TRPM8 Research Articles

Highly Ca2+-selective TRPM Channels Regulate IP3-dependent Oscillatory Ca2+Signaling in theC. elegansIntestine

Xing et al. 2008. J. Gen. Physiol. doi:10.1085/jgp.200709914 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1085%252Fjgp.200709914%26rft_id%253Dinfo%253Apmid%252F18299395%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%

One TRPM8 fits all

One TRPM8 fits all

The Expression of the Ca++Channel α2δ Subunit and TRPM8 in the Dorsal Root Ganglion of Sympathetically Maintained Pain and Sympathetic Independent Pain Rat Models

접수일:2008년 2월 14일, 승인일:2008년 3월 18일 책임저자:이윤우, (135-720) 서울시 강남구 도곡동 146-92 영동세브란스병원 마취통증의학과 Tel: 02-2019-3523, Fax: 02-3463-0940 E-mail: ywleepain@yuhs.ac 본 연구는 연세대학교 의과대학 2005년도 교수연구비에 의하여 이루어 졌음(과제번호: 2005-0036호). Received February 14, 2008, Accepted March 18, 2008 Correspondence to: Youn-Woo Lee Department of Anesthesiology and Pain Medicine, Yonsei University College of Medicine, Yongdong Severance Hospital, 146-92, Dogok-dong, Gangnam-gu, Seoul 135-720, Korea Tel: +82-2-2019-3523, Fax: +82-2-3463-0940 E-mail: ywleepain@yuhs.ac This study was supported by a faculty research grant of Yonsei University College of Medicine for 2005. 교감신경 의존적 및 비의존적 신경병증 통증 쥐 모델 후근신경 절에서 Ca Channel α2δ Subunit와 TRPM8 발현

Distribution profiles of transient receptor potential melastatin-related and vanilloid-related channels in prostatic tissue in rat

To investigate the expression and distribution of the members of the transient receptor potential (TRP) channel members of TRP melastatin (TRPM) and TRP vanilloid (TRPV) subfamilies in rat prostatic tissue. Prostate tissue was obtained from male Sprague-Dawley rats. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (PCR) were used to check the expression of all TRPM and TRPV channel members with specific primers. Immunohistochemistry staining for TRPM8 and TRPV1 were also performed in rat tissues. TRPM2, TRPM3, TRPM4, TRPM6, TRPM7, TRPM8, TRPV2 and TRPV4 mRNA were detected in all rat prostatic tissues. Very weak signals for TRPM1, TRPV1 and TRPV3 were also detected. The mRNA of TRPM5, TRPV5 and TRPV6 were not detected in all RT-PCR experiments. Quantitative real-time RT-PCR showed that TRPM2, TRPM3, TRPM4, TRPM8, TRPV2 and TRPV4 were the most abundantly expressed TRPM and TRPV subtypes, respectively. Fluorescence immunohistochemistry indicated that TRPM8 and TRPV1 are highly expressed in both epithelial and smooth muscle cells. Our results demonstrate that mRNA or protein for TRPM1, TRPM2, TRPM3, TRPM4, TRPM6, TRPM7, TRPM8, TRPV1, TRPV2, TRPV3 and TRPV4 exist in rat prostatic tissue. The data presented here assists in elucidating the physiological function of TRPM and TRPV channels.

