Putative chanzyme activity of TRPM2 cation channel is unrelated to pore gating

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable cation channel expressed in immune cells of phagocytic lineage, pancreatic β cells, and brain neurons and is activated under oxidative stress. TRPM2 activity is required for immune cell activation and insulin secretion and is responsible for postischemic neuronal cell death. TRPM2 is opened by binding of ADP ribose (ADPR) to its C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain, which, when expressed in isolation, cleaves ADPR into AMP and ribose-5-phosphate. A suggested coupling of this enzymatic activity to channel gating implied a potentially irreversible gating cycle, which is a unique feature of a small group of channel enzymes known to date. The significance of such a coupling lies in the conceptually distinct pharmacologic strategies for modulating the open probability of channels obeying equilibrium versus nonequilibrium gating mechanisms. Here we examine the potential coupling of TRPM2 enzymatic activity to pore gating. Mutation of several residues proposed to enhance or eliminate NUDT9-H catalytic activity all failed to affect channel gating kinetics. An ADPR analog, α-β-methylene-ADPR (AMPCPR), was shown to be entirely resistant to hydrolysis by NUDT9, but nevertheless supported TRPM2 channel gating, albeit with reduced apparent affinity. The rate of channel deactivation was not slowed but, rather, accelerated in AMPCPR. These findings, as well as detailed analyses of steady-state gating kinetics of single channels recorded in the presence of a range of concentrations of ADPR or AMPCPR, identify TRPM2 as a simple ligand-gated channel that obeys an equilibrium gating mechanism uncoupled from its enzymatic activity.Transient receptor potential melastatin 2 (TRPM2) belongs to the TRP protein family and is abundantly expressed in brain neurons, bone marrow, phagocytes, pancreatic β cells, and cardiomyocytes, where it forms Ca²⁺-permeable nonselective cation channels that open under oxidative stress. On contact with pathogens, phagocytic cells produce reactive oxygen species (ROS); the resulting activation of TRPM2 provides the Ca²⁺ influx necessary for cell migration and chemokine production (1). In pancreatic β cells, TRPM2 activity contributes to glucose-evoked insulin secretion; TRPM2 knock-out mice show higher resting blood glucose levels and impaired glucose tolerance (2).TRPM2 activity is also linked to several pathologic conditions that lead to apoptosis (3). Reperfusion after ischemia results in ROS generation; consequent Ca²⁺ influx through TRPM2 causes Ca²⁺ dysregulation and cell death. Certain neurodegenerative diseases, such as Alzheimer’s disease, also involve oxidative stress and TRPM2 activation. In contrast, a loss-of-function TRPM2 mutation identified in patients with amyotrophic lateral sclerosis and Parkinson''s disease dementia (4), as well as two TRPM2 mutations associated with bipolar disorder (5), suggest loss of TRPM2 activity can also cause disease.Similar to most TRP family ion channels, the TRPM2 channel is a homotetramer, and its transmembrane (TM) architecture resembles that of voltage-gated cation channels (6, 7). In addition to the TM domain and an N-terminal cytosolic domain of unknown function, TRPM2 contains an ∼270-residue C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain. The latter shows high (∼50%) sequence homology to the soluble mitochondrial enzyme NUDT9, an active ADP ribose (ADPR) pyrophosphatase (ADPRase) from the Nudix hydrolase family, which splits ADPR into AMP and ribose-5-phosphate (8). TRPM2 channels are coactivated by ADPR binding to NUDT9-H (9) and by Ca²⁺ binding to unidentified intracellular binding sites (10). ADPR is the key that links TRPM2 activation to oxidative stress; in living cells exposed to ROS, ADPR is released from mitochondria (9). In the past, studying TRPM2 channel gating at steady state has been limited by rapid deactivation of TRPM2 currents in cell-free patches (10). This rundown was recently shown to involve a conformational change of the ion selectivity filter, which could be completely prevented by a pore-loop substitution. This “T5L” TRPM2 variant, which shows no rundown but preserves intact regulation of gating by Ca²⁺ and ADPR (11), provides an unprecedented opportunity to study TRPM2 gating at steady state.Early studies reported slow (∼0.1 s⁻¹) but detectable ADPRase activity of isolated purified NUDT9-H (8, 12), classifying TRPM2 into the special group of channel-enzymes (“chanzymes”) that includes TRPM6 and TRPM7 (3) and the CFTR cystic fibrosis transmembrane conductance regulator (CFTR) chloride ion channel (13). TRPM2 pore opening/closure happens on the timescale of the reported ADPRase activity (11), which is consistent with coupling between gating and catalytic activity, as demonstrated for CFTR in which pore gating follows an irreversible cycle tightly linked to ATP binding and hydrolysis at conserved cytosolic domains (14).The involvement of TRPM2 in multiple diseases has made it an emerging therapeutic target. Depending on the disease, both inhibition (e.g., stroke, myocardial infarction, Alzheimer’s disease, chronic inflammation, hyperinsulinism) and stimulation (e.g., diabetes, amyotrophic lateral sclerosis, Parkinson''s disease dementia, bipolar disorder) of TRPM2 activity might be useful therapeutically. Because TRP family channels are involved in diverse processes (3), any useful TRPM2 agonists/antagonists will need to be highly selective. This singles out the NUDT9-H domain, the component unique to TRPM2, as the most attractive drug target. The significance of understanding whether ADPRase activity and gating are coupled is that optimal strategies for modulating fractional occupancy of a particular conformational state are profoundly different for equilibrium systems than for nonequilibrium systems. For most ion channels, pore gating is an equilibrium process, and open probability is modulated simply by energetic stabilization of either open (activators) or closed (inhibitors) channel ground states. In contrast, channels that gate by a nonequilibrium cycle are most efficiently accumulated in either open or closed states by manipulating the stability of transition states for rate-limiting irreversible steps (15). The aim of this study was to examine the tightness of coupling between the ADPRase cycle and specific gating transitions in TRPM2.     

