Transient receptor potential cation channel, subfamily C, member 5 (TRPC5) is a cold-transducer in the peripheral nervous system - PubMed (original) (raw)

Transient receptor potential cation channel, subfamily C, member 5 (TRPC5) is a cold-transducer in the peripheral nervous system

Katharina Zimmermann et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Detection and adaptation to cold temperature is crucial to survival. Cold sensing in the innocuous range of cold (>10-15 °C) in the mammalian peripheral nervous system is thought to rely primarily on transient receptor potential (TRP) ion channels, most notably the menthol receptor, TRPM8. Here we report that TRP cation channel, subfamily C member 5 (TRPC5), but not TRPC1/TRPC5 heteromeric channels, are highly cold sensitive in the temperature range 37-25 °C. We found that TRPC5 is present in mouse and human sensory neurons of dorsal root ganglia, a substantial number of peripheral nerves including intraepithelial endings, and in the dorsal lamina of the spinal cord that receives sensory input from the skin, consistent with a potential TRPC5 function as an innocuous cold transducer in nociceptive and thermosensory nerve endings. Although deletion of TRPC5 in 129S1/SvImJ mice resulted in no temperature-sensitive behavioral changes, TRPM8 and/or other menthol-sensitive channels appear to underpin a much larger component of noxious cold sensing after TRPC5 deletion and a shift in mechanosensitive C-fiber subtypes. These findings demonstrate that highly cold-sensitive TRPC5 channels are a molecular component for detection and regional adaptation to cold temperatures in the peripheral nervous system that is distinct from noxious cold sensing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Cooling potentiates TRPC5 current. (A) Whole-cell currents of an HM1-HEK293 cell expressing mouse TRPC5. Circles represent current amplitudes derived from 400-ms voltage ramps (−120 to +120 mV) applied at 2.4-s intervals (holding potential: gray circles, +80 mV; black circles, −40 mV; white circles, −80 mV). Cooling from 32–14 °C increased ITRPC5, whereas warming to 40 °C decreased ITRPC5. Carbachol (Cch; 100 μM) or La3+ (100 μM) activated and sensitized the current to cold. Bars indicate duration of agonist application. Numbers in circles correspond to I–V traces in B and C. (B and C) I–V relationship at three different temperatures for basal (B) and Cch-activated (C) currents. (D) Averaged Ca2+ in TRPC5-transfected (TRPC5+, red bars) and TRPC5-nontransfected (TRPC5−, black bars) HM1-HEK293 cells in response to cold and Cch, measured using fura2-AM (n = 45 each). (E) Representative images from calcium imaging. Top Left to Bottom Right: Cherry-FP signal illustrates transfection of HM1-HEK293 cells with TRPC5 (arrowheads); basal Ca2+ signal using fura2-AM; numbers 1–4 correspond to conditions as marked in the column pairs in D; ionomycin (IM) highlights maximum Ca2+ in all cells. (F) Averaged current densities at 40 mV. (G) A cell was cooled at a rate of 0.5 °C/s, and I/Imax was plotted vs. temperature. The highest temperature sensitivity was between 37–25 °C (Q10 ∼10). *P < 0.01; **P < 0.001; ***P < 0.00001, n = 8–22, Student's t test. See also

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. (H) Current–temperature relationship for TRPC5 at −40 mV. (I) Current–temperature relationship for TRPC1/C5 heteromeric channels at −40 mV. TRPC1/C5 heteromers are not cold sensitive. (Inset) Heteromeric current densities at 25 °C and 35 °C; n = 5.

Fig. 2.

Fig. 2.

TRPC5 in mouse and human peripheral nerves, DRG, and the spinal cord dorsal horn. (A) (Upper) TRPC5 is present as diffuse cytoplasmic staining in adult mouse DRG neurons. (Lower) TRPC5 staining in sections from _TRPC5_−/− DRGs. (B) Size–frequency distribution of TRPC5 in murine sensory neurons (TRPC5+/total neuron counts from eight DRGs: 290/687; 194/515; 169/482; 52/284; 158/451; 167/488; 37/408; and 88/350, respectively; mean diameter: 27.3 μm). Note: The majority of TRPC5+ neurons are small (<600 μm2) to medium-sized (<1,400 μm2). (C) TRPC5 is present in adult human skin intraepithelial nerves (arrows); dotted line indicates basement membrane. (Inset) TRPC5 channel. (D) Cross-section of a peripheral nerve: TRPC5 and TRPM8 are present in peripheral incoming processes from spinal nerve L5 (160 fibers TRPC5+, 146 fibers TRPM8+, 49 fibers colabeled, out of 325 fibers counted in peripheral incoming processes from three DRG neurons). (E) TRPC5 is present as diffuse cytoplasmic staining in adult human DRG neurons. LF, lipofuscin autofluorescence (arrows). (Inset) Human L5-DRG, double-labeled for TRPC5/TRPM8, contains three populations of neurons: 36% TRPM8+ (102/287; note the emerging fiber), 34% TRPC5+ (102/287 neurons), and 11% TRPM8/C5-colabeled neurons (32/287 neurons; n = 5 DRGs). (F) TRPC5+ is located in the adult human superficial dorsal horn, corresponding to lamina I/II/III (dotted lines). (Scale bars: A, C, F, and Inset E, 50 μm; E, 200 μm; D, 250 μm.)

