Pharmacology of transient receptor potential melastatin channels in the vasculature

Introduction

Adequate blood supply is critical for the proper function of all tissues and organs in the body, and cardiovascular diseases remain the leading cause of disability or death in industrial countries. Vascular smooth muscle cells (VSMC) regulate blood pressure and blood flow by adjusting their contractile state according to tissue metabolic or environmental needs (e.g. in thermoregulation). Ca²⁺-permeable ion channels initiate and maintain VSMC contraction, and hence the identification of their genes is important for our understanding of vascular function and, ultimately, for the treatment of human cardiovascular diseases. Evidence is also being accumulated for the important role of cation entry in slow phenotypic remodelling processes in the vascular system leading to hypertension, atherosclerosis, neointimal hyperplasia and other proliferative disorders.

Vascular smooth muscle cells are enriched with a multitude of ion channels of different types that act in concert to regulate cell membrane potential and smooth muscle excitability and excitation process. Calcium influx via various non-selective cation channels and voltage-dependent Ca²⁺ channels, an ultimate determinant of VSMC contractile state, is thus regulated in a very complex manner. As an added layer of complexity, same calcium signals that initiate VSMC contraction also often couple to ‘inhibitory’ Ca²⁺-dependent channels, such as Ca²⁺-activated potassium and, under certain conditions, chloride channels. For many channels, there also exist tight positive or negative feedback loops that control their activity, usually employing membrane potential and calcium level for activating or deactivating them. Microdomain organization of signal proteins and local efficient coupling between Ca²⁺ entry, Ca²⁺ release and activation of Ca²⁺-dependent channels (Bolton, 2006) makes identification of the roles of individual channel types an even more challenging task.

Transient receptor potential (TRP) cation channels (Montell, 2005; Nilius et al., 2007; Venkatachalam and Montell, 2007) are currently considered as the leading candidate proteins mediating diverse non-voltage-gated Ca²⁺ entry pathways in vascular and communicating endothelial cells (Beech, 2005; Yao and Garland, 2005; Albert and Large, 2006; Inoue et al., 2006; Simard et al., 2007; Saleh et al., 2008). However, in contrast to cells overexpressing a certain type of TRP channel, it is far more difficult to reveal its role in native cells such as VSMC. It also follows from these general considerations that even the net functional outcome of channel activation (e.g. VSMC contraction or relaxation) can be sometimes difficult to predict. For example, Ca²⁺-permeable non-selective cation channels, by elevating Ca²⁺ in the above mentioned signalling microdomains, can induce not only membrane depolarization but also spontaneous hyperpolarizations caused by the Ca²⁺‘sparks’/BK_Ca/STOCs mechanism as was shown for TRPV4 activation resulting in membrane hyperpolarization (and vasodilation) of cerebral artery mediated by the TRPV4/RyR/BK_Ca complex (Earley et al., 2005). Nevertheless, there has been considerable recent progress in decoding the function of several TRPs in VSMC, mostly from the family of ‘canonical’ TRP channels (TRPC), as reviewed elsewhere (Beech, 2005; Albert and Large, 2006; Inoue et al., 2006; Dietrich et al., 2007; Firth et al., 2007; Saleh et al., 2008). The purpose of this review is to outline the present state of knowledge of vascular ‘melastatin’ or long TRPs (TRPM), one of the most novel, largest and diverse TRP subfamilies, while emphasizing the usefulness and limitations of current TRPM pharmacological modulators that help reveal their roles in native cells.

TRPM channels

Based on structural homology, TRPMs belong to TRP group 1 that includes three other mammalian subfamilies (TRPC, TRPV and TRPA), as well as TRPN channels found only in invertebrates and zebrafish (Montell, 2005; Venkatachalam and Montell, 2007). The other two mammalian subfamilies, TRPP and TRPML, in group 2 are their much more distant relatives. Eight mammalian members of the TRPM subfamily are structurally and functionally diverse non-selective cation channels, which are involved in processes ranging from detection of cold, taste, osmolarity, redox state and pH to control of Mg²⁺ homeostasis and cell proliferation or death. Their phylogenetic analysis suggests subdivision into four groups, TRPM1/3, TRPM6/7, TRPM4/5 and TRPM2/8 (Harteneck, 2005). Remarkably, three out of eight TRPM members have connections with cancer development, but their role in vascular angiogenesis has not been investigated. The founding member melastatin (TRPM1) as well as TRPM5 and TRPM8 were identified by analysis of gene expression in several carcinomas (Kraft and Harteneck, 2005).