Characterisation of TRPM8 as a pharmacophore receptor

Some proteins of the transient receptor potential (TRP) family form temperature sensitive ion channels. One member of the melastatin (M) group, namely TRPM8 is activated by cold and cooling compounds such as menthol and icilin, and its gene is up-regulated in prostate cancer and other malignancies. Here we characterise the effects of the carboxamides WS-12, CPS-113, CPS-369, the carboxylic acid WS-30 and the phosphine oxide WS-148 by Ca 2+ imaging experiments and whole-cell patch-clamp recordings on TRPM8 expressing human embryonic kidney (HEK), lymph node prostate cancer (LNCaP) and dorsal root ganglia (DRG) cells. The cooling compounds introduced in this study, show a dose-dependent and reversible activation of TRPM8 with EC 50 values in the nM to low μM range. The carboxamide WS-12 is most potent in activating TRPM8. It is selective, since other TRP proteins are not stimulated at μM concentrations and its efficacy with respect to TRPM8 is similar to the one of icilin. In summary, the compounds described in this study represent new tools to dissect TRPM8 functions and may serve as chemical leads for the development of additional TRPM8 agonists and novel antagonists. Such compounds may be beneficial for preventing noxious cold perception. They could also be useful in diagnosis and treatment of most common cancers in which the TRPM8 gene is up-regulated in comparison to the corresponding normal tissue.

Determination of lipid‐ and protein‐protein interactions in the C‐terminal part of TRPM4

Determination of lipid‐ and protein‐protein interactions in the C‐terminal part of TRPM4

Molecular components of vertebrate Mg2+-homeostasis regulation.

Over the past decades, the clinical relevance and biological significance of Mg2+ have been thoroughly documented. Although multiple Mg2+-transport pathways have been biophysically characterized, the molecular identity of the postulated components of Mg2+-homeostasis regulation in vertebrates remain undefined. Recent advances in the fields of genetics, genomics and proteomics, and novel technologies such as cDNA microarrays have allowed for substantial progress in this area. The mitochondrial Mrs2 protein was the first human Mg2+ transporter characterized as such, and an important element for future analyses of the role of mitochondria in managing intracellular Mg2+. Several molecules with Mg2+ transport capabilities have been identified through a screen designed to find genes upregulated under hypomagnesic conditions. This includes SLC41A1 and 2, ACDP2 and MagT1. Finally, the elucidation of the molecular cause underlying two different hereditary diseases leading to hypomagnesemia resulted in the cloning and characterization of claudin 16 (paracellin-1), and TRPM6. Whereas claudin 16 plays a crucial role in paracellular Mg2+ transport, TRPM6 is involved in the transcellular pathway. TRPM6 and its closest relative TRPM7 are both puzzling ion channel-kinase fusions, and perhaps the most unexpected newly identified players in the regulation of Mg2+-homeostasis in vertebrates.

Hypomagnesemia with Secondary Hypocalcemia due to a Missense Mutation in the Putative Pore-forming Region of TRPM6

Hypomagnesemia with secondary hypocalcemia is an autosomal recessive disorder caused by mutations in the TRPM6 gene. Current experimental evidence suggests that TRPM6 may function in a specific association with TRPM7 by means of heterooligomeric channel complex formation. Here, we report the identification and functional characterization of a new hypomagnesemia with secondary hypocalcemia missense mutation in TRPM6. The affected subject presented with profound hypomagnesemia and hypocalcemia caused by compound heterozygous mutation in the TRPM6 gene: 1208(-1)G > A affecting the acceptor splice site preceding exon 11, and 3050C > G resulting in the amino acid change (P1017R) in the putative pore-forming region of TRPM6. To assess the functional consequences of the P1017R mutation, TRPM6(P1017R) and wild-type TRPM6 were co-expressed with TRPM7 in Xenopus oocytes and HEK 293 cells, and currents were assessed by two-electrode voltage clamp and whole cell patch clamp measurements, respectively. Co-expression of wild-type TRPM6 and TRPM7 resulted in a significant increase in the amplitude of TRPM7-like currents. In contrast, TRPM6(P1017R) suppressed TRPM7 channel activity. In line with these observations, TRPM7, containing the corresponding mutation P1040R, displayed a dominant-negative effect upon co-expression with wild-type TRPM7. Confocal microscopy and fluorescence resonance energy transfer recordings demonstrated that the P1017R mutation neither affects assembly of TRPM6 with TRPM7, nor co-trafficking of heteromultimeric channel complexes to the cell surface. We conclude that a functional defect in the putative pore of TRPM6/7 channel complexes is sufficient to impair body magnesium homeostasis.

