Function and pharmacology of TRPM cation channels
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- Harteneck, C. Naunyn-Schmiedeberg's Arch Pharmacol (2005) 371: 307. doi:10.1007/s00210-005-1034-x
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The physiological function and cellular role of some members of the TRPM family are poorly understood and still mysterious. Melastatin, the founding member of the TRPM group, is the most prominent example of the mysteries involved in understanding TRP channel function. Melastatin or TRPM1 was first cloned in 1998 and since then it has been suggested that it functions as a tumor suppressor protein in melanocytes. On the other hand, TRPM8 and TRPA1 have been described as cold receptors, TRPM4 and TRPM5 as calcium-activated nonselective cation channels, TRPM6 and TRPM7 as magnesium-permeable and magnesium-modulated cation channels, TRPM2 as an ADP-ribose-activated channel of macrophages, and TRPM3 as a hypo-osmolarity- and sphingosine-activated channel. There are many unsolved questions and many studies have to be performed to understand the overall function of the TRPM family. In addition to electrophysiological recordings and biochemical characterization, the use of compounds modulating TRPM channel function has often been helpful to study TRPM channels in a cellular context. Therefore, the review will summarize the known functions, activation mechanisms, and pharmacological modulations of the TRPM channels.
KeywordsCalcium homeostasisTRPM channel familyMagnesium homeostasisCold receptorOsmo-regulationImmune systemCalcium-activated nonselective cation channels
Organization of cells in a multicellular organism depends on rapid and accurate transmission of information. Electrical signals and chemical compounds induce changes of intracellular second messenger concentrations, e.g., calcium ions, cAMP and cGMP. In excitable cells, electrical signals induce increases in the intracellular calcium concentration necessary for cellular responses like hormone secretion, contraction of muscle cells and sensation, processing and responding to environmental stimuli. These functions are mediated by voltage-gated calcium channels and hyperpolarization-activated cyclic nucleotide-modulated channels. The molecular basis for the hormone-induced calcium transient in nonexcitable cells was unclear and began to be unraveled with the molecular characterization of the trp locus of the Drosophila genome. Montell and Rubin (1989) identified and cloned Drosophila TRP as a cation channel involved in Drosophila phototransduction. The sequencing of the different genomes accelerated the access to homologous proteins in worm and men, and soon the TRP channels grew up to a superfamily with more than 20 different genes coding for TRP-homologous channel proteins in men (Harteneck et al. 2000; Montell et al. 2002). The classification of the 20 TRP channels into at least three subfamilies (TRPC, TRPV, TRPM) was initially based on sequence comparison, later on functional data helped to structure the variety of proteins.
Functional and pharmacological properties of TRPM channels. BCTCN-(4-tert. butyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1 (2H)-carboxamide
Sensor of oxidative stress
ADP-ribose, hydrogen peroxide
Flufenamic acid, econazole, clotrimazole, poly(ADP-ribose) polymerase inhibitors (SB750139-B, DPQ, PJ34)
Hypo-osmolarity, d-erythro-sphingosine, dihydrosphingosine
Calcium-activated nonselective cation channel
ATP, ADP, AMP, polyamines (spermine), decavanadate
Calcium-activated nonselective cation channel
Mg2+ uptake in kidney and intestine
(Voets et al. 2004)
Cellular Mg2+ homeostasis
Mg-ATP, breakdown of PIP2, increase in cAMP concentrations
Mg2+, polycations (spermine), 2-APB, MnTBAP, La3+, Gd3+
Cold receptor (<28°C)
Cold, menthol, icilin, linalool, geraniol, hydroxycitronellal
Capsazepine, BCTC, thio BCTC,
Cold receptor (<17°C), mechanoreceptor
Icilin, gingerol, cinnamaldehyde allyl- or benzyl-isothyiocyanate, methyl salicylate, eugenol, Δ9-tetrahydrocannabinol
TRPM1 and TRPM3
In a differential display screen analyzing mRNAs of different melanoma cell lines, the mRNA of TRPM1 was identified and named melastatin because of the absence of TRPM1 mRNA in malignant transformed melanoma cell lines, suggesting a tumor suppressor function of the channel protein (Duncan et al. 1998; Hunter et al. 1998; Deeds et al. 2000). The putative role of TRPM1 in cellular differentiation and proliferation processes is further confirmed by studies of hexamethylene bisacetamide (HMBA)-treated human pigmented melanoma cell lines (Fang and Setaluri 2000). HMBA-treated cells differentiate and change pattern of expressed proteins. Pigmented metastatic melanocytes selectively transcribe TRPM1 mRNA. The level of TRPM1 mRNA could be enhanced by incubation in the presence of 5 mM HMBA (Fang and Setaluri 2000). Furthermore, the existence of a huge number of splice variants was described, a common phenomenon of TRPM1 and TRPM3. Analysis of signals from Northern hybridization studies revealed the presence of at least four different transcripts coding for TRPM1 proteins (1.3, 1.8, 4.5, 5.4 kb mRNA). The transcription of the individual mRNA species depends on melanoma cell lines and differentiation states. The available EST profiling of human EST indicates the presence of TRPM1 mRNA during all stages of embryonal development whereas in tissues of adults TRPM1 mRNA is restricted to skin and eye. Although melastatin is the first member of the TRPM family, little is known about its functional properties and cellular functions.
