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Pore dilation occurs in TRPA1 but not in TRPM8 channels
Jun Chen, Donghee Kim, Bruce R Bianchi, Eric J Cavanaugh, Connie R Faltynek, Philip R Kym, Regina M Reilly
Molecular Pain , 2009, DOI: 10.1186/1744-8069-5-3
Abstract: Abundantly expressed in sensory neurons, TRPV1, TRPA1 and TRPM8 are involved in sensory function, pain and neurogenic inflammation [1]. The function of these ion channels has been attributed to their ability to pass certain ion species across the plasma membrane. Once activated, TRPV1, TRPA1 and TRPM8 are permeable to small cations such as Ca2+, K+, Na+; hence, channel activation simultaneously depolarizes the plasma membrane and raises intracellular Ca2+, which subsequently triggers a variety of physiological processes. By analogy to voltage-gated K+ channels, it is assumed that ion selectivity of TRP channels should be an invariant signature to the respective channel. However, this notion has been challenged recently. When activated, TRPV1 exhibits time and agonist-dependent changes in ion selectivity [2]. In fact, TRPV1 undergoes pore dilation and allows permeation of large organic cations, including spermine (202.3 Da), NMDG (195.2 Da), Yo-Pro (376 Da), gentamycin (477.6 Da) and QX-314 [3-7]. Here we explored whether TRPA1 and TRPM8 undergo pore dilation by examining Yo-Pro uptake and changes in ion selectivity upon channel activation.Yo-Pro is a divalent cation impermeable to the plasma membrane. However, under certain conditions, it can enter cells, bind nucleic acids and emit fluorescence. Hence the uptake of Yo-Pro has been used previously as an indicator of pore dilation [2,8,9]. In HEK293-F cells transiently expressing rat TRPA1, allyl isothiocyanate (AITC) evoked robust increases in intracellular Ca2+ (Fig. 1A). Concomitantly, AITC also induced Yo-Pro uptake in a concentration-dependent manner (Fig. 1B). At higher concentrations of AITC (100 or 300 μM), the increase in fluorescence was immediately noticeable and continued to increase for about 50 min. In addition, AITC also induced Ca2+ influx and Yo-Pro uptake in cells expressing human TRPA1 and mouse TRPA1, but not in untransfected cells (data not shown). In cells expressing human TRPM8, menthol activat
Inorganic Polyphosphate Modulates TRPM8 Channels  [PDF]
Eleonora Zakharian, Baskaran Thyagarajan, Robert J. French, Evgeny Pavlov, Tibor Rohacs
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005404
Abstract: Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms complexes and modulates activities of many proteins including ion channels. Here we investigated the role of polyP in the function of the transient receptor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp and fluorescent calcium measurements we demonstrate that enzymatic breakdown of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in human embryonic kidney and F-11 neuronal cells expressing TRPM8. We demonstrate that the TRPM8 channel protein is associated with polyP. Furthermore, addition of scPPX1 altered the voltage-dependence and blocked the activity of the purified TRPM8 channels reconstituted into planar lipid bilayers, where the activity of the channel was initiated by cold and menthol in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2). The biochemical analysis of the TRPM8 protein also uncovered the presence of poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with polyP. We conclude that the TRPM8 protein forms a stable complex with polyP and its presence is essential for normal channel activity.