Transient receptor potential channels in native cells sometimes do not faithfully reproduce properties of their heterologously expressed counterparts, likely due to differences in the cellular milieu or heteromultimerization between different TRP subtypes. However, the knowledge of biophysical properties, interacting molecules and activation mechanisms of recombinant TRPs together with the analysis of the corresponding gene expression in native cells has significant predictive power in decoding native channel protein functions. Thus, Table 1 summarizes some key features of the individual members of the TRPM subfamily that have been reviewed in detail elsewhere (Fleig and Penner, 2004; Harteneck, 2005; Kraft and Harteneck, 2005; Ramsey et al., 2006; Nilius et al., 2007; Venkatachalam and Montell, 2007). Furthermore, pharmacological modulators of heterologously expressed TRP channels together with more selective molecular biology approaches such as gene silencing or gene knockout are invaluable tools for probing the functional roles of native channels. Therefore, Table 2 provides a summary of the pharmacological properties of TRPM channels.

Pharmacology of TRPM channels

Both agonists and antagonists are available for most TRPM subtypes, as summarized in Table 2. For some TRPMs, such as TRPM2/4/8, the list of known channel ligands is very extensive, but for others (e.g. TRPM1) pharmacological modulators are still lacking. Table 2 summarizes data on the available activators and their affinities [expressed as pD₂, or −log(EC₅₀)], including references; when this was not established the concentration range used is shown. Apparent affinities of antagonists are given as the IC₅₀ value; when single concentration is shown, this caused significant or complete inhibitory effect. Several pharmacological features of TRPM channels can be summarized as follows.

High-affinity selective and potent TRPM ligands are generally lacking, which is a common problem in TRP research. The effective concentrations are typically in the range of micromolar or even millimolar. For example, 2-APB, which is considered as a general inhibitor of TRP channels, commonly inhibits TRPM2/3/7/8 channels, although between individual members its apparent affinity varies from 1 to 200 µM. Several other blockers, such as SKF-96365, flufenamic acid, tetracaine, ruthenium red and spermine also inhibit other TRP and non-TRP channels, often with much higher potency (e.g. ruthenium red inhibits ryanodine receptors at nanomolar concentrations and it is a potent blocker of several TRPVs; flufenamic acid inhibits various chloride and potassium channels and SKF-96365 inhibits various ROC and, especially, SOC channels). However, there are also some exceptional examples, for example TRPM2 is insensitive to La³⁺ although lanthanides commonly inhibit TRP channels including TRPM3/4/7. ATP⁴⁻ allows pharmacological differentiation between TRPM4 and TRPM5. In the case of TRPM8 its most potent known antagonist clotrimazole also shows characteristic voltage dependency (e.g. higher potency at negative potentials explained by a positive shift of the activation curve caused by the blocker). Clotrimazole can be an especially useful pharmacological tool to discriminate between TRPM8- and TRPA1-mediated responses as it has the opposite, activating effect on TRPA1 (Meseguer et al., 2008). Another recently discovered antagonist AMTB [N-(3-aminopropyl)-2-([(3-methylphenyl) methyl]oxy)-N-(2-thienylmethyl)benzamide hydrochloride salt] also shows high affinity among other TRPM8 blockers; these include BCTC, thio-BCTC and capsazepine that are well-known TRPV1 antagonists. TRPM6/7 channels are inhibited by Mg²⁺ or Ca²⁺; in contrast, Ca²⁺ potentiates TRM2/4/5. Inhibition of the ubiquitously expressed TRPM7 by intracellular Mg²⁺ can be very useful for isolation of currents mediated by other TRPMs (e.g. Oancea et al., 2009).

TRPM agonists also offer several unique properties for characterization of individual channel subtypes. Oxidants, such as H₂O₂, β-NAD⁺, ADPR, cADPR and NAADP⁺ activate TRPM2. Interestingly, the activation by cADPR shows strong temperature sensitivity: cADPR does not activate TRPM2 at 25°C, but heat dramatically potentiates TRPM2 activation by this ligand (Togashi et al., 2006). TRPM3 is activated by d-erythrosphingosine and SPH analogues, but not by other lipids, including ceramides, S1P, AA and DAG. TRPM3 activation by the neurosteroid pregnelone sulphate allowed its recent identification as an essential component of an ionotropic steroid receptor in pancreatic β-cells (Wagner et al., 2008). The action of pregnelone sulphate and closely related substances (e.g. pregnenolone) indeed seems very specific to TRPM3 as other TRPs (TRPM2/7/8 and TRPV1/4/6) are insensitive to these steroids. TRPM4 can be activated by BTP2 (4-methy-4′-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide) in a voltage-dependent manner while decavanadate activates TRPM4 but not TRPM5. Interestingly, the related TRPM6 and TRPM7 channels are activated, rather than inhibited, by 2-APB that acts more potently on TRPM6. TRPM8 displays perhaps the most impressive array of known activators and their discovery was often guided by the analysis of compounds that are well known to produce cooling sensation, such as icilin, menthol and its derivatives. Interestingly, chemicals with diverse structures can act as TRPM8 agonists. Among them the carboxamides WS-12, CPS-369 and CPS-113 activate TRPM8 with EC₅₀ in sub- to low micromolar range. These new pharmacological tools to study TRPM8 show specificity as they do not activate TRPM3 and TRPV6. TRPM8 channel is also strongly activated by LPL, such as lysophosphatidylcholine and lysophosphatidylinositol but it is inhibited by polyunsaturated fatty acids such as arachidonic acid (Abeele et al., 2006; Andersson et al., 2007). Lysophosphatidylcholine is also known to activate a distant TRPC5 channel (Flemming et al., 2006). Moreover, two widely used TRPM8 agonists, menthol and icilin, can also activate another cold-sensitive TRPA1 channel, although for menthol the modulation can be bimodal or even species-specific (Story et al., 2003; Karashima et al., 2007; Xiao et al., 2008).

