The acquisition of cold sensitivity during TRPM8 ion channel evolution

Abstract

To cope with temperature fluctuations, molecular thermosensors in animals play a pivotal role in accurately sensing ambient temperature. Transient receptor potential melastatin 8 (TRPM8) is the most established cold sensor. In order to understand how the evolutionary forces bestowed TRPM8 with cold sensitivity, insights into both emergence of cold sensing during evolution and the thermodynamic basis of cold activation are needed. Here, we show that the trpm8 gene evolved by forming and regulating two domains (MHR1-3 and pore domains), thus determining distinct cold-sensitive properties among vertebrate TRPM8 orthologs. The young trpm8 gene without function can be observed in the closest living relatives of tetrapods (lobe-finned fishes), while the mature MHR1-3 domain with independent cold sensitivity has formed in TRPM8s of amphibians and reptiles to enable channel activation by cold. Furthermore, positive selection in the TRPM8 pore domain that tuned the efficacy of cold activation appeared late among more advanced terrestrial tetrapods. Interestingly, the mature MHR1-3 domain is necessary for the regulatory mechanism of the pore domain in TRPM8 cold activation. Our results reveal the domain-based evolution for TRPM8 functions and suggest that the acquisition of cold sensitivity in TRPM8 facilitated terrestrial adaptation during the water-to-land transition.

Keywords: MHR1-3 domain; TRPM8; cold sensitivity; water-to-land transition.

Fig. 1.

The functional origination of trpm8 genes. (A) To show the skeletal reconstruction (from pectoral fin to forelimb) of representative animals in water-to-land transition, bone and cartilage were stained in red (Alizarin red) and blue (Alcian blue), respectively. The location of trpm2 (black), trpm2-like (yellow), and trpm8 (red) genes on one of their chromosomes is indicated by solid arrows (Left). Phylogenetic relationships are shown among fishes, amphibians, and reptiles (Right). The presence or absence of trpm2 (black), trpm2-like (yellow), and trpm8 (red) genes is given. (B) Comparison of trpm2, trpm8, and trpm2-like gene structures in P. annectens. Solid boxes represent exons. Homologous exons are aligned and colored in gray. By comparing these exons of the three genes, the exons without sequence similarity are colored in black (trpm2), red (trpm8), or yellow (trpm2-like). (C) Representative temperature-driven activation of TRPM8 orthologs. The cold-activated currents were normalized to saturating menthol-induced activation at room temperature. (D) The maximum cold-induced activation of TRPM8 orthologs was normalized to the current amplitude induced by saturating menthol. Data are given as average ± SEM, n = 3. n.d., no data for the TRPM8 orthologs without menthol sensitivity; N.S., no significance. (E) Phylogenetic relationships among P. annectens (Pa), Geotrypetes seraphini (Gs), R. bivittatum (Rb), X. tropicalis (Xt), and C. mydas (Cm) (Top). The species highlighted in red represent the trpm8 gene under significant positive selection. Positive selection sites on TRPM8 are shown as dots in a structural diagram (Bottom). Dots within the N-terminal of TRPM8 are shown in red. (F) Representative TRPM8 currents activated by cold bathing solution (8 °C). Rb_Xt_(N) represents RbTRPM8 N-terminal substituted by the homologous region of XtTRPM8, and vice versa [Xt_Rb_(N)]. (G) Variance versus average current plot from TRPM8 current traces activated at different temperatures. The open probability was determined as the ratio between the macroscopic current (after correcting for temperature-dependent single-channel conductance) and the maximum current estimated using noise analysis.

Fig. 2.

