Christopher D. Benham

The diversity in the vanilloid (TRPV) receptor family of ion channels

Following cloning of the vanilloid receptor 1 (VR1) at least four other related proteins have been identified. Together, these form a distinct subgroup of the transient receptor potential (TRP) family of ion channels. Members of the vanilloid receptor family (TRPV) are activated by a diverse range of stimuli, including heat, protons, lipids, phorbols, phosphorylation, changes in extracellular osmolarity and/or pressure, and depletion of intracellular Ca2+ stores. However, VR1 remains the only channel activated by vanilloids such as capsaicin. These channels are excellent molecular candidates to fulfil a range of sensory and/or cellular roles that are well characterized physiologically. Furthermore, as novel pharmacological targets, the vanilloid receptors have potential for the development of many future disease treatments.

Cloning and functional expression of a human orthologue of rat vanilloid receptor-1

&NA; Capsaicin, resiniferatoxin, protons or heat have been shown to activate an ion channel, termed the rat vanilloid receptor‐1 (rVR1), originally isolated by expression cloning for a capsaicin sensitive phenotype. Here we describe the cloning of a human vanilloid receptor‐1 (hVR1) cDNA containing a 2517 bp open reading frame that encodes a protein with 92% homology to the rat vanilloid receptor‐1. Oocytes or mammalian cells expressing this cDNA respond to capsaicin, pH and temperature by generating inward membrane currents. Mammalian cells transfected with human VR1 respond to capsaicin with an increase in intracellular calcium. The human VR1 has a chromosomal location of 17p13 and is expressed in human dorsal root ganglia and also at low levels throughout a wide range of CNS and peripheral tissues. Together the sequence homology, similar expression profile and functional properties confirm that the cloned cDNA represents the human orthologue of rat VR1.

[3H]Resiniferatoxin autoradiography in the CNS of wild-type and TRPV1 null mice defines TRPV1 (VR-1) protein distribution

Knowledge of the distribution and function of the vanilloid receptor (VR-1 or TRPV1) in the CNS lacks the detailed appreciation of its role in the peripheral nervous system. The radiolabelled vanilloid agonist [3H]resiniferatoxin (RTX) has been used to indicate the presence of TRPV1 receptor protein in the brain but low specific binding has complicated interpretation of this data. Recently, support for a more widespread CNS distribution of TRPV1 mRNA and protein has been provided by RT-PCR and antibody data. We have exploited the availability of TRPV1 null mice and used [3H]RTX autoradiography in the CNS of TRPV1 wild-type and TRPV1 null mice to identify the component of [3H]RTX binding to TRPV1 receptor protein. In the brains of TRPV1+/+ mice, specific [3H]RTX binding was broadly localised with the greatest binding in the olfactory nuclei, the cerebral cortex, dentate gyrus, thalamus, hypothalamus, periaqueductal grey, superior colliculus, locus coeruleus and cerebellar cortex. Specific binding was also seen in the spinal cord and sensory (dorsal root and trigeminal) ganglia. This binding was much lower but not abolished in most regions in the TRPV1-/- mice. Nonspecific binding was low in all cases. The present study unequivocally demonstrates a widespread and discrete distribution pattern of the TRPV1 receptor protein in the rat central nervous system. The presence of TRPV1 receptors in several brain regions suggests that it may function as a cannabinoid-gated channel in the CNS.

TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP‐ribose) polymerase

TRPM2 (melastatin‐like transient receptor potential 2 channel) is a nonselective cation channel that is activated under conditions of oxidative stress leading to an increase in intracellular free Ca2+ concentration ([Ca2+]i) and cell death. We investigated the role of the DNA repair enzyme poly(ADP‐ribose) polymerase (PARP) on hydrogen peroxide (H2O2)‐mediated TRPM2 activation using a tetracycline‐inducible TRPM2‐expressing cell line. In whole‐cell patch‐clamp recordings, intracellular adenine 5′‐diphosphoribose (ADP‐ribose) triggered an inward current in tetracycline‐induced TRPM2‐human embryonic kidney (HEK293) cells, but not in uninduced cells. Similarly, H2O2 stimulated an increase in [Ca2+]i (pEC50 4.54±0.02) in Fluo‐4‐loaded TRPM2‐expressing HEK293 cells, but not in uninduced cells. Induction of TRPM2 expression caused an increase in susceptibility to plasma membrane damage and mitochondrial dysfunction in response to H2O2. These data demonstrate functional expression of TRPM2 following tetracycline induction in TRPM2‐HEK293 cells. PARP inhibitors SB750139‐B (patent number DE10039610‐A1 (Lubisch et al., 2001)), PJ34 (N‐(6‐oxo‐5,6‐dihydro‐phenanthridin‐2‐yl)‐N,N‐dimethylacetamide) and DPQ (3, 4‐dihydro‐5‐[4‐(1‐piperidinyl)butoxy]‐1(2H)‐isoquinolinone) inhibited H2O2‐mediated increases in [Ca2+]i (pIC50 vs 100 μM H2O2: 7.64±0.38; 6.68±0.28; 4.78±0.05, respectively), increases in mitochondrial dysfunction (pIC50 vs 300 μM H2O2: 7.32±0.23; 6.69±0.22; 5.44±0.09, respectively) and decreases in plasma membrane integrity (pIC50 vs 300 μM H2O2: 7.45±0.27; 6.35±0.18; 5.29±0.12, respectively). The order of potency of the PARP inhibitors in these assays (SB750139>PJ34>DPQ) was the same as for inhibition of isolated PARP enzyme. SB750139‐B, PJ34 and DPQ had no effect on inward currents elicited by intracellular ADP‐ribose in tetracycline‐induced TRPM2‐HEK293 cells, suggesting that PARP inhibitors are not interacting directly with the channel. SB750139‐B, PJ34 and DPQ inhibited increases in [Ca2+]i in a rat insulinoma cell line (CRI‐G1 cells) endogenously expressing TRPM2 (pIC50 vs 100 μM H2O2: 7.64±0.38; 6.68±0.28; 4.78±0.05, respectively). These data suggest that oxidative stress causes TRPM2 channel opening in both recombinant and endogenously expressing cell systems via activation of PARP enzymes.

