TRP channels as potential drug targets

Biology of the Cell 96 (2004) 47–54 www.elsevier.com/locate/biocell

Review

TRP channels as potential drug targets Ulrich Wissenbach, Barbara A. Niemeyer, Veit Flockerzi * Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, Gebäude 46, 66421 Homburg Germany Received 11 December 2003; accepted 15 December 2003

Abstract Calcium (Ca2+) is an ubiquitous intracellular signal that is responsible for a plethora of cellular processes including fertilization, secretion, contraction, neuronal signaling and learning. In addition, changes in intracellular Ca2+ have been known to influence cell proliferation and differentiation for more than three decades. Recent studies have indicated that members of the transient receptor potential (TRP) family of ion channels which respond to many different modes of stimulation both from within and outside the cell may be a primary mode of cation and Ca2+ entry into cells and may have roles in growth control. Moreover, changes in the expression of these channels may contribute to certain cancers. In the following, recent results concerning the expression and function of members of this family of ion channels are summarized. © 2003 Elsevier SAS. All rights reserved. Keywords: Prostate cancer; TRPV6; Prognostic marker; TRP cation channel

1. General characteristics of TRP channels TRP channels are a relatively new class of ion channels, named after their seminal member , the Drosophila melanogaster TRP protein. The complex eye of Drosophila trp mutants - unlike wildtype - is unable to sustain a steady-state response during prolonged light stimulation, thus showing a transient receptor potential. Although trp mutants were initially identified in 1969 (Cosens and Manning), the underlying gene defect was not characterized until 1989 (Montell and Rubin) and identified as an ion channel in 1992 (Hardie and Minke). Since then, many new ion channels of the TRP family of channels have been identified exclusively in ophistokonts (animals and fungi) but so far not in green plants, red alges, protists and bacteria. The general topology of TRP channels includes intracellular N- and C-terminal regions of variable length and six transmembrane spanning domains with a pore loop between transmembrane domain 5 and 6. In analogy to voltage gated potassium channels it is thought that four subunits need to assemble to form a functional channel (see also Hoenderop et al. 2003, for architecture of TRPV5 and TRPV6). In contrast to the voltage-gated potassium channel, the fourth transmembrane region of TRP channels does not contain the regular spacing of voltage sensing posi* Corresponding author E-mail address: [email protected] (V. Flockerzi). © 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.biolcel.2003.12.003

tively charged amino acids. So far, all identified channels are cation conducting channels. This rapidly growing important family can be subdivided into seven subgroups according to the degree of sequence homology (see Table 1): The classical (or canonical) TRPC-group is most closely related to the seminal Drosophila TRP and includes seven members, the TRPV group, named after their first member, the vanilloid receptor, includes six members and the TRPM group, named after the melastatin gene includes eight members. More distantly related are the TRPP group including one polycystic kidney disease protein (PKD2) and two PKD-like proteins (PKD2L1 and PKD2L2), the TRPML group containing three proteins related to the mucolipidosis Type IV protein (MCOLN1) and the TRPA group that – in mammals - so far only contains one member. A seventh group, the TRPN channels, have so far not been found in mammals. They include the mechanosensory transduction channel in Drosophila, NOMPC, and related channels in Caenorhabditis elegans and Danio rerio. Virtually all cell types tested so far contain at least one TRP channel. In many cells, several TRP transcripts are detectable and heteromeric combinations between subgroup family members are possible (Philipp et al., 2000, Strubing et al., 2003). Interestingly one laboratory has reported the association between TRPC1 and PKD2 (Tsiokas et al., 1999), members of two relatively unrelated subgroups. Many TRP sequences have been found by homology searches and functional characterization is often initially

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Table 1 Properties of TRP channel subunits Subgroup TRPC TRPC1

Physiological role

Possible pathophysiological role

References*

MGluR-I mediated slow EPSC Changes in endothelial permeability

LnCAP SOC downregulated in androgen independent prostate cancer...