Subunit interaction in channel assembly and functional regulation of transient receptor potential melastatin (TRPM) channels

Functional TRPM (transient receptor potential melastatin) ion channels are multimers, thought to be tetramers. Subunit interaction is the prerequisite step in channel assembly, and the specificity of subunit interaction is crucial in assembling channels with distinct functional properties. In addition, expression of short non-functional subunits and their interaction with full-length subunits serve as one of the post-translational mechanisms regulating the channel activity. This paper aims to provide an overview of the current knowledge of TRPM subunit interactions and their roles in assembly and functional regulation of the TRPM channels.

TRPM3, a biophysical enigma?

TRPM3 [TRP (transient receptor potential) melastatin 3] is one of the least investigated proteins of the TRP family of ion channels. Heterologously expressed TRPM3 channels are constitutively active, have an outwardly rectifying current-voltage relationship and are inhibited by intracellular Mg(2+) ions. Besides these rather common features, in which TRPM3 channels resemble the closely related channels TRPM6 and TRPM7, TRPM3 channels have several unique characteristics. The TRPM3 gene encodes a plethora of different proteins owing to alternative splicing and alternative exon usage. One site of alternative splicing affects the ion-conducting pore region and profoundly alters the pore properties of the encoded channels. The channels having the longer pore region efficiently conduct univalent cations, but are only poorly permeated by bivalent cations. Conversely, the channels with the shorter pore region are highly permeable to bivalent cations. Unusually, the short-pore TRPM3 channels are inhibited by extracellular Na(+) ions. At physiological sodium concentration, this block is very strong, making it difficult to envision a physiological function for these ion channels. Recently, pharmacological investigations have been initiated in order to identify substances that influence TRPM3 channel activity. With the use of such substances, it might be possible to identify TRPM3 channels in their native environment and to elucidate some of their physiological roles. Hopefully, TRPM3 channels will then no longer appear to be as enigmatic as they do right now.

Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides

We have developed an in situ hybridization procedure for the detection of microRNAs (miRNAs) in tissue sections from mouse embryos and adult organs. The method uses highly specific washing conditions for RNA oligonucleotide probes conjugated to a fluorescein hapten. We show that this method detects predominantly mature miRNAs rather than the miRNA precursors or primary transcripts. We have determined expression patterns for several miRNAs expressed in the developing and adult nervous system, including miR-124a, miR-9, miR-92, and miR-204. Whereas miR-124a is expressed in neurons, miR-9 is expressed in neural progenitors and some neurons, and miR-204 is expressed in the choroid plexus, retinal pigment epithelium, and ciliary body. miR-204 is located in an intron of the TRPM3 gene, and the TRPM3 mRNA is coexpressed with miR-204 in the choroid plexus. We also find that primary transcripts for miR-124a and miR-9 genes are expressed in patterns similar to their respective mature miRNAs. The ability to visualize expression of specific miRNAs in embryos and tissues should aid studies on miRNA function.

The Epithelial Mg2+ Channel Transient Receptor Potential Melastatin 6 Is Regulated by Dietary Mg2+ Content and Estrogens

