Abstract

Menthol, a secondary alcohol produced by the peppermint herb, Mentha piperita, is widely used in the food and pharmaceutical industries as a cooling/soothing compound and odorant. It induces Ca2+ influx in a subset of sensory neurons from dorsal root and trigeminal ganglia, due to activation of TRPM8, a Ca2+-permeable, cold-activated member of the TRP superfamily of cation channels. Menthol also induces Ca2+ release from intracellular stores in several TRPM8-expressing cell types, which has led to the suggestion that TRPM8 can function as an intracellular Ca2+-release channel. Here we show that menthol induces Ca2+ release from intracellular stores in four widely used cell lines (HEK293, lymph node carcinoma of the prostate (LNCaP), Chinese hamster ovary (CHO), and COS), and provide several lines of evidence indicating that this release pathway is TRPM8-independent: 1) menthol-induced Ca2+ release was potentiated at higher temperatures, which contrasts to the cold activation of TRPM8; 2) overexpression of TRPM8 did not enhance the menthol-induced Ca2+) release; 3) menthol-induced Ca2+ release was mimicked by geraniol and linalool, which are structurally related to menthol, but not by the more potent TRPM8 agonists icilin or eucalyptol; and 4) TRPM8 expression in HEK293 cells was undetectable at the protein and mRNA levels. Moreover, using a novel TRPM8-specific antibody we demonstrate that both heterologously expressed TRPM8 (in HEK293 cells) and endogenous TRPM8 (in LNCaP cells) are mainly localized in the plasma membrane, which contrast to previous localization studies using commercial anti-TRPM8 antibodies. Finally, aequorin-based measurements demonstrate that the TRPM8-independent menthol-induced Ca2+ release originates from both endoplasmic reticulum and Golgi compartments.

Highlights

  • Menthol is a naturally occurring compound responsible for the minty flavor and smell of the mint plant (Mentha piperita)

  • Our results demonstrate that this intracellular Ca2ϩ release originates from both ER and Golgi compartments, is not related to TRPM8 expression, and is observed in several other widely used cell types, pointing to a more universal TRPM8-independent menthol-activated release pathway

  • Note the delay in the time course for the observed Ca2ϩ rise: TRPM8 current is already desensitizing due to the high menthol concentration when [Ca2ϩ]i increases. As this set of experiments was performed in Ca2ϩ-free extracellular solution, these findings indicate that the menthol-induced rise in [Ca2ϩ]i originates from the release of Ca2ϩ from intracellular stores

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Summary

Introduction

Menthol is a naturally occurring compound responsible for the minty flavor and smell of the mint plant (Mentha piperita). Whereas performing simultaneous whole cell patch clamp recordings and intracellular Ca2ϩ measurements on HEK293 cells overexpressing human TRPM8, we observed that menthol activates plasma membrane TRPM8 currents, and induces Ca2ϩ release from intracellular stores.

Results
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