L type Ca2+ channel blockers prevent oxaliplatin-induced cold hyperalgesia and TRPM8 overexpression in rats
Takehiro Kawashiri, Nobuaki Egashira, Kentaro Kurobe, Kuniaki Tsutsumi, Yuji Yamashita, Soichiro Ushio, Takahisa Yano, Ryozo Oishi
Molecular Pain , 2012, DOI: 10.1186/1744-8069-8-7
Abstract: Cold hyperalgesia was assessed by the acetone test. Oxaliplatin (4 mg/kg), sodium oxalate (1.3 mg/kg) or vehicle was injected i.p. on days 1 and 2. Ca2+ (diltiazem, nifedipine and ethosuximide) and Na+ (mexiletine) channel blockers were administered p.o. simultaneously with oxaliplatin or oxalate on days 1 and 2.Oxaliplatin (4 mg/kg) induced cold hyperalgesia and increased in the transient receptor potential melastatin 8 (TRPM8) mRNA levels in the dorsal root ganglia (DRG). Furthermore, oxalate (1.3 mg/kg) significantly induced the increase in TRPM8 protein in the DRG. Treatment with oxaliplatin and oxalate (500 μM for each) also increased the TRPM8 mRNA levels and induced Ca2+ influx and nuclear factor of activated T-cell (NFAT) nuclear translocation in cultured DRG cells. These changes induced by oxalate were inhibited by nifedipine, diltiazem and mexiletine. Interestingly, co-administration with nifedipine, diltiazem or mexiletine prevented the oxaliplatin-induced cold hyperalgesia and increase in the TRPM8 mRNA levels in the DRG.These data suggest that the L type Ca2+ channels/NFAT/TRPM8 pathway is a downstream mediator for oxaliplatin-induced cold hyperalgesia, and that Ca2+ channel blockers have prophylactic potential for acute neuropathy.Oxaliplatin, a platinum-based chemotherapeutic agent, is widely used for treatment of colorectal cancer. However, oxaliplatin frequently causes severe acute and chronic peripheral neuropathies. Acute neuropathy is peculiar to oxaliplatin and includes acral paresthesias enhanced by exposure to cold [1-4]; the acute neuropathy is not attributed to morphological damage to the nerve [5,6]. On the other hand, the chronic neuropathy is characterized by loss of sensory and motor function after long-term oxaliplatin treatment, and it is similar to cisplatin-induced neurological symptoms [4]. Recently, we reported that repeated administration of oxaliplatin induced cold hyperalgesia in the early phase and mechanical allodynia in the l
NGF – the TrkA to successful pain treatment
Kumar V, Mahal BA
Journal of Pain Research , 2012, DOI: http://dx.doi.org/10.2147/JPR.S33408
Abstract: he TrkA to successful pain treatment Review (1997) Total Article Views Authors: Kumar V, Mahal BA Published Date August 2012 Volume 2012:5 Pages 279 - 287 DOI: http://dx.doi.org/10.2147/JPR.S33408 Received: 29 April 2012 Accepted: 30 May 2012 Published: 17 August 2012 Vinayak Kumar,1 Brandon A Mahal2 1Department of Chemistry, College of Arts and Sciences, University of Pennsylvania Philadelphia, PA, USA; 2Beth Israel Deaconess Medical Center, Harvard Medical School, Harvard University, Cambridge, MA, USA Abstract: Chronic pain arising from various pathological conditions such as osteoarthritis, low back or spinal injuries, cancer, and urological chronic pelvic pain syndromes presents significant challenges in diagnosis and treatment. Specifically, since the underlying cause of these pain syndromes is unknown or heterogeneous, physicians diagnose and treat patients based on the symptoms presented. Nerve growth factor (NGF) has been recognized as an important mediator of chronic pain in many pathological conditions, and has been shown to be upregulated in a subset of individuals suffering from such pain syndromes. These findings have led to the development of anti-NGF monoclonal antibodies such as tanezumab as potentially effective therapeutics for chronic pain. Although tanezumab has reached Phase II and III clinical trials, the trials of anti-NGF antibodies were halted due to safety concerns. Some of these trials of anti-NGF treatment have had statistically significant decreases in pain, while others have yielded inconclusive results. These findings are suggestive of, though do not prove, target (NGF) neutralization in chronic pain syndromes. A biomarker-driven anti-NGF clinical study layout is proposed that incorporates NGF measurements in the relevant samples before and after treatment, in addition to collecting the pain scores. This approach might not only confirm the mechanism of tanezumab's action in these chronic pain patients, but should establish NGF levels as a predictive biomarker for patients who can benefit from anti-NGF treatment, thereby creating a personalized approach to pain treatment.
