Figure 1.
Differences in temperature preference for frog versus rat are reflected in differential cold sensitivity of primary afferent neurons.
(A) Dissociated DRG neurons from X. laevis frogs or rats were exposed to a cold ramp (33°C–7°C) and responses measured by ratiometric calcium imaging. After a recovery period at 25°C, cells were challenged with menthol (500 µM), followed by high extracellular potassium (70 mM KCl) to depolarize and identify all excitable cells. Note differential sensitivity of frog and rat neurons to 20°C stimulus. (B) Averaged trace of calcium signals from menthol-resopnsive rat or frog DRG neurons stimulated as described in (A). Dotted lines indicate respective cold activation thresholds (9.6±0.6°C for frog and 25.4±1.3°C for rat) (n = 30–40 cells). Cells showing an increase in intracellular calcium greater than five standard deviations above baseline fluctuations were taken as positive responders.
Figure 2.
Sequence comparison of TRPM8 species orthologs.
(A) Previously described rat, human, and chicken TRPM8 sequences were aligned with the full-length sequences of X. tropicalis and X. laevis TRPM8 (this study) using MultAlin and ESPript. The locations of predicted transmembrane helices [6] and the C-terminal coiled-coiled assembly domain [25] are shown as black and gray bars, respectively. The asterisk indicates the polymorphic residue previously shown to determine TRPM8 icilin sensitivity [17]. (B) Phylogenetic tree indicating the evolutionary relationship between TRPM8 ortholog sequences.
Figure 3.
Cloned Xenopus TRPM8 channels show alterations in thermosensitivity compared to their mammalian and avian counterparts.
(A) Current-voltage relation from a two-electrode voltage clamp recording of xlTRPM8-expressing oocytes under basal or cold-stimulated conditions. Note the strong outward rectification of the cold-evoked current. (B) Normalized inward (−60 mV) and outward (+80 mV) cold-evoked currents for xlTRPM8. The current obtained at a given temperature was normalized to the maximum cold-evoked current (obtained at 6°C) obtained at each potential. (C) Normalized current-temperature plots (at +80 mV) for chicken (blue), rat (red), X. laevis (green) and X. tropicalis (orange) TRPM8, yielding half-maximal activation temperatures of 29.35±0.21°C (chicken), 24.00±0.43°C (rat), 13.89±0.39°C (X. laevis), and 13.90±0.44°C (X. trop.). (D) Plot of species core body temperature (obtained from previously published measurements) versus experimentally determined temperature of half-maximal TRPM8 cold activation. Core temperatures were based on previously published values (for Xenopus, core temperatures were estimated as the arithmetic mean and error bars indicate the full range of tolerated temperatures) [14], [18], [19].
Figure 4.
X. laevis TRPM8 is activated by menthol but not icilin.
(A) In oocytes expressing xlTRPM8, application of menthol (1 mM) evoked inward currents (green trace) that were suppressed by a rise in bath temperature (gray trace). Holding potential was −60 mV. (B) Cell-attached patches from oocytes expressing xlTRPM8 (n = 4) but not uninjected oocytes (n = 3) displayed a strongly rectifying current (green trace) when the pipette solution contained 500 µM menthol. Formation of the inside-out configuration resulted in rapid current rundown (black trace, 90 seconds after patch excision.) (C) Concentration-response relation for menthol-evoked currents (at +80 mV) from rat (red) or X. laevis (green) TRPM8-expressing oocytes. (D) Application of 10 µM icilin in the presence of 2 mM extracellular Ca2+ failed to activate xlTRPM8, while 1 mM menthol evoked robust inward currents.