Like for TRPM1, the transcription of the TRPM3 gene results in a vast number of different mRNA species. In Northern Blot analyses of mouse brain at least three transcripts of different lengths were detectable (Grimm et al. 2003), whereas Lee et al. (2003) cloned six variants from human kidney, which vary in short deletions and insertions indistinguishable by Northern Blot analysis. The variability of TRPM3 transcripts is further enhanced by the presence of two or probably three alternative start positions and two different C-terminal ends. At this time, it is unclear whether mRNA species of all possible variations are transcribed and result in missense proteins, which are subsequently degraded prior to translocation to the plasma membrane, or whether the different TRPM3 mRNA species are transcribed in a cell type-specific manner. TRPM3 protein has been shown to be expressed in human brain and human kidney, whereas in mouse kidney TRPM3 is undetectable.
TRPM6 and TRPM7
TRPM6 and TRPM7 are the closest relatives of TRPM1 and TRPM3 (see Fig. 1). The four proteins share many identical amino acids within their N-terminal sequences, the transmembrane domains, the putative pore and a small cytosolic domain following the sixth transmembrane domain. However, the C-termini are different between both groups. Whereas TRPM6 and TRPM7 share a protein kinase domain at their C-termini, the function of the long C-terminal ends of TRPM1 and TRPM3 is unclear. Delineated from the chimeric structure of a pore-forming transmembrane domain and a putative C-terminal domain with enzymatic activity, TRPM6 and TRPM7 were described as chanzymes or channel kinases (Drennan and Ryazanov 2004; Chubanov et al. 2005).
Within the human kinome, both channel kinases can be classified as atypical alpha protein kinases, which have homologies with the elongation factor 2 kinase and the heart, lymphocyte and muscle alpha kinases (Manning et al. 2002). Introduction of mutations in the alpha kinase domain results in loss of function, therefore it is believed that the protein kinase domain is involved in the activation process (Runnels et al. 2001). However, the relationship between TRPM7 channel activity and kinase activity remain unclear.
Experiments characterizing the heterologously expressed kinase domain of TRPM7 showed that the protein fragment is able to mediate autophosphorylation and to phosphorylate prototypic kinase substrates like myelin basic protein and histone H3 (Ryazanova et al. 2004). Furthermore, it was shown that annexine I is phosphorylated by the kinase domain of TRPM7 (Dorovkov and Ryazanov 2004). The kinase activity of TRPM7 depends on ATP and requires magnesium (optimum 4–10 mM) (Dorovkov and Ryazanov 2004). On the other hand, TRPM7 is inhibited by magnesium concentrations ∼0.6 mM (Nadler et al. 2001; Runnels et al. 2001). The discrepancies in sensitivity to the magnesium concentration could result from differences in the experimental approach (bacterial vs. eukaryotic expression system and holoprotein vs. protein fragment) or from spatially distributed magnesium concentrations, which may differ between cytosolic concentrations and local concentrations next to the magnesium-transducing mouth of the pore. Despite these discrepancies, it is accepted that TRPM7 is modulated by the intracellular magnesium concentration and by ATP, forms a pore permeable for divalent cations (permeability Zn2+∼Ni2+≫Ba2+>Co2+>Mg2+≥Mn2+≥Sr2+≥Cd2+≥Ca2+), is blocked by magnesium, polycations like spermine, 2-aminophenoxyborate (2-APB), Mn (III) tetrakis (4–benzoic acid) porphyrin chloride (MnTBAP), lanthanum and gadolinium ions (see Table 1; Nadler et al. 2001; Runnels et al. 2001; Hermosura et al. 2002; Aarts et al. 2003; Kerschbaum et al. 2003).