Comparison of aortic and carotid arterial stiffness parameters in patients with verified coronary artery disease

Background:

Arterial stiffness parameters are commonly used to determine the development of atherosclerotic disease. The independent predictive value of aortic stiffness has been demonstrated for coronary events.

Hypothesis:

The aim of our study was to compare regional and local arterial functional parameters measured by 2 different noninvasive methods in patients with verified coronary artery disease (CAD). We also compared and contrasted these stiffness parameters to the coronary SYNTAX score in patients who had undergone coronary angiography.

Methods:

In this study, 125 CAD patients were involved, and similar noninvasive measurements were performed on 125 healthy subjects. The regional velocity of the aortic pulse wave (PWVao) was measured by a novel oscillometric device, and the common carotid artery was studied by a Doppler echo‐tracking system to determine the local carotid pulse wave velocity (PWVcar). The augmentation index (AIx), which varies proportionately with the resistance of the small arteries, was recorded simultaneously.

Results:

In the CAD group, the PWVao and aortic augmentation index (Alxao) values increased significantly (10.1 ± 2.3 m/sec and 34.2% ± 14.6%) compared to the control group (9.6 ± 1.5 m/sec and 30.9% ± 12%; P < 0.05). We observed similar significant increases in the local stiffness parameters (PWVcar and carotid augmentation index [Alxcar]) in patients with verified CAD. Further, we found a strong correlation for PWV and AIx values that were measured with the Arteriograph and those obtained using the echo‐tracking method (r = 0.57, P < 0.001 for PWV; and r = 0.65, P < 0.001 for AIx values).

Conclusions:

Our results indicate that local and regional arterial stiffness parameters provide similar information on impaired arterial stiffening in patients with verified CAD. © 2011 Wiley Periodicals, Inc. This study was supported by the Hungarian National Research Foundation (OTKA) No.78480. The authors have no other funding, financial relationships, or conflicts of interest to disclose.     

A case of cellulitis causing tissue defect during etanercept therapy

Tumor necrosis factor-alpha (TNF-α) antagonists are employed increasingly during recent years in patients with active rheumatoid arthritis who do not respond to disease-modifying anti-rheumatic drugs. Contraindications such as infections, auto-antibody formation and hypersensitive reactions can be observed during the treatment with TNF-α antagonist drugs. Our case was a 52-year-old woman, followed by several centers for a period of 21 years with a seropositive, erosive and nodular RA diagnosis. Anti TNF-α treatment was commenced due to the failure to control the disease. During the treatment, a serious cellulite developed, which required hospitalization and surgical debridement as well as intravenous antibiotics treatment. Through the present case, we aimed to draw attention to the skin infection during the use of etanercept in a patient with RA.     