The kidney is the principal organ responsible for the regulation of the body Mg(2+) balance. Identification of the gene defect in hypomagnesemia with secondary hypocalcemia recently elucidated transient receptor potential melastatin 6 (TRPM6) as the gatekeeper in transepithelial Mg(2+) transport, whereas its homolog, TRPM7, is implicated in cellular Mg(2+) homeostasis. The aim of this study was to determine the tissue distribution in mouse and regulation of TRPM6 and TRPM7 by dietary Mg(2+) and hormones. This study demonstrates that TRPM6 is expressed predominantly in kidney, lung, cecum, and colon, whereas TRPM7 is distributed ubiquitously. Dietary Mg(2+) restriction in mice resulted in hypomagnesemia and renal Mg(2+) and Ca(2+) conservation, whereas a Mg(2+)-enriched diet led to increased urinary Mg(2+) and Ca(2+) excretion. Conversely, Mg(2+) restriction significantly upregulated renal TRPM6 mRNA levels, whereas a Mg(2+) enriched diet increased TRPM6 mRNA expression in colon. Dietary Mg(2+) did not alter TRPM7 mRNA expression in mouse kidney and colon. In addition, it was demonstrated that 17beta-estradiol but not 1,25-dihydroxyvitamin D(3) or parathyroid hormone regulates TRPM6 renal mRNA levels. Renal TRPM7 mRNA abundance remained unaltered under these conditions. The renal TRPM6 mRNA level in ovariectomized rats was significantly reduced, whereas 17beta-estradiol treatment normalized TRPM6 mRNA levels. In conclusion, kidney, lung, cecum, and colon likely constitute the main sites of active Mg(2+) (re)absorption in the mouse. In addition, Mg(2+) restriction and 17beta-estradiol upregulated renal TRPM6 mRNA levels, whereas a Mg(2+)-enriched diet stimulated TRPM6 mRNA expression in colon, supporting the gatekeeper function of TRPM6 in transepithelial Mg(2+) transport.

Physiological genomics in PG and beyond: October to December 2005

physiological genomics ( PG ) publishes, on a quarterly basis, brief editorial perspectives entitled “Physiological genomics in PG and beyond.” These editorial perspectives provide brief, accessible highlights of the studies published in PG in the most recent quarter, and relate those studies to

Genetic background of HSH in three Polish families and a patient with an X;9 translocation

Hypomagnesemia with secondary hypocalcemia (HSH) is a rare inherited disease, characterised by neurological symptoms, such as tetany, muscle spasms and seizures, due to hypocalcemia. It has been suggested that HSH is genetically heterogeneous, but only one causative gene, TRPM6, on chromosome 9 has so far been isolated. We have now studied the genetic background of HSH in four Polish patients belonging to three families, and a HSH patient carrying an apparently balanced X;9 translocation. The translocation patient has long been considered as an example of the X-linked form of HSH. We identified six TRPM6 gene mutations, of which five were novel, in the Polish patients. All the alterations were either nonsense/splicing or missense mutations. The clinical picture of the patients was similar to the HSH patients reported earlier. No genotype-phenotype correlation could be detected. Sequencing did not reveal any TRPM6 or TRPM7 gene mutations in the female HSH patient with an X;9 translocation. Isolation of the translocation breakpoint showed that the chromosome 9 specific breakpoint mapped within satellite III repeat sequence. The X-chromosomal breakpoint was localised to the first intron of the vascular endothelial growth factor gene, VEGFD. No other sequence alterations were observed within the VEGFD gene. Even though the VEGFD gene was interrupted by the X;9 translocation, it seems unlikely that VEGFD is causing the translocation patient's HSH-like phenotype. Furthermore, re-evaluation of patient's clinical symptoms suggests that she did not have a typical HSH.

Novel Role of Cold/Menthol-sensitive Transient Receptor Potential Melastatine Family Member 8 (TRPM8) in the Activation of Store-operated Channels in LNCaP Human Prostate Cancer Epithelial Cells

Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (I(cold/menthol)) that shows inward rectification and high Ca(2+) selectivity, which are dramatically different properties from "classical" TRPM8-mediated I(cold/menthol). Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both I(cold/menthol) and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca(2+) release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca(2+) release channel, potentially involved in a number of Ca(2+)- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis.

