues in TRPV channels. A, sequence alignment of the human thermosensitive TRPV channels. Rat TRPV1 (rTRPV1) is included for comparison with the cryo-EM structure in B. Trp residues in the transmembrane region of hTRPV3 are colored yellow, and homologous positions in rTRPV1 are shown in red. B, schematic representations of the rat TRPV1 cryo-EM structure (32), seen along the 4-fold symmetric axis (top left) and in a side view (bottom left). Ball-and-stick representations depict rTRPV1 residues, homologous to Trp residues in hTRPV3. Numbers corresponding to Trp residues in hTRPV3 are shown in yellow, and numbers corresponding to residues in homologous positions in rTRPV1 are shown in red.