Both intracellular Ca²⁺ measurements and more direct patch-clamp measurements of ion currents have been extensively used in evaluating affinity and potency of pharmacological modulators of TRPM channels. In some cases, large, up to 2–3 orders, differences in the apparent ligand affinity are also notable, which may arise from the differences in the techniques or cell expression system used in different studies. WS-12 well exemplifies such differences in pharmacological properties: its apparent affinity (EC₅₀) for TRPM8 varies from 39 nM in current measurements in TRPM8 expressing HEK293 cells (Beck et al., 2007) to 193 nM in Fura-2 calcium measurements or 680 nM in current measurements in the same study using TRPM8 expressing HEK293 cells (Bodding et al., 2007). A much higher value (12 µM) was reported in TRPM8 current measurements when the channel was expressed in Xenopus laevis oocytes (Sherkheli et al., 2008). Such possible differences in channel pharmacological properties depending on cell environment are further highlighted by findings that indicate substantial differences in the biophysical properties of native and recombinant channels (e.g. about 140 mV difference in the potential of half-maximal activation of TRPM8) and their stronger modulation by the same drug in native cells (Malkia et al., 2007).

TRPM channel in vascular function and disease

The above-discussed diverse mechanisms of channel activation are paralleled by diverse vascular functions of TRPM channels, as is increasingly recognized during the last 5 years. These range from mechanosensory transduction and regulation of the arterial myogenic response to magnesium transport in hypertension, and from cold sensitivity to vascular inflammation (Figure 1).

TRPM2 channels are likely to be involved in a range of pathophysiological processes in oxidant-induced vascular injury, cerebral ischemia and stroke (Simard et al., 2007; Hecquet and Malik, 2009). In endothelial cells, TRPM2 activation by H₂O₂ causes Ca²⁺ entry thus increasing endothelial permeability (Hecquet et al., 2008). In monocytes, ROS-evoked Ca²⁺ entry via TRPM2 is a key trigger of chemokine production in inflammation. These processes are attenuated by the Trpm2 gene disruption in mice (Yamamoto et al., 2008). The functional roles of TRPM2 channels expressed in VSMCs are not known, but they may contribute to atherosclerosis development, which is associated with mitochondrial dysfunction, ROS production and inflammation. The related TRPM8 channel is the most highly expressed TRPM member in blood vessels (Yang et al., 2006). Notably, the heat-sensitive TRPV4 is the most highly expressed TRPV channel and the presence of these thermoTRPs in blood vessels raises intriguing questions as to their functional roles. Although most blood vessels are not exposed to any essential temperature variations, thermal control of cutaneous vessels is physiologically important, but not completely understood (Roosterman et al., 2006). Cooling of peripheral blood vessels causes vasoconstriction that is important for heat conservation, but excessive or prolonged cooling causes vasodilatation [this includes a poorly understood non-neurogenic component (Johnson et al., 2005)] and rapid heat loss. We found that TRPM8 channels are both expressed and functional in several rat arteries. Both in isolated cells and in blood vessels in situ menthol produced Ca²⁺ transients that consisted of an initial ‘phasic’ component, followed by a sustained component. The ‘phasic’ component appeared as asynchronous intracellular propagating Ca²⁺ waves associated with asynchronous mechanical oscillations that integrated into a small contraction of the vessel segment. Both components were resistant to nifedipine suggesting little role for voltage-gated Ca²⁺ channels (Borisova et al., 2008). In contractile studies, the major effect of TRPM8 activation was seen in precontracted vessels, where TRPM agonists, menthol and icilin, caused a profound vasodilation and similar effects were also observed in human forearm cutaneous vessels (Johnson et al., 2009). Interestingly, in different microvessels TRPV1 activation can also cause vasoconstriction or vasodilation (Kark et al., 2008) while TRPV4 activation, especially in endothelial cells, causes vasodilatation (Earley et al., 2005; Zhang et al., 2009). TRPM8 can also be activated by Ca²⁺ store depletion (Thebault et al., 2005; Abeele et al., 2006) raising the possibility that it can function as a component of SOC channel in VSMC. Importantly, this pathway involves iPLA₂ activation and LPLs generation (Smani et al., 2004; Abeele et al., 2006) making TRPM8 a likely factor in the development of atherosclerosis, but these roles are awaiting further investigation.