The Selectivity Filter of the Cation Channel TRPM4

Transient receptor potential channel melastatin subfamily (TRPM) 4 and its close homologue, TRPM5, are the only two members of the large transient receptor potential superfamily of cation channels that are impermeable to Ca(2+). In this study, we located the TRPM4 selectivity filter and investigated possible structural elements that render it Ca(2+)-impermeable. Based on homology with known cation channel pores, we identified an acidic stretch of six amino acids in the loop between transmembrane helices TM5 and TM6 ((981)EDMDVA(986)) as a potential selectivity filter. Substitution of this six-amino acid stretch with the selectivity filter of TRPV6 (TIIDGP) resulted in a functional channel that combined the gating hallmarks of TRPM4 (activation by Ca(2+), voltage dependence) with TRPV6-like sensitivity to block by extracellular Ca(2+) and Mg(2+) as well as Ca(2+) permeation. Neutralization of Glu(981) resulted in a channel with normal permeability properties but a strongly reduced sensitivity to block by intracellular spermine. Neutralization of Asp(982) yielded a functional channel that exhibited extremely fast desensitization (tau < 5 s), possibly indicating destabilization of the pore. Neutralization of Asp(984) resulted in a non-functional channel with a dominant negative phenotype when coexpressed with wild type TRPM4. Combined neutralization of all three acidic residues resulted in a functional channel whose voltage dependence was shifted toward very positive potentials. Substitution of Gln(977) by a glutamate, the corresponding residue in divalent cation-permeable TRPM channels, altered the monovalent cation permeability sequence and resulted in a pore with moderate Ca(2+) permeability. Our findings delineate the selectivity filter of TRPM channels and provide the first insight into the molecular basis of monovalent cation selectivity.

Evidence for specific TRPM8 expression in human prostate secretory epithelial cells: functional androgen receptor requirement

TRPM8 (melastatine-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily of cation channels, has been shown to be a calcium-channel protein. TRPM8 mRNA has also been shown to be overexpressed in prostate cancer and is considered to play an important role in prostate physiology. This study was designed to determine the androgen-regulation mechanisms for TRPM8 mRNA expression and to identify the phenotype of TRPM8-expressing cells in the human prostate. Our findings show that trpm8 gene expression requires a functional androgen receptor. Furthermore, this article argues strongly in favour of the fact that the trpm8 gene is a primary androgen-responsive gene. Single-cell reverse transcriptase PCR and immunohistochemical experiments also showed that the trpm8 gene was mainly expressed in the apical secretory epithelial cells of the human prostate and trpm8 down-regulation occurred during the loss of the apical differentiated phenotype of the primary cultured human prostate epithelial cells. The androgen-regulated trpm8 expression mechanisms are important in understanding the progression of prostate cancer to androgen-independence. These findings may contribute to design a strategy to predict prostate cancer status from the TRPM8 mRNA level. Furthermore, as the TRPM8 channel is localized in human prostate cells, it will be interesting to understand its physiological function in the normal prostate and its potential role in prostate cancer development.

A Potential Rearrangement between CYP19 and TRPM7 Genes on Chromosome 15q21.2 as a Cause of Aromatase Excess Syndrome

Aromatase excess syndrome (AES) is a rare hereditary autosomal dominant disorder characterized by increased extraglandular aromatization of steroids and presented with heterosexual precocity in males and isosexual precocity in females. The objective was to study the molecular basis of AES in a kindred with 16 affected subjects, both males and females. The propositus, currently a 17-year-old boy, presented with breast enlargement in the first year of life, which persisted thereafter. Investigations at the age of 7.5 yr revealed growth acceleration (height sd score, 2.8), puberty staging Tanner P1B3, testicular volume 6 ml, and bone age 13 yr. The hormonal data were compatible with increased conversion of androgens to estrogens, which was independent of gonadotropin secretion. In the affected adults, there were short stature (height sd score ranged from -3.7 to -2), gynecomastia in males, and macromastia in females. Linkage analysis was performed using a polymorphic tetranucleotide (TTTA) repeat marker at nucleotide position 682 of CYP gene, as well as two additional STS markers, D15S123 (CA)n and D15S209 (CA)n, located within genetic distance of less than 5 cM from CYP19 gene. Using RNA extracted from the breast tissue of the propositus, a 5'-rapid amplification of cDNA ends (RACE) was performed with gene-specific primers corresponding to exon 2 of CYP19 gene. Linkage analysis with (TTTA)n, D15S123 (CA)n, and D15S209 (CA)n markers produced LOD scores 0.85, 1.5, and 1.17, respectively. 5'-RACE revealed a novel untranslated exon 1 composed by exon 1 of TRPM7 gene (Transient Receptor Potential Cation Channel, Subfamily M, member 7), which has ubiquitous expression. 5'-RACE finding points to a potential rearrangement between CYP19 and TRPM7 genes on chromosome 15q21.2 as a cause of AES.

