Abstract
TRPM8 (Transient Receptor Potential Melastatin-8) is a cold- and menthol-gated ion channel necessary for the detection of cold temperatures in the mammalian peripheral nervous system. Functioning TRPM8 channels are required for behavioral responses to innocuous cool, noxious cold, injury-evoked cold hypersensitivity, cooling-mediated analgesia, and thermoregulation. Because of these various roles, the ability to pharmacologically manipulate TRPM8 function to alter the excitability of cold-sensing neurons may have broad impact clinically. Here we examined a novel compound, PBMC (1-phenylethyl-4-(benzyloxy)-3-methoxybenzyl(2-aminoethyl)carbamate) which robustly and selectively inhibited TRPM8 channels in vitro with sub-nanomolar affinity, as determined by calcium microfluorimetry and electrophysiology. The actions of PBMC were selective for TRPM8, with no functional effects observed for the sensory ion channels TRPV1 and TRPA1. PBMC altered TRPM8 gating by shifting the voltage-dependence of menthol-evoked currents towards positive membrane potentials. When administered systemically to mice, PBMC treatment produced a dose-dependent hypothermia in wildtype animals while TRPM8-knockout mice remained unaffected. This hypothermic response was reduced at lower doses, whereas responses to evaporative cooling were still significantly attenuated. Lastly, systemic PBMC also diminished cold hypersensitivity in inflammatory and nerve-injury pain models, but was ineffective against oxaliplatin-induced neuropathic cold hypersensitivity, despite our findings that TRPM8 is required for the cold-related symptoms of this pathology. Thus PBMC is an attractive compound that serves as a template for the formulation of highly specific and potent TRPM8 antagonists that will have utility both in vitro and in vivo.
Highlights
The cold and menthol-gated ion channel TRPM8 [1,2] serves as a neuronal sensor of cold temperatures and is essential for innocuous cool and noxious cold sensations [3,4,5,6,7]
PBMC selectively blocks TRPM8 activity We first tested the effects of PBMC on menthol-induced responses in heterologous cells expressing TRPM8 channels using calcium microfluorimetry [32]
In HEK293T cells transiently transfected with the mouse orthologue of TRPM8, brief and repeated exposure to 200 mM menthol evoked a robust increase in intracellular calcium, measured as a change in the Fura-2 fluorescence signal ratio (Figure 2A,B)
Summary
The cold and menthol-gated ion channel TRPM8 [1,2] serves as a neuronal sensor of cold temperatures and is essential for innocuous cool and noxious cold sensations [3,4,5,6,7]. Mice lacking functional TRPM8 channels are unable to discriminate between mildly warm and mildly cool temperatures, and do not show normal aversion to temperatures in the noxious cold range [6]. Recent evidence suggests that the channel is necessary for increased cold sensitivity associated with injury [4,8,9]. TRPM8 has recently been reported to be involved in thermoregulation [11], a role that is not entirely unexpected given that other temperature sensitive ion channels, the heat-gated TRPV1, have been implicated in regulating body temperature [12]. Several studies have shown that TRPV1-null mice display attenuated fever responses, and TRPV1 antagonism induces thermogenesis in rats and humans [12,13,14,15]
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