Dissection of the components for PIP2 activation and thermosensation in TRP channels

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in the activation of several transient receptor potential (TRP) channels. The role of PIP2 on temperature gating of thermoTRP channels has not been explored in detail, and the process of temperature activation is largely unexplained. In this work, we have exchanged different segments of the C-terminal region between cold-sensitive (TRPM8) and heat-sensitive (TRPV1) channels, trying to understand the role of the segment in PIP2 and temperature activation. A chimera in which the proximal part of the C-terminal of TRPV1 replaces an equivalent section of TRPM8 C-terminal is activated by PIP2 and confers the phenotype of heat activation. PIP2, but not temperature sensitivity, disappears when positively charged residues contained in the exchanged region are neutralized. Shortening the exchanged segment to a length of 11 aa produces voltage-dependent and temperature-insensitive channels. Our findings suggest the existence of different activation domains for temperature, PIP2, and voltage. We provide an interpretation for channel-PIP2 interaction using a full-atom molecular model of TRPV1 and PIP2 docking analysis.

Fig. 1.

PIP₂ effect is conserved among thermoTRP channel chimeras. (A) Schematic alignments between rTRPV1 and rTRPM8. The cut–paste limit for chimera construction is marked by different colors: blue corresponds to TRPM8 original sequence, and red corresponds to TRPV1 swapped sequence. Important features are highlighted in the scheme: the TRP domain, the TRP-box, TRPM8 charges R998 and R1008 are those involved in PIP₂ sensitivity. These charges are conserved in TRPV1 (R701 and K710). (B, D, and F) Representative whole-cell current recordings at two different temperatures from cells expressing TRPM8 (686-752 V1) chimera and the mutants TRPM8 (686-752 V1/K710A) and TRPM8 (686-752 V1/R701A), respectively. See Methods for the voltage protocol. (C, E, and G) Plots showing the whole-cell current as a function of voltage at the indicated temperatures for the chimeras TRPM8 (686-752 V1), TRPM8 (686-752 V1/K710A), and TRPM8 (686-752 V1/R701A), respectively. (H) PIP₂ Dose–response curve for WT TRPM8, TRPM8 (686-752 V1) chimera, and the mutants TRPM8 (686-752 V1/K710A) and TRPM8 (686-752 V1/R701A). Curves were fitted to a Hill equation (solid lines). A Hill coefficient of 1.2 was obtained for WT TRPM8 and TRPM8 (686-752 V1) chimera. Each point represents an average of at least four different experiments. Error bars indicate SE.

Fig. 2.

A small region inside the C-terminal tail of TRPV1 confers heat sensitivity. (A and B) Schematic alignments between rTRPV1 and rTRPM8. The cut–paste limit for chimera construction is marked by different colors, and the corresponding amino acid number for each sequence boundaries is highlighted. (C and E) Representative whole-cell recordings of cells expressing TRPM8 (727-752 V1) and TRPM8 (741-752 V1) chimeras, respectively. Cells were exposed to different temperatures to compare their heat responsiveness. See Methods for the voltage protocol. (D) Whole-cell current as a function of voltage at the indicated temperatures for TRPM8 (727-752 V1) chimera. (F) Whole-cell current as a function of voltage at the indicated temperatures for TRPM8 (741-752 V1) chimera. This 11-aa chimera lacks the temperature responsiveness but retains voltage dependence.

Fig. 3.

Voltage-dependence, temperature-dependence and PIP₂ effect on chimeric channels. (A) Averaged G/G_max vs. V curves for chimeric channels. Solid lines correspond to the best fit to Boltzmann functions. Fit parameters are: V0.5 = 124.96 ± 2 mV, z = 0.83 [TRPM8(686-752V1)]; V0.5 = 137.22 ± 3 mV, z = 0.91 [TRPM8(686-752V1/K710A)]; V0.5 = 130.64 ± 2 mV, z = 0.86 [TRPM8(686-752V1/R701A)];V0.5 = 111.75 ± 2 mV, z = 0.77 [TRPM8(727-752V1)]; V0.5 = 113.04 ± 3 mV, z = 0.75 [TRPM8(741-752V1)]. Each curve represents the average of at least four different experiments performed at 22°C. (B) comparative Q₁₀ bar plot for the chimeric channels used in this work. Q₁₀ was obtained from the ratio of the ionic currents (I) obtained a two different temperatures, I_T/I_T+10°C at a fixed voltage. Each bar represents the average of at least four different experiments. Chimeras have a lower Q₁₀ (≈10) compared with wild-type TRPM8 (Q₁₀ = 23). TRPM8 (741-752 V1) chimera forms temperature-insensitive channels (Q₁₀ = 3). (C) Effect of PIP₂ (10 μM) and menthol (300 μM). Gray bars indicate menthol, and white bars indicate PIP₂ channel activation. Current records were obtained at +100 mV. Notice that 10 μM PIP₂ is unable to activate the neutralization chimeras. Each point represents an average of at least four different experiments. Error bars indicate SE.

Fig. 4.

Homology model for the TRPV1 channel reveals a PIP₂-binding site. (A) Side view of the solvated TRPV1–PIP₂-bound channel embedded into a POPC lipid bilayer. Three of the four channel-bound PIP₂ molecules in surface representation can be seen. Blue spheres are Na⁺, and green spheres are Cl⁻ ions (140 mM NaCl). Water molecules (TIP₃) are represented as the transparent red spheres conforming the background. (B) Ribbon diagram of the TRPV1 channel depicting one subunit in yellow and one bound PIP₂ molecule. The two positively (R701 and K710) charged amino acid residues involved in the apparent PIP₂ binding are shown in stick representation. Notice the cluster of positively charged residues contained in the proximal part of the C terminus. (C) Two channel subunits (purple and yellow) and one PIP₂ molecule are highlighted to describe the interactions between the aliphatic chains and the polar head of PIP₂ with the channel. The aliphatic chains of PIP₂ are making contact with the S6 and S5 transmembrane domains of one subunit [S6(A) and S5(A)] and with the S6 segment of the adjacent subunit [S6(B)]. (D) Structures defining the PIP₂ binding (α-helix comprising residues 696–722) and the channel temperature sensitivity (α-helix comprising residues 696–722). Residues 777–810 define the structure proposed as the PIP₂ inhibitory binding site (13).