TRPC2 §

Pheremone sensory signaling, sperm acrosome reaction

TRPC3

BDNF mediated neuronal differentiation Lymphocyte signalling Vasorelaxation, Microvascular permeability, GABA release

Zhu et al., 1995, Wes et al, 1995 Tozzi et al., 2003 Kim et al., 2003 Jungnickel et al., 2001, Vannier et al., 1999, Liman et al., 1999, Wissenbach et al., 1998 Zhu et al., 1996, Philipp et al., 2003 Munsch et al., 2003 Tiruppathi et al., 2002 Freichel et al., 2001 Philipp et al., 1996 Greka et al., 2003 Philipp et al., 1998 Boulay et al., 1997 Okada et al., 1999

TRPC4

TRPC5

Neuronal extension

TRPC6 TRPC7 TRPV TRPV1

? ?

TRPV2

Noxiuos thermosensation, Nociception Noxiuos thermosensation

Caterina et al., 1997

TRPV3

Warm-sensation

TRPV4

Osmo-sensation Warm-sensation

TRPV5

Epithelial Ca2+ uptake in kidney ?

TRPV6

Epithelial Ca2+ uptake induodenum / placenta ?

Upregulated in prostate cancer and endometrial cancer, involvement in metastasis ?

TRPM TRPM1

?

Downregulated in Melanom, Tumorsuppressor ?

TRPM2 TRPM3 TRPM4

? ? ?

TRPM5

Taste transduction

Located on critical locus responsible for Beckwidth-Wiedemann-Syndrome

TRPM6

Intestinal and renal Mg2+ absorption

Hypomagnesemia

TRPM7

Mg-Homeostasis

TRPM8

Cold-sensation

TRPA TRPA1

Cold-sensation

Upregulated in several types of cancer, breast colon, prostate, lung, skin

Caterina et al., 1999 Kanzaki et al., 1999 Peier et al., 2002, Smith et al., 2002, Xu et al., 2002 Guler et al., 2002, Liedtke et al., 2000, Strothmann et al., 2000, Wissenbach et al., 2000 Hoenderop et al., 2000 Hoenderop et al., 1999 Wissenbach et al., in press Fixemer et al., 2003 Wissenbach et al., 2000 Peng et al., 1999,

Duncan et al., 2001 Duncan et al., 1998 Nagamine et al., 1998 Grimm et al., 2003 Nilius et al., 2003, Launay et al., 2002, Xu et al., 2001 Zhang et al., 2003 Perez et al., 2002, Prawitt et al., 2000 Schlingmann et al., 2002 Voets et al., 2003 Schmitz et al., 2003 Runnels et al., 2001 Peier et al., 2002, McKemy et al., 2002 Tsavaler et al., 2001 Story et al., 2003 Jaquemar et al., 1999 (continued on next page)

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Table 1 (continued) Subgroup TRPML MNL1

MNL2 MNL3 TRPP PKD2 PKD2L1 PKD2L2 TRPN $ NOMPC zTRPN1 CNOMPC°

Physiological role

Possible pathophysiological role

References*

?

Mucolipidosis type IV

Bassi et al., 2000 Bargal et al., 2000 Sun et al., 2000 Di Palma et al., 2002 Di Palma et al., 2002

Varitint-waddler deafness ?

Autosomal dominant polycystic kidney disease

Mechanosensory transduction Mechanosensory transduction

Cai et al., 1999 Mochizuki et al., 1996 Nomura et al., 1998 Guo et al., 2000 Walker et al., 2000 Sidi et al., 2003 NP_493429