Fig. 3.

Fig. 3.

Functional and immunophenotypic characterization of thermo-TRPs in DRG neurons from WT and TRPC5_−/_− mice. (A) Percentage of isolated DRG neurons identified by anti-TRPM8, -TRPV1, -CGRP, -peripherin, -NF200, and -IB4 antibodies. Only the percentage of TRPM8+ neurons was significantly different (**P < 0.005). (B) DRG were immunostained with anti-TRPM8 (green) or -peripherin (red) antibodies and DAPI nuclear staining (blue). TRPM8 expression is decreased in TRPC5−/− mouse DRG. (Scale bars: 50 μm.) (C) Comparison of cold sensitivity in WT and TRPC5−**/**− DRG neurons. WT neurons: 16% (105/655) of neurons were activated by cold, and 40% (42/105) of cold-sensitive neurons demonstrated menthol sensitivity. TRPC5−**/**− neurons: 4.6% (32/690) of neurons were activated by cold, and 50% (16/16) of cold-sensitive neurons were menthol-sensitive (***P < 0.00001). Note: Despite a striking reduction of cold-sensitive TRPC5−**/**− neurons, the fraction of cold-sensitive neurons that demonstrate menthol sensitivity was maintained (40% vs. 50%). (D) Percentage of WT (gray) and TRPC5−**/**− (red) neurons with responses to menthol (TRPM8 activator), allyl isothiocyanate (AITC) (TRPA1 activator), or capsaicin (TRPV1 activator). Fewer neurons in TRPC5−**/**− mice than in WT mice responded to menthol or allyl isothiacyanate (AITC) (**P < 0.005; *P < 0.05).

Fig. 4.

Fig. 4.

Skin-nerve recordings of WT and TRPC5_−/− mice: gain of function in C-cold nociceptors (CMC). C-cold nociceptors of TRPC5_−**/− mice had larger cold responses than those of WT mice as measured by action potential firing rates (A), action potentials (APs) per cold stimulus (B), and peak firing rates (C) but exhibited similar temperature thresholds (D). (E) Four-way distribution pattern of the subclasses of mechanosensitive C-fibers in WT and _TRPC5−/−_ mice, identified by mechanostimulation and subsequent thermal characterization. The numbers of fibers in WT and _TRPC5−/−_ cells are given in parentheses. CM, mechano-sensitive nociceptor; CMC, mechano-cold nociceptor; CMCH, mechano-cold and heat nociceptor; CMH, mechano-heat nociceptor. (F) _TRPC5−/−_ mice exhibited more menthol-sensitive CMCs and fired more cold- and menthol-activated action potentials than WT mice (500 μM menthol). Numbers in bars indicate number of fibers (Left) and number of action potentials (Right). (G) Original cold response of WT and _TRPC5−/−_ CMC-fibers before and after treatment with menthol (arrow); I.f.p. s−1, instantaneous frequency plot. (Insets) Extracellular potentials; y = 200 μV, x = 1 ms. (H) Four-way distribution pattern of the subclasses of mechanosensitive C-fibers in WT and TRPM8−/− mice; compare to E. _TRPM8−/−_ mice lack CMC-fibers and exhibit more CMH-fibers. (I) Menthol-insensitive Aδ-cold nociceptors were observed infrequently in _TRPC5−/−_ mice, and responses were small (n = 65 each genotype). Note: The fraction of cold-sensitive fibers that demonstrate menthol sensitivity (WT: 13.3% vs. _TRPC5_−/−: 33.3%) as well as responses (WT: 13.7% vs. _TRPC5_−/−: 58.2%) was increased. (J) Representative responses of _TRPC5−/**−_ and WT Aδ-cold nociceptors; (Insets) extracellular potentials; y = 200 μV, x = 1 ms. *P < 0.05; **P < 0.0005; Mann–Whitney U test (B_–_D) and χ2 test (F and I). See also

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References

    1. Bessou P, Perl ER. Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. J Neurophysiol. 1969;32:1025–1043. - PubMed
    1. Zotterman Y. Specific action potentials in the lingual nerve of the cat. Skand Arch Physiol. 1936;75:105–119.
    1. Kang D, Choe C, Kim D. Thermosensitivity of the two-pore domain K+ channels TREK-2 and TRAAK. J Physiol. 2005;564:103–116. - PMC - PubMed
    1. Ramsey IS, Moran MM, Chong JA, Clapham DE. A voltage-gated proton-selective channel lacking the pore domain. Nature. 2006;440:1213–1216. - PMC - PubMed
    1. Caterina MJ, Rosen TA, Tominaga M, Brake AJ, Julius D. A capsaicin-receptor homologue with a high threshold for noxious heat. Nature. 1999;398:436–441. - PubMed

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