TRPM7: channeling the future of cellular magnesium homeostasis?

A recent paper by Schmitz and colleagues provides persuasive evidence that the ion channel transient receptor potential melastatin 7 (TRPM7) may be the long-sought regulator of magnesium (Mg) homeostasis in mammalian cells. This finding is not unexpected, because TRPM channels contain a kinase domain that allows them to participate in signal transduction pathways and regulatory networks. However, these studies introduce an exciting new twist into our understanding of Mg homeostasis; TRPM7 facilitates Mg entry into the cell, whereas other putative Mg transporters apparently operate in the opposite direction. By combining electrophysiological, biochemical, and genetic approaches, Schmitz and colleagues characterized most of the key features that demonstrate a well-defined and biologically plausible regulator of Mg homeostasis. TRPM7 genetics are well in hand, its regulation by intracellular free Mg2+ unravels the mechanisms of regulatory feedback loops, and its kinase domain modulates its sensitivity to free Mg2+. These findings are discussed in light of the indirect and descriptive information we had about Mg regulation before this rigorous characterization of TRPM7 brought it to the center of the Mg stage. Although the molecular events downstream of TRPM7 phosphorylation and dephosphorylation await in-depth elucidation, these results open up exciting perspectives in Mg research and may provide a much-needed tool with which to reexamine the role of Mg in cell proliferation and other important pathophysiologic events. Likewise, these findings will offer guidelines for research on disease states that are characterized by Mg imbalance.

The effects of spinal cord injury on the status of messenger ribonucleic acid for TRPM 2 and androgen receptor in the prostate of the rat.

The prostate is one of the male accessory sex glands that produce fluid components of the seminal plasma. In addition to androgen, a normal innervation of the prostate is believed to be important for maintaining normal function of the prostate. Previously we noted that, in the rat, the weight of the prostate decreased following surgically induced spinal cord injury (SCI). This observation suggests that growth, and possibly function, of the prostate may be compromised after SCI. To explore this possibility, we examined the effects of SCI on the androgen-related biochemical properties and morphology of the prostate in the rat at various times after surgically induced SCI. SCI resulted in an acute decrease in prostate weight and an increase in steady state level of mRNA for testosterone-repressed prostate message 2 (TRPM 2) during the first 2 weeks postinjury. These changes perhaps relate to an increase in cell death or a decrease in secretory activity due to an acute suppression of serum testosterone after the injury. Concomitantly, there was a transient, but significant, decrease in the steady state level of androgen receptor (AR) mRNA in the prostate during the first 2 weeks after SCI, an indication of an altered autoregulation of AR by its own ligand. Despite the fact that growth of the prostate, as indicated by weight increase, in SCI rats resumed 2 weeks postinjury, prostate weights were persistently lower in SCI rats than sham-operated controls for at least 3 months. Furthermore, prostate TRPM 2 mRNA levels remained elevated throughout the recovery period even after a normal prostate weight had been restored. In addition, a decrease in the height of ventral prostate epithelial cells was noted in SCI rats 28 and 90 days postinjury. These results demonstrate a prolonged effect of SCI on prostate function. These findings and our unreported observation of persistently smaller seminal vesicles in the same groups of SCI rats suggest that functions of male accessory sex glands may also be compromised after SCI. These changes may affect biochemical properties of the secretory products of these glands and may provide some explanation for the reported changes in the composition of the seminal plasma and abnormal sperm motility seen in the semen of SCI men.