*

Cited were the most relevant papers concerning cloning, function and pathophysiological evidence. TRPC2 appears to be a pseudogene in humans. $ Members of the TRPN group have not been found in mammals. ° Database entry of the National Center for Biotechnology information, NCBI. §

conducted in heterologous expression studies. This has led to controversial results concerning selectivities and modes of activation, especially for the TRPC group of channels. As most of the TRP channels found in mice are conserved in humans, creating targeted gene deletions within the mouse may help to define their physiological function. So far, five knock-out mice have been published and we will first discuss how the analysis of these mice relates to results obtained from heterologous expression studies. In rodents, TRPC2 channels are expressed in the vomeronasal organ (VNO) and thought to be involved in pheromone sensory signaling (Liman et al., 1999). Indeed, TRPC2 deficient animals are not able to distinguish male from female counterparts (Leypold et al., 2002, Stowers et al., 2002). It has recently been shown that TRPC2 channels function directly as the DAG gated pheromone transduction channel in VNO neuron dendrites (Lucas et al., 2003). Analysis of the knock-out mice questioned previous models about the importance of TRPC2 in the sperm acrosome reaction (Jungnickel et al., 2001) as TRPC2 deficient mice can still reproduce. The TRPC2 knock-out mice also demonstrate that not all the aquired knowledge about their physiological role is applicable to humans: Here, partner finding must follow other rules than pheromone sensory signaling as TRPC2 is a nonfunctional pseudogene. TRPC4 transcripts are expressed predominantly in brain, uterus, ovary and kidney cells as well as in endothelial cells. Primary cultured mouse vascular endothelial cells (MAECs) express TRPC4 transcripts and protein, and Ca2+ permeable channels can be activated by store-depletion protocols in these cells (store-operated channels: SOC). After deleting the gene in the mouse, TRPC4 -/- MAECs lack store-operated Ca2+ currents, indicating that the TRPC4 protein is functionally expressed in wild-type MAECs and is responsible for the expression of the store-operated channels in these cells. As a

consequence of TRPC4 deletion and lack of SOC, agonistinduced Ca2+ entry is reduced markedly, resulting in a significant decrease of endothelium-dependent vasorelaxation of blood vessels. Therefore, TRPC4 is an indispensable component of store-operated channels in native endothelial cells (Freichel et al., 2001). TRPC4 -/- mice also show a decreased microvascular permeability to protease activated receptors (PAR) (Tiruppathi et al., 2002). Most recently, it has been shown that TRPC4 -/- mice show altered GABA transmitter release from thalamic interneurons (Munsch et al., 2003). TRPV channels were first identified in a search for proteins involved in the neuronal response to pain (Caterina et al., 1997). TRPV1 channels, heterologously expressed, activate by binding capsaicin, a vanilloid compound of hot chili and were therefore termed vanilloid receptors (VR1, now TRPV1). TRPV1 is expressed in afferent neurons of the nociceptive pathway and, besides capsaicin, can also be activated by noxious heat (more than 43°C) and a decrease in pH. TRPV1 K.O. mice are relatively insensitive to noxious heat but do not completely lack thermosensation (Caterina et al., 2000). This led to the identification of additional temperature gated channels such as TRPV2, TRPV3 and TRPV4. The TRPV4 protein is, in addition to heat, activated by hypoosmolarity, phorbolesters and derivates of the arachidonic acid path (Liedtke et al., 2000, Strotman et al., 2000, Wissenbach et al., 2000, Watanabe et al., 2002, Watanabe et al., 2003, Alessandri-Haber et al., 2003). Expression studies of the TRPV4 transcripts revealed expression in kidney, brain and cells of the inner ear. In brain TRPV4 transcripts are found in regions not controlled by the blood brain barrier and it was speculated that TRPV4 channels might be activated by changes in the osmolarity of the blood. In addition, it is imaginable that a similar mechanism occurs in the kidney. Contrary results have recently been published on the physiological function of TRPV4 channels: Two groups have cre-

