Electrophysiological approach to examine a putative cold- and menthol-sensitive channel in the liverwort Conocephalum conicum

Abstract

The mechanism of cold perception by plants is still poorly understood. It was found that temperature drop evokes changes in the activity of ion pumps and channels, which leads to plasma membrane depolarization.^1,2 The nature of the primary step of its action (alteration in membrane composition,³ transient influx of Ca²⁺ etc.,²) has not been elicited yet. Our electrophysiological experiments conducted on the liverwort Conocephalum conicum showed that its cells respond not only to sudden cooling⁴ but also to menthol, generating depolarization of the plasma membrane and action potentials (APs). Similar results are well documented in mammals; cold or “cooling compounds” including menthol cause activation of thermosenstitive channel TRPM8 permeable to Ca²⁺ and generation of AP series.⁵ TRP receptors are detected, among others, in green and brown algae. Possible existence of TRPM8-like channel-receptor in Conocephalum conicum is discussed here.

TRPM8 and Other Types of TRP Channels in Mammals

Transient receptor potential (TRP) channels belong to a large superfamily of nonselective cation channels. The most intensively examined are mammalian TRP channels, placed in peripherial dendrites involved in many sensing processes. They are thought to play a crucial role in recognizing and responding to such stimuli as temperature, touch, pain, taste etc. All mammalian TRP receptors are divided into seven groups in terms of their homology: TRPC (classical or canonical), TRPV (vanilloid), TRPA (ankyrin-like), TRPPP (polycysteine), TRPM (melastatin), TRPML (mucolipin) and TRPN (no mechanoreceptor potential C, NOMPC).^6–9 Even within one subfamily, the receptors exhibit different selectivities toward cations, various ranges of thermal sensitivity or distinct mechanism of activation and signal transduction. On the other hand, the low homology of members belonging to distant families does not exclude similar functions and mechansims.¹⁰

Channels participating in thermosensation, belonging mostly to TRPV- or to TRPM-class are intensively studied. The TRPM8 receptor is activated by temperature drops in the range between 28-8°C or by menthol and other “cooling” compounds.^5,11,12 By binding to the TRPM8 channel, menthol initially sensitizes the receptor to low temperature, but repetitive application thereof results in desensitization (dependent on the extracellular calcium concentration).^7,13 The desensizing tendency of TRPM8 is used in treatment of pain.¹⁴ Menthol is commonly used in cosmetic and pharmaceutical industry ensuring impression of cold and freshness.

Electrophysiological Exploration of TRPM8-Like Channel in the Liverwort

Existence of a putative TRPM8-like channel in the liverwort was examined on the basis of electrophysiological responses evoked by cooling and menthol.¹⁵ In mammals, reactions to both stimuli are the same and are manifested in depolarization of membrane potential and firing of vanishing AP series.^5,16,17 The results obtained from our intracellular measurements in Conocephalum conicum show both similarities and differences between sudden cooling and menthol.¹⁵ A slow decrease in temperature or application of at least 5 mM menthol caused initial depolarization of the resting potential and, in consequence, generation of AP. In the case of cold treatment there was always only one AP, irrespective of the rate of temperature drop, while the number of menthol-evoked APs depended on its concentration. It was always possible to evoke AP after either successive cooling or by menthol which, however, had to be used in a higher concentration than the initial one, which mimicks desensitization. After blockage of anion and potassium channels by A9C and TEACl, cooling evoked voltage transients (VTs) regarded as the calcium component of APs. VTs last much shorther than APs, their amplitude depends on the stimulus strenght and they do not propagate.¹⁸ Menthol in the presense of the ion channel inhibitors evoked responses similar to APs (lasting even longer). They were called AP-like responses. Application of proton pump blockers caused vanishing of AP-like responses trigerred by menthol but only dampened the amplitude of cold-evoked VTs.

The results mentioned above pointed out to differences in the ion mechanism of responses to cooling and menthol. The changes refer to diverse pools of Ca²⁺ entering the cytosol and different role of the proton pump in responses to these two kinds of stimuli. Differences in dose-dependencies and ion mechanisms of APs evoked by cooling and by menthol, and lack of (−)- and (+) menthol discrimination distinguish electrophysiological reaction of C. conicum from this in mammalian neurons bearing TRPM8 receptors. The concentration range of menthol constitutes another difference between mammals and the liverwort: tens of micromol per liter are sufficient to evoke APs in the former, whereas a milimolar concentrations are required in the latter. The discussion about the identity or just functional homology of TRP-like receptors in C. conicum and TRPM8 in animals is still open. The role of cold sensors in C. conicum may be fulfilled, for instance, by non-selective cation channels which can carry Ca²⁺ into the cytosol and initiate APs. Our preliminary experiments showed that, in addition to menthol, geraniol and eucalyptol—other plant originating agonists of TRPM8 receptor evoke series of APs in C. conicum.

Why TRPM8-Like Channel in Conocephalum conicum?

To date, the genome of Conocephalum conicum has not been identified yet, so we cannot check if any amino acid homolog of the TRPM8 channel exists in the liverwort. Although there is neither a structure related to TRPM8 nor any other type of TRP channels in sequenced plant organisms (TRPM8 blasted at blast.ncbi.nlm.nih.gov/Blast.cgi), they do occur in some species of green and brown algea, oomycetes and fungi. Interestingly, Ostreococcus tauri is equipped with the osmotically activated TRP channel with 93% coverage of human TRPV3, activated by warm temperatures. Similarly, one of the TRP channels of Chlamydomonas reinhardti, named TRPL, covers TRPV3 in 86% (TRP blasted at blast.ncbi.nlm.nih.gov/Blast.cgi). It seems that despite high homology of channels existing in different organisms, they fulfill different functions. One can assume that some receptor belonging to the TRP family, even though different from TRPM8, may have its function. Moreover, as liverworts are phylogenetically the oldest terrestrial plants, located between green algae and higher plants, we cannot exclude that such putative TRP-like channels exist in Conocephalum conicum. The TRP-like receptor or some other cold sensor(s), functionally similar to TRPM8, would enable C. conicum to survive severe winters.

Addendum to: Kupisz K, Trebacz K. Effect of cold and menthol on membrane potential in plants. Physiol Plant. 2011;141:352–360. doi: 10.1111/j.1399-3054.