Tissue Distribution Profiles of the Human TRPM Cation Channel Family

Eight members of the TRP-melastatin (TRPM) subfamily have been identified, whose physiological functions and distribution are poorly characterized. Although tissue expression and distribution patterns have been reported for individual TRPM channels, comparisons between individual studies are not possible because of variations in analysis techniques and tissue selection. We report here a comparative analysis of the expression patterns of all of the human TRPM channels in selected peripheral tissues and the central nervous system (CNS) using two distinct but complimentary approaches: TaqMan and SYBR Green real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). These techniques generated comparative distribution profiles and demonstrated tissue-specific co-expression of TRPM mRNA species, indicating significant potential for the formation of heteromeric channels. TRPM channels 2, 4, 5, 6, and 7 in contrast to 1, 3, and 8 are widely distributed in the CNS and periphery. The tissues demonstrating highest expression for individual family members were brain (TRPM1), brain and bone marrow (TRPM2), brain and pituitary (TRPM3), intestine and prostate (TRPM4), intestine, pancreas, and prostate (TRPM5), intestine and brain (TRPM6), heart, pituitary, bone, and adipose tissue (TRPM7), and prostate and liver (TRPM8). The data reported here will guide the elucidation of TRPM channel physiological functions.

Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy

BackgroundTransient receptor potential (TRP) receptors expressed by primary sensory neurons mediate thermosensitivity, and may play a role in sensory pathophysiology. We previously reported that human dorsal root ganglion (DRG) sensory neurons co-expressed TRPV1 and TRPV3, and that these were increased in injured human DRG. Related receptors TRPV4, activated by warmth and eicosanoids, and TRPM8, activated by cool and menthol, have been characterised in pre-clinical models. However, the role of TRPs in common clinical sensory neuropathies needs to be established.MethodsWe have studied TRPV1, TRPV3, TRPV4, and TRPM8 in nerves (n = 14) and skin from patients with nerve injury, avulsed dorsal root ganglia (DRG) (n = 11), injured spinal nerve roots (n = 9), diabetic neuropathy skin (n = 8), non-diabetic neuropathic nerve biopsies (n = 6), their respective control tissues, and human post mortem spinal cord, using immunohistological methods.ResultsTRPV1 and TRPV3 were significantly increased in injured brachial plexus nerves, and TRPV1 in hypersensitive skin after nerve repair, whilst TRPV4 was unchanged. TRPM8 was detected in a few medium diameter DRG neurons, and was unchanged in DRG after avulsion injury, but was reduced in axons and myelin in injured nerves. In diabetic neuropathy skin, TRPV1 expressing sub- and intra-epidermal fibres were decreased, as was expression in surviving fibres. TRPV1 was also decreased in non-diabetic neuropathic nerves. Immunoreactivity for TRPV3 was detected in basal keratinocytes, with a significant decrease of TRPV3 in diabetic skin. TRPV1-immunoreactive nerves were present in injured dorsal spinal roots and dorsal horn of control spinal cord, but not in ventral roots, while TRPV3 and TRPV4 were detected in spinal cord motor neurons.ConclusionThe accumulation of TRPV1 and TRPV3 in peripheral nerves after injury, in spared axons, matches our previously reported changes in avulsed DRG. Reduction of TRPV1 levels in nerve fibres in diabetic neuropathy skin may result from the known decrease of nerve growth factor (NGF) levels. The role of TRPs in keratinocytes is unknown, but a relationship to changes in NGF levels, which is produced by keratinocytes, deserves investigation. TRPV1 represents a more selective therapeutic target than other TRPs for pain and hypersensitivity, particularly in post-traumatic neuropathy.