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ated independent knock-out mice and investigated mechanisms of osmoregulation. Interestingly, while one group found that their knock-out mice show exaggerated secretion of the antidiuretic hormone (ADH) in response to salt ingestion (Mizuno et al., 2003), Liedtke and Friedman, (2003) show a decreased secretion of ADH in response to hypertonic stimulation and decreased fluid uptake behavior implicating a physiological function of TRPV4 as a component of the osmosensoric system. The reason for this discrepancy is unclear. The TRPM5 (MTR1) gene was originally cloned from a critical region which might be involved in the development of the Beckwidth-Wiedemann syndrome, a predisposition of childhood tumors (Prawitt et al., 2000). Interestingly, in mice TRPM5 has been found as a taste-cell specific protein (Perez et al., 2002) and analysis of TRPM5 -/- mice revealed that reception of sweet, bitter and umami compounds is abolished (Zhang et a l., 2003). Recent heterologous expression data reveals that TRPM5 can “read” rate changes in intracellular calcium concentration, thus linking store depletion in a novel way to plasmamembrane conductance (Prawitt et al., 2003). The latter group also finds expression of TRPM5 in pancreatic beta cells and other tissues. Currently there are no other published knockout mice of TRP channels and the following data was derived from localization and heterologous expression data. In general, TRPC, transcripts are predominantly expressed in the brain; in addition TRPC1 and TRPC3 are detectable in many other tissues (Zhu et al., 1996). In some cell types several TRPC transcripts are expressed (Philipp et al., 2000) and are believed to form heteromultimeric channels (Hofmann et al., 2002, Strubing, 2001, 2003). In many overexpression studies, TRPC channels can be activated by protocols that deplete internal calcium stores (for review see Trebak et al., 2003). However, there is growing evidence that TRPC channels might be involved in other modes of cellular signaling processes. Kim and coworkers (2003) recently reported direct association of TRPC1 channels with metabotropic glutamate receptors type I. Stimulation of mGluRI results in a mixed cation slow excitatory postsynaptic conductance (EPSC) which appears to depend on TRPC1 channels: A dominant negative TRPC1 construct blocks this current. Hofmann et al., (1999) demonstrated activation of TRPC3 and TRPC6 channels independent of store depletion by activation with diacylglycerol. TRPC5 was shown to influence hippocampal neurite extension and growth-cone morphology (Greka et al., 2003), in addition TRPC5 can form heteromultimeric channels with TRPC1 in these neurons (Strubing et al., 2001). TRPV1, TRPV2, TRPV3 and TRPV4 are channels gated by temperature changes (for review see Benham et al., 2003). TRPV1 and TRPV2 channels open at noxious temperatures above 43°C and 53°C, respectively. In contrast, recombinant TRPV3 and TRPV4 channels are activated by temperature changes within the physiological range and desensitize at noxious temperatures (Smith et al., 2002, Xu et al., 2002,

Peier et al., 2002). In addition to their expression in sensory neurons, TRPV2 channels are also expressed in vascular smooth muscle cells and can be activated by cell swelling caused by hypotonic solutions (Muraki et al., 2003). Another group reported that stimulation with insulin like growth factor translocates TRPV2 channels from intracellular pools into the plasma membrane in transfected CHO cells (Kanzaki et al., 1999). Besides their expression in the CNS and sensory neurons, TRPV3 channels are expressed in keratinocytes of the skin implicating that a similar temperature sensing mechanism as in neurons occurs in these cells (Peier et al., 2002). Despite some similarity of TRPV5 and TRPV6 to other TRPVs, these channels are not gated by temperature changes. The TRPV5 channel is expressed predominantly in epithelial cells of the distal tubule in the kidney and is thought to be involved in calcium reabsorption. Furthermore, TRPV5 (and TRPV6) are expressed in human placenta and could be part of the transplacental calcium transport during pregnancy (Care et al., 1991, Wissenbach et al., 2001). In addition, the TRPV6 channel is expressed in parts of the small intestine of the rat and could also be involved in intestinal calcium absorption (Peng et al., 1999) . The first identified member of the TRPM family (melastatin, TRPM1, Duncan et al., 1998) was cloned from murine melanoma cells and it was demonstrated that expression is inversely correlated with the aggressiveness of melanoma cells and the channel was therefore named melastatin. The physiological function of melastatin which is predominantly expressed in the retina in humans remains unknown as well as the roles of TRPM2, TRPM3 and TRPM4. In overexpression systems TRPM4 and TRPM5 are the first channels of the TRP group described which are voltage-modulated although a typical voltage sensor known from classical voltage gated channels was not described (Hofmann et al., 2003, Nilius et al., 2003). The TRPM7 channel has a kinase domain which phosphorylates the channel itself in vitro (Runnels et al., 2001). TRPM4 channels are expressed in all organs or cell types tested so far. Interestingly, TRPM4 is, although activated by intracellular calcium, impermeable for calcium ions. TRPM6 is expressed in epithelia of the intestine and kidney and permeable for calcium and magnesium ions and is implicated in the absorption of magnesium ions in these tissues (Schlingmann et al., 2002). TRPM6 and TRPM7 channels are different from the other members within their subgroup as their sequence not only shows the transmembrane and pore domains of an ion channel but also contains a C-terminal protein kinase domain. The channels are essential for Mg 2+ homeostasis and an inducible TRPM7 knock-out B-cell line is lethal after knock-out of TRPM7 (Nadler et al., 2001, Schmitz et al., 2003). In addition, the TRPM7 channel is permeable for metal ions such as zinc, nickel, cobalt, magnesium and manganese. The C-terminal domain of the TRPM2 channel exhibits an activity catalysing the hydrolysis of ADP-ribose (Perraud et al., 2001). The TRPM8 channels were found to be expressed very specifically in dorsal

