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.     

The impact of menstrual cycle phase on cardiac autonomic regulation

This study investigated menstrual cycle phase differences in heart rate (HR) and RR interval variability (RRV) in 49 healthy, premenopausal, eumenorrheic women (age 30.2±6.2 years). HR and RRV were computed from ambulatory 24-h electrocardiogram, collected for up to 6 days, with at least 1 day each during early to midfollicular and midluteal menstrual phases. Phase effects on HR and RRV were assessed using linear mixed effects models with a random intercept to account for the correlation of observations within each subject as well as intrasubject variation. During follicular phase monitoring, women had significantly lower average HR (−2.33 bpm), and higher standard deviation, the root mean squared successive difference, and high frequency (0.04–0.15 Hz) and low frequency (0.15–0.40 Hz) RRV than during the luteal phase. These results provide strong support for the influence of menstrual phase on cardiac autonomic regulation in premenopausal women.     

Independent initiation of primary electron transfer in the two branches of the photosystem I reaction center

Photosystem I (PSI) is a large pigment-protein complex that unites a reaction center (RC) at the core with ∼100 core antenna chlorophylls surrounding it. The RC is composed of two cofactor branches related by a pseudo-C2 symmetry axis. The ultimate electron donor, P₇₀₀ (a pair of chlorophylls), and the tertiary acceptor, F_X (a Fe₄S₄ cluster), are both located on this axis, while each of the two branches is made up of a pair of chlorophylls (ec2 and ec3) and a phylloquinone (PhQ). Based on the observed biphasic reduction of F_X, it has been suggested that both branches in PSI are competent for electron transfer (ET), but the nature and rate of the initial electron transfer steps have not been established. We report an ultrafast transient absorption study of Chlamydomonas reinhardtii mutants in which specific amino acids donating H-bonds to the 13¹-keto oxygen of either ec3_A (PsaA-Tyr696) or ec3_B (PsaB-Tyr676) are converted to Phe, thus breaking the H-bond to a specific ec3 cofactor. We find that the rate of primary charge separation (CS) is lowered in both mutants, providing direct evidence that the primary ET event can be initiated independently in each branch. Furthermore, the data provide further support for the previously published model in which the initial CS event occurs within an ec2/ec3 pair, generating a primary ec2⁺ec3^- radical pair, followed by rapid reduction by P₇₀₀ in the second ET step. A unique kinetic modeling approach allows estimation of the individual ET rates within the two cofactor branches.     

Metabolic cycling in single yeast cells from unsynchronized steady-state populations limited on glucose or phosphate

Oscillations in patterns of expression of a large fraction of yeast genes are associated with the “metabolic cycle,” usually seen only in prestarved, continuous cultures of yeast. We used FISH of mRNA in individual cells to test the hypothesis that these oscillations happen in single cells drawn from unsynchronized cultures growing exponentially in chemostats. Gene-expression data from synchronized cultures were used to predict coincident appearance of mRNAs from pairs of genes in the unsynchronized cells. Quantitative analysis of the FISH results shows that individual unsynchronized cells growing slowly because of glucose limitation or phosphate limitation show the predicted oscillations. We conclude that the yeast metabolic cycle is an intrinsic property of yeast metabolism and does not depend on either synchronization or external limitation of growth by the carbon source.     

Systolic blood pressure, not BIS, is associated with movement during laryngoscopy and intubation

OBJECTIVE: To compare bispectral index (BIS) values to hemodynamic variations, in order to evaluate adequacy of anesthesia during orotracheal intubation with muscle relaxants. METHODS: Forty-one patients ASA I-II, scheduled for elective peripheral surgery under general anesthesia with tracheal intubation were enrolled in the study. Fentanyl/thiopental followed by vecuronium were used for induction. Onset of relaxation was monitored at the orbicularis occuli (OO) muscle using train-of-four stimulation. Intubation was performed when no response at the OO was detected visually. Intubating conditions were noted. The "isolated forearm" technique was used to detect movement during laryngoscopy/intubation. BIS values, pulse rate (PR), and systolic pressure were recorded before induction, during laryngoscopy/intubation and 60 sec after intubation. RESULTS: Although intubating conditions were clinically adequate for all patients, ten out of 41 had movement of the isolated arm during laryngoscopy/intubation. BIS values were not significantly different for these patients: 67 (55-83) compared to those who had no movement: 60 (35-80), P = 0.6. During laryngoscopy, PR increased for all patients while systolic pressure increased significantly only in patients who moved: 125 (100-136) mmHg vs those who did not: 108 (67-140), P < 0.05. CONCLUSION: Systolic pressure elevations were associated with inadequate anesthesia as evaluated by the "isolated forearm" technique, during laryngoscopy/intubation. BIS values were not different between groups, suggesting that systolic blood pressure may be a better predictor of inadequate anesthesia under the circumstances described.     