Spectroscopic Investigations of Pentobarbital Interaction with Transthyretin  [PDF]
Saqer M. Darwish,Jafar Ghithan,Musa M. Abuteir,Mariam Faroun,Mahmoud M. Abu-hadid
Journal of Spectroscopy , 2013, DOI: 10.1155/2013/927962
Abstract: Transthyretin (TTR) aggregation has been characterized to be responsible for several amyloid diseases. Fourier transform infrared (FTIR) spectroscopy, fluorescence, and atomic force microscopy (AFM) are used to investigate secondary structure changes in transthyretin, induced upon thermal denaturation and interaction with pentobarbital. Spectral analysis revealed a strong static quenching of the intrinsic fluorescence of TTR by pentobarbital with a binding constant (K) estimated at . Fourier self-deconvolution (FSD) technique is used to evaluates intensity changes in the spectra of the component bands in the amide I and amide II regions due to the changes in pentobarbital concentration in the protein complex. The increases of the relative intensities of the peaks at 1614?cm?1 and 1507?cm?1 are due to the increase of pentobarbital concentrations which is linked to the formation of oligomers in the protein. 1. Introduction Transthyretin (TTR) is a plasma protein composed of 127-residue subunits mainly composed of β-sheet structures [1]. It is present in both human plasma and cerebrospinal fluid (CSF) with concentrations of (0.1–0.4?mg/mL) in human plasma and (0.017?mg/mL) in CSF [2]. X-ray crystal structure studies have shown that human TTR have a molecular weight of 55?kDa in a tetramer form with four identical subunits [3]. TTR is synthesized by the liver and released in the plasma, while the TTR in CSF is mostly produced by the choroid plexus [4–6]. It is considered to be the primary transporter of thyroid hormones in the form of thyroxine in the CSF and it carries retinol via interaction with the retinol-binding protein (RBP) [7]. Other additional function of TTR has been detected in the development of the central nervous system due to the high concentration during the prenatal and postnatal life [8]. Several research groups have shown cerebral TTR expression to rise during the course of experimental Alzheimer disease (AD) in mice and in response to the intake of some drug or mixtures of compounds such as gingko extracts or dietary fatty acids [9–11]. The process of transthyretin amyloidogenesis or amyloid fibril formation seems to be associated with some amyloid diseases. It is not understood precisely how TTR forms amyloids, but several biophysical studies on wild-type (WT) TTR reveals that tetramer dissociation is rate limiting for amyloidogenesis [12–14]. All amyloid diseases are characterized by misfolded proteins that undergo aggregation causing a deposition of insoluble amyloid fibrils either systemically or in specific organs as the brain
NGF Causes TrkA to Specifically Attract Microtubules to Lipid Rafts  [PDF]
Shona Pryor, Gretchen McCaffrey, Lindsay R. Young, Mark L. Grimes
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0035163
Abstract: Membrane protein sorting is mediated by interactions between proteins and lipids. One mechanism that contributes to sorting involves patches of lipids, termed lipid rafts, which are different from their surroundings in lipid and protein composition. Although the nerve growth factor (NGF) receptors, TrkA and p75NTR collaborate with each other at the plasma membrane to bind NGF, these two receptors are endocytosed separately and activate different cellular responses. We hypothesized that receptor localization in membrane rafts may play a role in endocytic sorting. TrkA and p75NTR both reside in detergent-resistant membranes (DRMs), yet they responded differently to a variety of conditions. The ganglioside, GM1, caused increased association of NGF, TrkA, and microtubules with DRMs, but a decrease in p75NTR. When microtubules were induced to polymerize and attach to DRMs by in vitro reactions, TrkA, but not p75NTR, was bound to microtubules in DRMs and in a detergent-resistant endosomal fraction. NGF enhanced the interaction between TrkA and microtubules in DRMs, yet tyrosine phosphorylated TrkA was entirely absent in DRMs under conditions where activated TrkA was detected in detergent-sensitive membranes and endosomes. These data indicate that TrkA and p75NTR partition into membrane rafts by different mechanisms, and that the fraction of TrkA that associates with DRMs is internalized but does not directly form signaling endosomes. Rather, by attracting microtubules to lipid rafts, TrkA may mediate other processes such as axon guidance.
Guideline of transthyretin-related hereditary amyloidosis for clinicians  [cached]
Ando Yukio,Coelho Teresa,Berk John L,Cruz Márcia Waddington
Orphanet Journal of Rare Diseases , 2013, DOI: 10.1186/1750-1172-8-31
Abstract: Transthyretin amyloidosis is a progressive and eventually fatal disease primarily characterized by sensory, motor, and autonomic neuropathy and/or cardiomyopathy. Given its phenotypic unpredictability and variability, transthyretin amyloidosis can be difficult to recognize and manage. Misdiagnosis is common, and patients may wait several years before accurate diagnosis, risking additional significant irreversible deterioration. This article aims to help physicians better understand transthyretin amyloidosis—and, specifically, familial amyloidotic polyneuropathy—so they can recognize and manage the disease more easily and discuss it with their patients. We provide guidance on making a definitive diagnosis, explain methods for disease staging and evaluation of disease progression, and discuss symptom mitigation and treatment strategies, including liver transplant and several pharmacotherapies that have shown promise in clinical trials.