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root ganglia but in no other healthy tissue tested so far (Peier et al., 2002, McKemy et al., 2002). The TRPM8 protein was shown to be a receptor stimulated by cold temperatures in afferent neurons. In addition, stimulation by cold-mimicking compounds such as menthol, icillin and eucalyptol activated TRPM8 channels using micromolar amounts of these compounds, a concentration that is unable to cool the surrounding bath solution. This indicates that TRPM8 channels may directly be gated by these compounds rather than by physical cooling. Furthermore, the TRPA1 channel, also known as ANKTM1, cloned by Jaqemar et al. (1999), was shown to be activated by noxious cold (Story et al., 2003). It seems that thermosensation in mammals depends on neurons expressing cold receptors and heat receptors alone or together (polymodal neurons). TRPA1 channels can be activated by temperatures of 10°C or lower but not with menthol whereas the TRPM8 protein can be activated by temperatures lower than 25°C degree. In mammals at least two cold sensing mechanism exist and it is of interest to note that TRPM8 and TRPA1 show no overlapping expression pattern in neurons. Channels of the TRPN group have so far not been found in mammals, but occur in insects, nematodes and fish. The NOMPC channel of the fruit fly and the homologous gene in the nematode are expressed in mechanosensory organs. Walker et al. (2000) demonstrated that loss of function of the NOMPC protein virtually abolishes mechanosensory transduction in flies. In mammals mechanosensory mechanisms are not thoroughly understood on a molecular level. In overexpression systems, some members of the TRPV family show increased activity after stretch of the cell membrane.

2. Pathophysiological roles of TRP-Proteins First evidence of the involvement of a TRP channel with diseases came from the TRPM1 protein (melastatin). The melastatin mRNA is suppressed in highly aggressive melanoma cells compared to melanoma cells with less metastatic potential and in benign cutaneous nevi (Duncan et al., 1998). Loss of melastatin expression correlated inversely with the disease free survival of patients with localized melanoma (Duncan et al., 2001). The authors concluded that TRPM1 expression is an indicator for melanoma aggressiveness and that TRPM1 is a tumor suppressor gene. As mentioned above, there exists an association between the genomic region harboring the TRPM5 locus and a region which might be involved in the development of the BeckwidthWiedemann syndrome, a predisposition of childhood tumors. However, a functional link between TRPM5 and Beckwidth– Wiedemann syndrome has not been demonstrated (Prawitt et al., 2000). A positional cloning approach led to the identification of TRPM6 as a potential candidate gene in patients with an autosomal-recessive hypomagnesemia disorder (hypomagnesemia with secondary hypocalcemia, HSH). Several affected individuals show frame shift mutations, deletions and point mutations in translated exons of the TRPM6