Enhanced bone regeneration in rat calvarial defects implanted with surface-modified and BMP-loaded bioactive glass (13-93) scaffolds

The repair of large bone defects, such as segmental defects in the long bones of the limbs, is a challenging clinical problem. Our recent work has shown the ability to create porous scaffolds of silicate 13-93 bioactive glass by robocasting which have compressive strengths comparable to human cortical bone. The objective of this study was to evaluate the capacity of those strong porous scaffolds with a grid-like microstructure (porosity = 50%; filament width = 330 μm; pore width = 300 μm) to regenerate bone in a rat calvarial defect model. Six weeks post-implantation, the amount of new bone formed within the implants was evaluated using histomorphometric analysis. The amount of new bone formed in implants composed of the as-fabricated scaffolds was 32% of the available pore space (area). Pretreating the as-fabricated scaffolds in an aqueous phosphate solution for 1, 3 and 6 days to convert a surface layer to hydroxyapatite prior to implantation enhanced new bone formation to 46%, 57% and 45%, respectively. New bone formation in scaffolds pretreated for 1, 3 and 6 days and loaded with bone morphogenetic protein-2 (BMP-2) (1 μg per defect) was 65%, 61% and 64%, respectively. The results show that converting a surface layer of the glass to hydroxyapatite or loading the surface-treated scaffolds with BMP-2 can significantly improve the capacity of 13-93 bioactive glass scaffolds to regenerate bone in an osseous defect. Based on their mechanical properties evaluated previously and their capacity to regenerate bone found in this study, these 13-93 bioactive glass scaffolds, pretreated or loaded with BMP-2, are promising in structural bone repair.     

Mechanical properties of bioactive glass (13-93) scaffolds fabricated by robotic deposition for structural bone repair

There is a need to develop synthetic scaffolds to repair large defects in load-bearing bones. Bioactive glasses have attractive properties as a scaffold material for bone repair, but data on their mechanical properties are limited. The objective of the present study was to comprehensively evaluate the mechanical properties of strong porous scaffolds of silicate 13-93 bioactive glass fabricated by robocasting. As-fabricated scaffolds with a grid-like microstructure (porosity 47%, filament diameter 330 μm, pore width 300 μm) were tested in compressive and flexural loading to determine their strength, elastic modulus, Weibull modulus, fatigue resistance, and fracture toughness. Scaffolds were also tested in compression after they were immersed in simulated body fluid (SBF) in vitro or implanted in a rat subcutaneous model in vivo. As fabricated, the scaffolds had a strength of 86 ± 9 MPa, elastic modulus of 13 ± 2 GPa, and a Weibull modulus of 12 when tested in compression. In flexural loading the strength, elastic modulus, and Weibull modulus were 11 ± 3 MPa, 13 ± 2 GPa, and 6, respectively. In compression, the as-fabricated scaffolds had a mean fatigue life of ～10⁶ cycles when tested in air at room temperature or in phosphate-buffered saline at 37 °C under cyclic stresses of 1–10 or 2–20 MPa. The compressive strength of the scaffolds decreased markedly during the first 2 weeks of immersion in SBF or implantation in vivo, but more slowly thereafter. The brittle mechanical response of the scaffolds in vitro changed to an elasto-plastic response after implantation for longer than 2–4 weeks in vivo. In addition to providing critically needed data for designing bioactive glass scaffolds, the results are promising for the application of these strong porous scaffolds in loaded bone repair.     

Solid pseudopapillary tumor of the pancreas: ‘Experiences’ and ‘Lessons’ at a tertiary‐care oncology center

Solid pseudopapillary tumor of the pancreas (SPT) is a rare and fascinating entity of elusive histogenesis and unpredictable biology. It has a peculiar proclivity to afflict young females and involve the pancreatic body‐tail region. Cytology diagnosis of these rare neoplasms remains a challenge. We analyzed the cytology features of all SPT cases diagnosed on fine needle aspiration cytology (FNAC) from 2003 to 2009 along with their histopathology slides. Nineteen consecutive cases were diagnosed as SPT on FNAC. Fifteen out of nineteen cases were confirmed as true SPT on histopathology. Amongst the true SPT, all except one occurred in females. Age ranged from 14 to 50 years. Pseudopapillae bearing stout branches terminating in bulbous tips and enclosing transgressing vessels, separated from a collar of tumor cells by a clear zone of myxohyaline coat were pathognomonic of SPT. Singly dispersed monomorphic tumor cells with bland chromatin formed the second diagnostic component of SPT. Nuclear grooves and hyaline globules were in addition helpful in segregating SPT from its close differentials. In four cases diagnosed as SPT on FNAC, histopathology revealed a different final diagnosis (one case each of paraganglioma, extragastrointestinal stromal tumor, metastatic papillary renal cell carcinoma and inflammatory myofibroblastic tumor). Conversely, one case of SPT had been erroneously diagnosed as neuroendocrine tumor on FNAC. Six cases (40%) developed metastasis; commonest site being liver. In conclusion, cytology in conjunction with clinico‐radiologic findings plays a key role in making a correct diagnosis. Awareness of unique cytomorphological features is important in distinguishing this tumor from its diverse mimics. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.