Although TRPM3 shows mechanosensitive properties, it was the TRPM4 channel that received much attention as the channel involved in myogenic constriction that can offer new insights in the molecular nature of myogenic tone control by Ca²⁺ and PKC (Earley et al., 2004; Earley et al., 2007; Brayden et al., 2008). The authors used antisense technology to suppress TRPM4 expression in cerebral arteries [this was necessary due to the lack of selective TRPM4 blockers (Table 2)] and found reduction of pressure- and protein kinase C (PKC)-induced VSMC depolarization as well as pressure-induced vasoconstriction. Thus, both TRPM4 and the earlier studied TRPC6 have now been implicated in generation of the myogenic response due to their direct or indirect mechanosensitivity. Functional properties of TRPM4/5 channels also make them excellent candidates for various poorly understood CAN channels in VSMC, but any insight here is still missing and TRPM5 does not seem to be expressed in VSMC.

TRPM6 and TRPM7 channels regulate Mg²⁺ homeostasis that is reflected in their major roles in vascular Mg²⁺ transport and implicates them in hypertension (He et al., 2005; Hamaguchi et al., 2008; Touyz, 2008; Paravicini et al., 2009). Furthermore, TRPM7 may be a novel mechanosensor in VSMC, the function of which can be altered in hypertension as reviewed by Touyz (2008). In VSMC, but not in endothelial cells, fluid flow increases TRPM7 current as the channel is translocated to the plasma membrane suggesting a TRPM7 role in cellular response to vessel injury (Oancea et al., 2006). In addition, silencing TRPM7 by siRNA or its inhibition by 2-APB or Gd³⁺ promoted growth and proliferation of vascular endothelial cells as well as production of nitric oxide, the critically important endogenous vasodilator (Inoue and Xiong, 2009).

Challenges of studying TRPMs and future perspectives

Multifunctional non-selective TRPM cation channels are the important players regulating vascular function and potential new targets for treating vascular disease. Numerous pharmacological tools are available for the study of TRPM subfamily members, but there are important concerns considering that: (i) most ligands have limited selectivity and/or potency; (ii) most studies have been performed in artificial cell systems, with notable examples of differences in the pharmacological properties of recombinant TRPMs depending on the expression system; and (iii) heteromultimerization of TRPM isoforms and their interaction with non-TRP proteins in native vascular cells can also alter their pharmacological properties. Thus, any robust identification of functional roles of TRPM subtypes in native cells requires a combination of various approaches, the use of several ligands to characterize the pharmacological profile of the channel in question as well as its biophysical ‘signature’.

Molecular biology approaches such as antisense or siRNA technologies have been widely used in TRP vascular research attempting to overcome the problems of limited selectivity of current pharmacological tools. Antibodies targeting extracellular loops near the channel pore region are also being introduced (Naylor et al., 2008) and will undoubtedly aid this research. Genetic studies of hereditary disorders (exemplified by TRPM6 defect in HSH) and knockout mouse models are revealing specific roles of TRPMs in diverse biological processes, such as magnesium homeostasis, mast cell migration, inflammatory responses and sensing of cold and taste. These models will be indispensible in decoding specific roles of TRPM channels in the vasculature in future studies, with caution regarding possible compensatory up- or down-regulation of other ion channels or altered expression of transcription and growth factors.

With all this effort, there have been several exciting developments in the area of vascular roles of TRPM channels in recent years. These roles often conformed to the expectations based on the knowledge of the activation mechanisms and functional properties of heterologously expressed TRPM channels, but we have also seen several novel and unexpected developments, including the identification of mechanosensory roles of vascular TRPM4 and TRPM7 channels. Numerous other vascular functions of TRPMs can be envisaged based on their known properties as indicated by the question marks in Figure 1. This area of research presents many challenging tasks, and likely holds many new surprises. TRPM pharmacological tools discussed here will remain an invaluable resource in this continuing research.

Conflict of interest

The author has no conflict of interests.