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gene locus which is located on the human chromosome 9 (Schlingmann et al., 2002). The polycystic kidney disease gene PKD2 which is distantly related to TRP channels is mutated in about 10-15 % of patients undergoing autosomal dominant polycystic kidney disease (Li et al., 2003; Koptides et al., 1999). PKD2 is a non selective cation channel and loss of function has been implied to cause polycystic kidney disease (Luo et al., 2003). The activity of the channel is influenced by the gene product of PKD1 which directly interacts with the C-terminal of PKD2 (Tsiokas et al., 1997; Xu et al., 2003, Vandorpe et al., 2002). Whether channel activity or trafficking is modulated by PKD1 / PKD2 interaction is not known. Another TRP related gene, the Mucolipidosis Typ IV (MCOLN1) gene causes a neurodegenerative lysosomal storage disorder characterized by psychomotor retardation and ophthalmological abnormalities (Sun et al., 2000, Bach, 2001). Two defined mutations in the MCOLN1 gene are found in about 95% of patients of the Ashkenazi Jewish alleles (Bargal et al., 2001; Slaugenhaupt, 2002). Whether these mutations affect function, trafficking or regulation of the MCOLN1 protein is not known. Further, no data are available whether or whether not MCOLN1 is involved in any activity or transport processes. Recently an approach was undertaken to identify differentially expressed genes from prostate tissue. This approach led to the identification of the TRPM8 cDNA (Trp-p8) which is upregulated in carcinomas of the prostate, breast, colon, lung and skin. TRPM8 transcripts are most abundantly expressed in prostate cancer. In addition low levels of TRPM8 transcripts occur in healthy prostate, whereas in benign prostate tissue TRPM8 levels were also elevated. Whether TRPM8 expression correlates with pathological stages and grades in prostate cancer was not reported. A homology based screen to identify new members of the TRP-family led to the identification of the human TRPV6 cDNA (CaT-Like). Extensive expression studies revealed that TRPV6 is expressed in healthy placenta, pancreas and salivary glands as well as in prostate cancer (Wissenbach et al., 2001). In contrast to the TRPM8 expression pattern no detectable levels of TRPV6 transcripts were found in healthy and benign prostate tissue. Furthermore, no transcripts are found in PIN lesions of the prostate (prostate intraepithelial neoplasia) which may consist of premalign cells. TRPV6 expression correlates with Gleason grading and tumor staging (Fixemer et al., 2003). Using in situ hybridization techniques, no TRPV6 transcripts were detectable in very small tumors and tumors of stage PT1 which are confined to the prostate. A fraction of tumors (20%) of the stage PT2 which is also confined to the prostate exhibits TRPV6 transcripts. In about 79% of PT3a tumors which extend out of the prostate capsule, TRPV6 transcripts were detected. This amount increases to more than 90% of PT3b tumors which have invaded into the seminal vesicles. These results clearly indicate that prostate cancers which have the potency to spread out of the prostate express TRPV6 transcripts. Since patients with

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extraprostatic extension have a bad prognosis, TRPV6 monitoring may be useful to predict the clinical outcome. Several malign tissues including melanoma and carcinoma of the pancreas do not express TRPV6 transcripts, this implicates that TRPV6 overexpression is not a common feature of all cancers. TRPV6 is, when overexpressed, a spontaneously active calcium selective ion channel which is located within the plasma membrane of human cells. The localization and function of the TRPV6 protein offers the possibility to use TRPV6 as a drug target to establish pharmacological treatment of prostate cancer. So far, no specific inhibitors are available for TRP channels in general and most of the TRP channels do not have a high conductance which limits their use to identify channel inhibitors by high throughput screening.

Acknowledgements This work was supported by the Wilhelm Sander-Stiftung, the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

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