Whereas the ubiquitously expressed TRPM7 is involved in the regulation of cellular magnesium homeostasis, TRPM6 is selectively expressed in intestinal and renal epithelia and involved in the body magnesium homeostasis (Konrad et al. 2004). Mutations in the TRPM6 gene are responsible for the hereditary disease of familial hypomagnesemia with secondary hypocalcemia (Schlingmann et al. 2002; Walder et al. 2002). The mutations found in patients often result in truncated proteins or in variants with deficits in the translocation process to the plasma membrane (Chubanov et al. 2004). Heterologously expressed TRPM6 was found to form a magnesium- and calcium-permeable cation channel, which is regulated by magnesium and blocked by ruthenium red in a voltage-dependent manner (see Table 1; Voets et al. 2004). In summary, TRPM6 is involved in intestinal uptake and renal reabsorption of magnesium, whereas TRPM7 regulates cellular magnesium homeostasis.
TRPM4 and TRPM5
TRPM2 and TRPM8
The Nudix domain of TRPM2 cleaves ADP-ribose, a breakdown product of NAD and cyclic ADP-ribose, representing an intracellular second messenger stimulating calcium release mediated by ryanodine receptors. While ADP-ribose is hydrolyzed by TRPM2, it also activates TRPM2 and induces TRPM2 currents during infusion of ADP-ribose by the patch pipette. The activity of TRPM2 depends on the presence of intracellular calcium and is induced by extracellular application of hydrogen peroxide (see Fig. 5; Wehage et al. 2002; McHugh et al. 2003). The activation of TRPM2 by hydrogen peroxide is probably linked to the activity of the poly(ADP-ribose) polymerase, an enzyme transferring multiple ADP-ribose groups to proteins. Oxidative stress and other stimuli causing DNA damage enhance poly(ADP-ribose) polymerase activity (Davidovic et al. 2001). The large and branched structure of the poly(ADP-ribose) modifications are reduced prior to protein degradation to mono(ADP-ribose) by the poly(ADP-ribose) glycohydrolase, a process releasing ADP-ribose. Evidence for this intracellular pathway resulting in TRPM2 activation has been confirmed by the use of inhibitors of poly(ADP-ribose) polymerase, which were able to interfer with the hydrogen peroxide-induced TRPM2 activation (see Fig. 5; Fonfria et al. 2004).
Whereas TRPM2 is insensitive to lanthanum and gadolinium ions, it was recently shown that TRPM2 currents are blocked by flufenamic acid, a compound known to block a great variety of channel proteins (Hill et al. 2004a). Flufenamic acid has been characterized as an open-channel blocker of TRPM2. Activity of flufenamic acid depends on the pH with enhanced effect at acidic conditions. Flufenamic acid will allow analyzing TRPM2-mediated cellular effects in cells of the immune system, where TRPM2 is expressed especially in cells of the monocytic lineage including various cultured macrophage cell lines, peripheral blood monocytes and neutrophils (Perraud et al. 2001; Sano et al. 2001; Hara et al. 2002). TRPM2 expression was detected in pancreas and cell lines derived form pancreatic islet cells (Inamura et al. 2003). In brain, TRPM2 is mainly expressed in the immune cells of the brain, the microglia (Kraft et al. 2004). TRPM2 sensitivity to hydrogen peroxide depends on the activation state of the microglia (Kraft et al. 2004). This suggests that TRPM2 is involved in the regulation of intracellular calcium depending on the developmental state of the microglia. In summary TRPM2 is a hydrogen peroxide-activated cation channel involved in the host-defense system of the body.
