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. 2014 Jan;143(1):91-103.
doi: 10.1085/jgp.201311024. Epub 2013 Dec 16.

Divalent Cations Activate TRPV1 Through Promoting Conformational Change of the Extracellular Region

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Free PMC article

Divalent Cations Activate TRPV1 Through Promoting Conformational Change of the Extracellular Region

Fan Yang et al. J Gen Physiol. .
Free PMC article

Abstract

Divalent cations Mg(2+) and Ba(2+) selectively and directly potentiate transient receptor potential vanilloid type 1 heat activation by lowering the activation threshold into the room temperature range. We found that Mg(2+) potentiates channel activation only from the extracellular side; on the intracellular side, Mg(2+) inhibits channel current. By dividing the extracellularly accessible region of the channel protein into small segments and perturbing the structure of each segment with sequence replacement mutations, we observed that the S1-S2 linker, the S3-S4 linker, and the pore turret are all required for Mg(2+) potentiation. Sequence replacements at these regions substantially reduced or eliminated Mg(2+)-induced activation at room temperature while sparing capsaicin activation. Heat activation was affected by many, but not all, of these structural alternations. These observations indicate that extracellular linkers and the turret may interact with each other. Site-directed fluorescence resonance energy transfer measurements further revealed that, like heat, Mg(2+) also induces structural changes in the pore turret. Interestingly, turret movement induced by Mg(2+) precedes channel activation, suggesting that Mg(2+)-induced conformational change in the extracellular region most likely serves as the cause of channel activation instead of a coincidental or accommodating structural adjustment.