The evolved MHR1-3 domain bestows TRPM8 orthologs with cold sensitivity. (A) Representative TRPM8 currents activated by 5 mM menthol. Rb, R. bivittatum; Cm, C. mydas. (B) Variance versus average current plots from TRPM8 currents activated by menthol at different concentrations. Open probability was determined as the ratio between the macroscopic current and the maximum current estimated using noise analysis. (C) Schematic representation of the chimeras (using CmTRPM8 amino acid number) between CmTRPM8 (gray) and XtTRPM8 (red). The responses of chimeras to cold and menthol are shown (n.d., no data for the channels without menthol sensitivity). (D–F) Representative temperature-driven activation of wild-type TRPM8 orthologs and chimeric channels. The chimeric channels are named X1_X2. X represents the species-specific TRPM8 orthologs. X1_X2_(M) represents the chimeric channel that the MHR1-3 domain of X1TRPM8 was substituted by the homologous region of X2TRPM8. The cold-activated currents were normalized to saturating menthol-induced activation. (G) Summary of the maximum cold response in wild-type TRPM8 orthologs and the chimeric channels (average ± SEM; n = 3; N.S., no significance).

Fig. 3.

The MHR1-3 domain exhibits conformational changes upon cooling. (A) Summary of the shifts in emission peak of ANAP mutants in each domain (average ± SEM; n = 3). The columns colored in red represent the ANAP emission peak shifted significantly upon cooling (larger than 4 nm). AP represents the ANAP substitution. (B) Gel filtration chromatography of the MHR1-3 domain of XtTRPM8 (Top) or CmTRPM8 (Bottom). The temperatures of the column are given. Glycine was used as a control. (C) Circular dichroism (CD) spectra of the MHR1-3 domain of XtTRPM8 (Top) or CmTRPM8 (Bottom) at cold (10 °C) and moderate temperature (30 °C), respectively. (D) Representative currents and the ANAP emission peak induced by a temperature ramp were recorded from HEK293 cells expressing ANAP-incorporated XtTRPM8. (E) Summary of the shifts in emission peak of ANAP mutants in full-length XtTRPM8 (average ± SEM; n = 3). (F) Structural alignment of the XtTRPM8 model in the close state (gray) and cold-activated state (cyan). The pore region (S5–S6 segment) and MHR1-3 domain exhibited discernible conformational rearrangements between these two states. The key sites with the shifts in emission peak of ANAP are shown in these structures.

Fig. 4.

The evolved MHR1-3 domain is required for pore domain function in tuning cold activation. (A) Phylogenetic relationship of TRPM8 orthologs with cold activation. The species-specific TRPM8s under significant positive selection are shown in red. (B) Positive selection sites of TRPM8 observed in Camelus bactrianus and Rattus norvegicus were mapped on a structural diagram of TRPM8. The residue 915 (in CbTRPM8 number) is highlighted in red. (C) Normalized maximum cold-induced currents of CmTRPM8 and the chimeric channels (average ± SEM; n = 3). The chimeric channels are named as X1_X2. X represents the species-specific TRPM8. X1_X2_(p) means the chimeric channel that the pore domain of X1TRPM8 was substituted by the homologous region of X2TRPM8. X1_X2_(M)_X3_(p) means that the MHR1-3 and pore domain of X1TRPM8 were substituted by the MHR1-3 domain of X2TRPM8 and the pore domain of X3TRPM8, respectively. The chimeric channels based on CmTRPM8 were constructed by homologous recombination using the Xt(_M) segment (1 to 540, in XtTRPM8 number) and/or the Cb(_P) segment (856 to 976, in CbTRPM8 number). (D) Representative currents of chimeric TRPM8 channels activated by cold bathing solution (8 °C) and 5 mM menthol. (E) Representative temperature-driven activation of CmTRPM8 point mutations (residue 906, in CmTRPM8 number) with or without the MHR1-3 domain of XtTRPM8. The cold-activated currents were normalized to saturating menthol-induced activation (Left). Temperature threshold of the chimeric channels is shown with the MHR1-3 domain of XtTRPM8 (Middle). Q₁₀ value of the chimeric channels is shown (Right). Data are given as average ± SEM, n = 3, *P < 0.01. N.S., no significance. (F) Representative temperature-driven activation of CbTRPM8 and chimeric channels (Left). Temperature threshold (Middle) and Q₁₀ value (Right) of CbTRPM8 and chimeric channels. Data are given as average ± SEM, n = 3, *P < 0.01. (G) The summary of TRPM8 functional evolution. The cryo-electron microscopy structure of TRPM8 (PDB: 6O77) was used (38).