The next relative to TRPM2 is TRPM8, a channel protein activated by a quite different mechanism. The cDNA of TRPM8 was isolated from prostate cancer cells, and the function of TRPM8 was initially linked to progression of cancer cells (Tsavaler et al. 2001). The physiological role of TRPM8 as a cold receptor of the body was revealed by an expression cloning approach to identify a menthol receptor from trigeminal neurons (McKemy et al. 2002; Peier et al. 2002). The isolated cDNA codes for TRPM8 and forms a calcium-permeable cation channel. In TRPM8-expressing cells, application of menthol, icilin or other cooling agents induce TRPM8 currents, which are comparable to activation of TRPM8 by temperatures lower than 28°C. Furthermore, TRPM8 is activated by many other odorant agents isolated from plants, e.g., linalool, geraniol, and hydroxycitronellal (Behrendt et al. 2004). Like TRPV1, TRPM8 is inhibited by capsazepine, N-(4-tert. butyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1 (2H)-carboxamide (BCTC) and a thio-derivative of BCTC. TRPM8 expression is detected in prostate and other tissues of the urogenital tract, but high TRPM8 expression is specifically found in a subset of pain- and temperature-sensing neurons (McKemy et al. 2002; Peier et al. 2002; Stein et al. 2004). In summary, TRPM8 displays a cold receptor of the body, and its activation can be modulated by many cooling compounds and odorants.
Noxious cold is transduced by ANKTM1, another channel protein with many properties of TRP channels, which is therefore classified as TRPA1. Like TRPM8, which was initially found in prostate cancer cells, ANKTM1 was initially identified in a cancer cell line (Jaquemar et al. 1999). ANKTM1 was characterized as a protein with six putative transmembrane domains and many N-terminal ankyrin repeats. As the Drosophila mechanosensor channel protein NOMPC also carries 29 ankyrin repeats, the accumulation of 18 ankyrin repeats in the N terminus of ANKTM1 made it likely that ANKTM1 is involved in mechanotransduction. This has recently been confirmed (Corey et al. 2004). ANKTM1 is highly expressed in the hair cell epithelia of the cochlea, which are responsible for mechanotransduction. Down-regulation of highly expressed ANKTM1 resulted in a loss of function and reduced ANKTM1 currents. On the other hand, ANKTM1 is expressed in a subset of pain- and temperature-sensing neurons, where ANKTM1 transduces noxious cold (Story et al. 2003; Bandell et al. 2004; Jordt et al. 2004). Like TRPM8, ANKTM1 is activated by icilin and cold temperatures (lower than 17°C), whereas ANKTM1 is insensitive to menthol and can be blocked by ruthenium red (see Table 1; Story et al. 2003). Furthermore, ANKTM1 is activated by a wide range of naturally occurring compounds isolated from plants. ANKTM1 can be stimulated by pungent compounds of mustard, cinnamon, wintergreen, ginger and clove containing allyl- or benzyl-isothyiocyanate, cinnamaldehyde, methyl salicylate, gingerol and eugenol, respectively (see Table 1; Bandell et al. 2004). In summary, pungent agents activate ANKTM1, a channel involved in mechanotransduction and sensation of noxious cold.
The mysteries of TRPM function slowly solve resulting in many unexpected functions, such as involvement in magnesium homeostasis and in plasma membrane depolarization, cold sensation or osmo-regulation. This process of understanding is accelerated by the use of pharmacological tools and will probably end in a picture of channels proteins regulated by highly different activation mechanisms. The phenomenon of dually activated cation channels has already been described for other TRP channel proteins e.g., for TRPA1 involved in cold- and mechanosensation, for TRPV4 activated by heat, small lipid compounds and cell-swelling or hypo-osmolarity. Dual regulation will probably become a common property of TRPM channels as well.
Our own work reviewed here was supported by the Deutsche Forschungsgemeinschaft, Fonds der Chemischen Industrie, and Sonnenfeld-Stiftung.