Pharmacokinetics and pharmacodynamics of rapacuronium in patients with cirrhosis.

BACKGROUND: Delayed elimination kinetics of steroidal neuromuscular blocking agents have been observed in patients with cirrhosis. Like other steroidal muscle relaxants, rapacuronium may, in part, be eliminated by the liver. To determine the influence of liver disease on its neuromuscular blocking effect, we studied the pharmacokinetics and pharmacodynamics of rapacuronium in patients with cirrhosis. METHODS: Sixteen patients undergoing elective surgery or endoscopy with general anesthesia, eight with cirrhosis and eight with normal liver function, were studied. Anesthesia was induced with fentanyl 2 microg/kg and thiopental 5-7 mg/kg and maintained with 60% nitrous oxide and 0.6-0.8% isoflurane in oxygen and repeated doses of fentanyl 1 microg/kg. Rapacuronium 1.5 mg/kg was administered intravenously before tracheal intubation. Thumb adduction force evoked by supramaximal ulnar nerve stimulation was recorded in 16 patients. Venous blood was sampled at frequent intervals for 8 h. Rapacuronium and its breakdown product Org 9488 were measured in plasma by high-pressure liquid chromatography. Values are reported as median (range). RESULTS: The central volume of distribution was increased to 131 (104-141) ml/kg in patients with cirrhosis (P < 0.01), compared with 75 (47-146) ml/kg in controls. The total apparent volume of distribution was also increased (P < 0.05) to 331 (284-488) ml/kg in patients with cirrhosis, compared with 221 (124-285) ml/kg in controls. The elimination half-life was 88 (77-102) min in controls and 90 (76-117) min in patients with cirrhosis. Plasma clearance was increased (P < 0.05) to 6.9 (6.1-8.9) ml x min(-1) x kg(-1) in patients with cirrhosis, compared with 5.3 (4.2-8.4) ml x min(-1) x kg(-1) in controls. Rapacuronium neuromuscular blocking effect was similar between the two groups. Onset time was 65 (40-110) s in controls and of 60 (52-240) s in patients with cirrhosis. Time to return to 90% of thumb adduction force control value was of 49 (28-80) min in controls and 47 (28-71) min in patients with cirrhosis. CONCLUSION: The neuromuscular blocking effect of a single bolus dose of rapacuronium in patients with cirrhosis is not different from that of patients with normal hepatic function. No decrease in plasma clearance of rapacuronium was observed in patients with cirrhosis.     

The combined IGG, IGM and IGA peroxidase conjugate can facilitate determination of immune complexes by CIF-ELISA and anti-C3 ELISA.

To determine the levels of circulating immune complexes (CIC) in normal and patients sera, CIF-ELISA and anti-C3 ELISA were performed. Immune complexes containing different antibody isotypes were detected simultaneously by the combined anti human IgG, IgM and IgA peroxidase conjugate as detecting antibody. The results obtained confirm the higher CIF-ELISA sensitivity, specificity and reproductivity compared to anti-C3 ELISA and provide good evidence to justify the use of CIF-ELISA as a screening test for CIC assessment.     

Proof‐of‐concept evidence for trimodal simultaneous investigation of human brain function

The link between spatial (where) and temporal (when) aspects of the neural correlates of most psychological phenomena is not clear. Elucidation of this relation, which is crucial to fully understand human brain function, requires integration across multiple brain imaging modalities and cognitive tasks that reliably modulate the engagement of the brain systems of interest. By overcoming the methodological challenges posed by simultaneous recordings, the present report provides proof‐of‐concept evidence for a novel approach using three complementary imaging modalities: functional magnetic resonance imaging (fMRI), event‐related potentials (ERPs), and event‐related optical signals (EROS). Using the emotional oddball task, a paradigm that taps into both cognitive and affective aspects of processing, we show the feasibility of capturing converging and complementary measures of brain function that are not currently attainable using traditional unimodal or other multimodal approaches. This opens up unprecedented possibilities to clarify spatiotemporal integration of brain function.