Transthyretin (prealbumin) in eye structures and variation of vitreous-transthyretin in diseases  [cached]
Ramakrishnan S,Sulochana K,Parikh Sunil,Punitham R
Indian Journal of Ophthalmology , 1999,
Abstract: Purpose: To evaluate the presence of transthyretin (TTR, prealbumin) a protein which binds retinol to retinol-binding protein in various ocular tissues and to study its quantitative changes in the vitreous humor in various diseases Method: Estimation of TTR was done by electrophoresis of 10 mg protein in each sample of tears, aqueous humor, vitreous, retina, and lens by an Imaging Densitometer using prealbumin as the standard. Results: TTR was present in all the eye structures except the lens and tear. The retina and the vitreous had relatively higher amounts of TTR compared with aqueous. The identity of TTR was confirmed by immuno-electrophoresis using anti-human TTR. Two bands in SDS electrophoresis revealed that this protein is a heterodimer. There was a significant decrease in vitreous TTR in diabetes with hypertension and increase in one case each of diabetes with hypertension associated with leukaemia or carcinoma with hepato-splenomegaly. Conclusion: Vitreous TTR is probably from retina and retinal pigment epithelium. The level of vitreous TTR is likely to have diagnostic significance in some retinal diseases.
Transthyretin and Amyloid in the Islets of Langerhans in Type-2 Diabetes  [PDF]
Gunilla T. Westermark,Per Westermark
Experimental Diabetes Research , 2008, DOI: 10.1155/2008/429274
Abstract: Transthyretin (TTR) is a major amyloid fibril protein in certain systemic forms of amyloidosis. It is a plasma protein, mainly synthesized by the liver but expression occurs also at certain minor locations, including the endocrine cells in the islets of Langerhans. With the use of immunohistochemistry and in situ hybridization, we have studied the distribution of transthyretin-containing cells in islets of Langerhans in type-2 diabetic and nondiabetic individuals. TTR expression was particularly seen in alpha (glucagon) cells. Islets from type-2 diabetic patients had proportionally more transthyretin-reactive islet cells, including beta cells. A weak transthyretin immunoreaction in IAPP-derived amyloid occurred in some specimens. In seeding experiments in vitro, we found that TTR fibrils did not seed IAPP while IAPP fibrils seeded TTR. It is suggested that islet expression of transthyretin may be altered in type-2 diabetes.
Cooling-Sensitive TRPM8 Is Thermostat of Skin Temperature against Cooling  [PDF]
Koji Tajino,Hiroshi Hosokawa,Shingo Maegawa,Kiyoshi Matsumura,Ajay Dhaka,Shigeo Kobayashi
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017504
Abstract: We have shown that cutaneous cooling-sensitive receptors can work as thermostats of skin temperature against cooling. However, molecule of the thermostat is not known. Here, we studied whether cooling-sensitive TRPM8 channels act as thermostats. TRPM8 in HEK293 cells generated output (y) when temperature (T) was below threshold of 28.4°C. Output (y) is given by two equations: At T >28.4°C, y = 0; At T <28.4°C, y = -k(T – 28.4°C). These equations show that TRPM8 is directional comparator to elicits output (y) depending on negative value of thermal difference (ΔT = T – 28.4°C). If negative ΔT-dependent output of TRPM8 in the skin induces responses to warm the skin for minimizing ΔT recursively, TRPM8 acts as thermostats against cooling. With TRPM8-deficient mice, we explored whether TRPM8 induces responses to warm the skin against cooling. In behavioral regulation, when room temperature was 10°C, TRPM8 induced behavior to move to heated floor (35°C) for warming the sole skin. In autonomic regulation, TRPM8 induced activities of thermogenic brown adipose tissue (BAT) against cooling. When menthol was applied to the whole trunk skin at neutral room temperature (27°C), TRPM8 induced a rise in core temperature, which warmed the trunk skin slightly. In contrast, when room was cooled from 27 to 10°C, TRPM8 induced a small rise in core temperature, but skin temperature was severely reduced in both TRPM8-deficient and wild-type mice by a large heat leak to the surroundings. This shows that TRPM8-driven endothermic system is less effective for maintenance of skin temperature against cooling. In conclusion, we found that TRPM8 is molecule of thermostat of skin temperature against cooling.
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