Figures

Figure 1.
Figure 1.
Schematic diagram illustrating the TRPV1 channel topology and highlighting regions previously indicated to be involved in heat and Mg2+ activation. Regions tested by mutations in this study are highlighted in purple.
Figure 2.
Figure 2.
Intracellular Mg2+ does not potentiate TRPV1. (A) Representative current traces from whole-cell recording at +80 mV with extracellularly applied Mg2+ and capsaicin (top) and from an inside-out patch exposed to an increasing amount of intracellular Mg2+ and capsaicin (bottom). (B) Summary of the effect of extracellular (black bars; n = 5) and intracellular (red bars; n = 5–6) Mg2+. (C) Representative current trace demonstrating that intracellular Mg2+ blocks, instead of potentiates, current induced by a low concentration of capsaicin. (D) Single-channel current traces recorded at +80 mV in the presence of intracellular Mg2+ at the indicated concentrations. (E) All-point histograms of single-channel events at the indicated intracellular Mg2+ concentration. The superimposed curve represents a fit of a double-Gaussian function. (F) Box-and-whisker plot of the single-channel conductance versus the corresponding concentration of intracellular Mg2+. The whisker top, box top, line inside the box, box bottom, and whisker bottom represent the maximum, 75th percentile, median, 25th percentile, and minimum value of each pool of conductance measurements, respectively. n = 3–4.
Figure 3.
Figure 3.
Two out of three S1–S2 linker mutants exhibit a lack of Mg2+ potentiation. (A) Diagram illustrating the design of mutations. (B) Representative Ca2+ imaging traces. (C) Summary of the amplitude ratio between Mg2+- and capsaicin-induced fluorescence changes in Ca2+ imaging experiments. n = 48 (wild type), 42 (Rs1s2_1), 55 (Rs1s2_2), and 89 (Rs1s2_3). (D) Representative current traces from Rs1s2_1 (top) and Rs1s2_2 (bottom) in response to 130 mM Mg2+ and 10 µM capsaicin. (E) Summary of the amplitude ratio between 130 mM Mg2+-induced current and 10 µM capsaicin-induced current. n = 5 (wild type), 4 (Rs1s2_1), and 3 (Rs1s2_2). (F) Representative current trace (top) and comparison of the amplitude ratio (bottom) between Mg2+- and capsaicin-induced currents for wild type (black bars) and Rs1s2_3 (blue bars). n = 5 (wild type) and 3–6 (Rs1s2_3). *, P < 0.05; ***, P < 0.001; N.S., not significant.
Figure 4.
Figure 4.
Two out of three S1–S2 linker mutants do not respond to heat. (A) Representative Ca2+ imaging traces. (B) Summary of the amplitude ratios between heat- and capsaicin-induced responses. n = 59 (wild type), 37 (Rs1s2_1), 45 (Rs1s2_2), and 52 (Rs1s2_3). *, P < 0.05; ***, P < 0.001. (C and D) Representative single-channel traces recorded at +80 mV from Rs1s2_1 (C) and Rs1s2_2 (D) at room temperature (RT), elevated temperatures, and in the presence of 10 µM capsaicin. The recording shown in D was from a patch containing two channels. (E) Representative current trace recorded from Rs1s2_3 in response to heating and 3 µM capsaicin. (F) Current–temperature relationship of Rs1s2_3. Dotted lines represent linear fits to the leak and channel currents.
Figure 5.
Figure 5.
Mutation of the S3–S4 linker eliminates Mg2+ response. (A) Diagram illustrating the design of mutation. (B) Representative Ca2+ imaging trace. (C) Summary of the amplitude ratio between Mg2+- and capsaicin-induced fluorescence responses for wild type (black bar; n = 48) and Rs3s4 (blue bar; n = 65). (D) Representative current trace recorded at +80 mV in response to 130 mM Mg2+ and 3 µM capsaicin. (E) Summary of amplitude ratio between Mg2+- and capsaicin-induced current for wild type (black bar; n = 5) and Rs3s4 (blue bar; n = 4). ***, P < 0.001.
Figure 6.
Figure 6.
Rs3s4 mutant exhibits heat-induced current activation. (A) Representative Ca2+ imaging trace (top) upon heat (bottom) and capsaicin challenges. (B) Summary of amplitude ratio between heat- and capsaicin-induced currents for wild type (black bar; n = 59) and Rs3s4 (blue bar; n = 55). ***, P < 0.001. (C) Representative single-channel traces recorded at +80 mV at varying temperatures and in the presence of 3 µM capsaicin. (D) Relationship between open probability (Po) and temperature. Open circles represent average Po of individual sweeps, and the red trace represents average Po over a 3.5°C range. (E) Representative single-channel traces demonstrating rapid deactivation transition upon voltage step from +80 to −80 mV.
Figure 7.
Figure 7.
Pore turret mutants exhibit diminished Mg2+ response. (A) Diagram illustrating the design of mutations. (B) Representative Ca2+ imaging traces. (C) Summary of amplitude ratio between Mg2+- and capsaicin-induced fluorescence responses. n = 48 (wild type), 38 (R2), and 66 (R3). (D and E) Representative current trace of wild type (D) and R3 (E) challenged by Mg2+ and capsaicin. (F) Summary of amplitude ratio between Mg2+- and capsaicin-induced currents. n = 5 (wild type), 9 (R2), and 5 (R3). (G and H) Representative current trace of wild type (G) and R3 (H) challenged by Ba2+ and capsaicin. (I) Summary of amplitude ratio between Ba2+- and capsaicin-induced currents. n = 5 (wild type), 6 (R2), and 4 (R3). **, P < 0.01; ***, P < 0.001.
Figure 8.
Figure 8.
Mg2+ induces pore turret movement. (A) Bright-field and fluorescence images of a patch-clamped fluorescently labeled cell. Red dashed lines mark the position of the spectrograph input slit. Fluorescence signal from the area covered by the slit was collected by spectral imaging (right). (B; left) Simultaneous recordings of current (open circles) and TMRM/FM fluorescence intensity ratio (red circles) upon the application of 130 mM Mg2+. (Right) Spectral images and spectra taken at time points 1 and 2 labeled in the left panel. (C) Control experiments with untransfected cells labeled with FM or TMRM (n = 6 each). No change in relative fluorescence intensity could be detected when various concentrations of Mg2+ were added. (D) Fluorescence signal recorded at N467C (which is close to the end of the S1–S2 linker) did not change upon Mg2+-induced activation. (E) Background fluorescence did not change upon Mg2+-induced activation of mutant channel without any extracellularly accessible cysteine.
Figure 9.
Figure 9.
Mg2+ induced FRET changes at the pore turret. (A) Mg2+ did not affect RatioA0 of TMRM-labeled cells (left) but increased RatioA of FM/TMRM-labeled cells (right), indicating an increase in FRET efficiency. (B) Statistical analysis of Mg2+ effects on RatioA0 (left) and RatioA (right). Dotted lines on the right link measurements from the same cells; RatioA0 measurements were not paired. (C) Summary of Mg2+-induced changes in FRET efficiency, which is defined as the FRET efficiency values in the presence of 100 mM Mg2+ subtracted by the FRET efficiency value in the presence of Na+. *, P < 0.05; **, P < 0.01. n = 3–6.
Figure 10.
Figure 10.
Divalent cations activate TRPV1 through conformational changes of the extracellular region. (A) Crystal structure of the Kv1.2-Kv2.1 chimera (Protein Data Bank accession no. 2R9R), with the pore turret, S1–S2 linker, and S3–S4 linker highlighted in surface-plot mode in different colors. The red circle highlights the S1–turret interaction that supports voltage-dependent gating. (B) Rosetta-generated structural model for TRPV1. (C) Schematic diagram summarizing the gating process of TRPV1 by divalent cations, heat, and capsaicin. Mg2+/Ba2+ and heat work from the extracellular side to induce a conformational rearrangement of channel structures exposed to the aqueous environment, which is coupled to pore opening (first horizontal transition). In the presence of Mg2+/Ba2+ or heat, the extracellular structure settles down over time to a more stable conformation that does not support the open pore conformation, leading to desensitization (second horizontal transition). Capsaicin binding to the intracellular S2–S3 linker region also leads to pore opening. This can happen to resting channels (left vertical transition) as well as to channels already desensitized to divalent cations (right vertical transition).

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