TRPV channels as temperature sensors.

The past year has seen a doubling in the number of heat-sensitive ion channels to six, and four of these channels are from the TRPV family. These channels characteristically have Q(10) values of >10 above the thermal threshold, very different from the Q(10) values of 1.5-2.0 seen in most ion channels. Cells expressing TRPV1 show similar temperature sensitivity to small capsaicin-sensitive nociceptor neurons, consistent with these neurons expressing homomers of TRPV1. A-delta fibres exhibit properties that may be explained by TRPV2 containing channels which is present in large diameter sensory neurons that do not express TRPV1. TRPV3 has a lower temperature threshold and may contribute to warm-sensitive channels together with TRPV1. Warm sensation may also be transduced by TRPV4 expressing sensory neurons and hypothalamic neurons. We can now look forward to further work defining the properties of the recombinant channels in more detail and a re-analysis of endogenous i(heat) currents in thermosensitive neurons and other cells. Data from the study of mice in which TRPV2, TRPV3 or TRPV4 have been deleted are also eagerly awaited.

Amyloid β-peptide(1–42) and hydrogen peroxide-induced toxicity are mediated by TRPM2 in rat primary striatal cultures

Amyloid β‐peptide (Aβ) is the main component of senile plaques which characterize Alzheimers disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin‐like transient receptor potential 2 (TRPM2) is a Ca2+‐permeant non‐selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Aβ and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2‐S) inhibited both hydrogen peroxide‐ and Aβ‐induced increases in intracellular‐free Ca2+ and cell death. Functional inhibition of TRPM2 activation by the poly(ADP‐ribose)polymerase inhibitor SB‐750139, a modulator of intracellular pathways activating TRPM2, attenuated hydrogen peroxide‐ and Aβ‐induced cell death. Furthermore, a small interfering RNA which targets TRPM2, reduced TRPM2 mRNA levels and the toxicity induced by hydrogen peroxide and Aβ. These data demonstrate that activation of TRPM2, functionally expressed in primary cultures of rat striatum, contributes to Aβ‐ and oxidative stress‐induced striatal cell death.

Vanilloid and TRP channels: a family of lipid-gated cation channels.

The emergence of the TRP (C) and vanilloid (TRPV) receptor family of Ca(2+) permeable channels has started to provide molecular focus to a linked group of ion channels whose common feature is activation primarily by intracellular ligands. These channels have a central role in Ca(2+) homeostasis in virtually all cells and in particular those that lack voltage-gated Ca(2+) channels. We will discuss recent work that is more precisely defining both molecular form and physiological function of this important group of Ca(2+) permeable channels with particular focus on the intracellular ligands that gate and modulate channel activity.

Cool and menthol receptor TRPM8 in human urinary bladder disorders and clinical correlations.

BackgroundThe recent identification of the cold-menthol sensory receptor (TRPM8; CMR1), provides us with an opportunity to advance our understanding of its role in the pathophysiology of bladder dysfunction, and its potential mediation of the bladder cooling reflex. In this study, we report the distribution of the cool and menthol receptor TRPM8 in the urinary bladder in patients with overactive and painful bladder syndromes, and its relationship with clinical symptoms.MethodsBladder specimens obtained from patients with painful bladder syndrome (PBS, n = 16), idiopathic detrusor overactivity (IDO, n = 14), and asymptomatic microscopic hematuria (controls, n = 17), were immunostained using specific antibodies to TRPM8; nerve fibre and urothelial immunostaining were analysed using fibre counts and computerized image analysis respectively. The results of immunohistochemistry were compared between the groups and correlated with the Pain, Frequency and Urgency scores.ResultsTRPM8-immunoreactive staining was observed in the urothelium and nerve fibres scattered in the suburothelium. The nerve fibre staining was seen in fine-calibre axons and thick (myelinated) fibres. There was marked increase of TRPM8-immunoreactive nerve fibres in IDO (P = 0.0249) and PBS (P < 0.0001) specimens, compared with controls. A significantly higher number of TRPM8-immunoreactive axons were also seen in the IDO (P = 0.0246) and PBS (P < 0.0001) groups. Urothelial TRPM8 and TRPM8-immunoreactive thick myelinated fibres appeared unchanged in IDO and PBS. The relative density of TRPM8-immunoreactive nerve fibres significantly correlated with the Frequency (r = 0.5487, P = 0.0004) and Pain (r = 0.6582, P < 0.0001) scores, but not Urgency score.ConclusionThis study demonstrates increased TRPM8 in nerve fibres of overactive and painful bladders, and its relationship with clinical symptoms. TRPM8 may play a role in the symptomatology and pathophysiology of these disorders, and may provide an additional target for future overactive and painful bladder pharmacotherapy.