Activation Of Nonselective Cation Channels Research Articles

Enhancement of muscarinic receptor-mediated excitation in spontaneously hypertensive rat adrenal medullary chromaffin cells.

One of the mechanisms for hypertension is an increase in blood catecholamines due to increased secretion from sympathetic nerve terminals and adrenal medullary chromaffin (AMC) cells. Spontaneously hypertensive rats (SHRs) are used as an animal model of hypertension. Catecholamine secretion in AMC cells occurs in response to humoral factors and neuronal inputs from the sympathetic nerve fibres. Acetylcholine (ACh) released from the nerve terminals activates nicotinic as well as muscarinic ACh receptors. The present experiment aimed to elucidate whether muscarinic receptor-mediated excitation is altered in SHR AMC cells and, if it is, how. Compared with normotensive rat AMC cells, muscarinic stimulation induced greater catecholamine secretion and larger depolarising inward currents in SHR AMC cells. In contrast to normotensive rat AMC cells, the muscarine-induced current consisted of quinine-sensitive and quinine-insensitive components. The former and the latter are possibly ascribed to nonselective cation channel activation and TWIK-related acid-sensitive K+ (TASK) channel inhibition, as noted in guinea pig AMC cells. In fact, immunoreactive material for TASK1 and several isoforms of transient receptor potential canonical (TRPC) channels was detected in SHR AMC cells. Stromal interaction molecule 1 (STIM1), which plays an essential role for heteromeric TRPC1-TRPC4 channel formation and is not expressed in normotensive rat AMC cells, was detected in the cytoplasm and co-localised with TRPC1. The expression of muscarinic M1 receptors was enhanced in SHR AMC cells compared with normotensive rats. The results indicate that muscarinic excitation is enhanced in SHR AMC cells, probably through facilitation of TRPC channel signalling.

Extracellular histone proteins activate P2XR7 channel current.

Extracellular histone proteins are elevated in circulation after injury or activation of the innate immune response. In resistance-size arteries, extracellular histone proteins increased endothelial cell (EC) Ca2+ influx and propidium iodide (PI) labeling, but paradoxically decreased vasodilation. These observations could be explained by the activation of an EC resident non-selective cation channel. We tested the hypothesis that the ionotropic purinergic receptor 7 (P2XR7), a non-selective cation channel associated with cationic dye uptake, is activated by histone proteins. We expressed mouse P2XR7 (C57BL/6J variant 451L) in heterologous cells and measured inward cation current using two-electrode voltage clamp (TEVC). Cells expressing mouse P2XR7 had robust ATP- and histone-evoked inward cation currents. ATP- and histone-evoked currents reversed approximately at the same potential. Current decay with agonist removal was slower for histone-evoked than ATP- or BzATP-evoked currents. As with ATP-evoked P2XR7 currents, histone-evoked currents were inhibited by non-selective P2XR7 antagonists (Suramin, PPADS, and TNP-ATP). Selective P2XR7 antagonists, AZ10606120, A438079, GW791343, and AZ11645373, inhibited ATP-evoked P2XR7 currents but did not inhibit histone-evoked P2XR7 currents. As previously reported with ATP-evoked currents, histone-evoked P2XR7 currents were also increased in conditions of low extracellular Ca2+. These data demonstrate that P2XR7 is necessary and sufficient for histone-evoked inward cation currents in a heterologous expression system. These results provide insight into a new allosteric mechanism of P2XR7 activation by histone proteins.

A wheat resistosome defines common principles of immune receptor channels

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses1. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity2–4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr355 in complex with the effector AvrSr356 of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35–AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.

Ionotropic P2X4 and P2X7 receptors in the regulation of ion transport across rat colon.

ATP plays an important role as an extracellular messenger acting via different types of purinoceptors. Whereas most of the actions of ATP at intestinal epithelia are thought to be mediated by metabotropic P2Y receptors, the role of ionotropic P2X receptors remains unclear. Consequently, we investigated the role of P2X4 and P2X7 receptors on ion transport across rat colonic epithelia by using BzATP, a potent agonist at P2X7 (and weak agonist at P2X4). Ussing chamber and Ca2+ imaging experiments were performed on rat colonic epithelia, combined with P2X receptor expression studies. Ussing chamber experiments revealed that serosal BzATP induced a neuronally mediated increase in short-circuit current caused by Cl- secretion. In contrast, the effect of mucosal BzATP was smaller, insensitive to tetrodotoxin and Cl- -independent. When epithelia were basolaterally depolarized to measure currents across the apical membrane, BzATP stimulated a cation current consistent with the activation of apical nonselective cation channels. Experiments with isolated colonic crypts revealed a BzATP-induced increase in the cytosolic Ca2+ concentration. Sensitivity to antagonists indicates stimulation of P2X4 and P2X7 receptors by serosal BzATP and of P2X7 receptors by mucosal BzATP. A similar pattern was observed with native ATP, which induced larger transepithelial currents in comparison to BzATP. RT-PCR and immunohistochemistry experiments confirmed the expression of P2X4 and P2X7 receptors in the colon localized in the epithelium and in submucosal ganglia. Epithelial and neuronal ionotropic P2X receptors are involved in the regulation of intestinal ion transport.

Polycystin-2 (TRPP2): Ion channel properties and regulation.

Polycystin-2 (TRPP2, PKD2, PC2) is the product of the PKD2 gene, whose mutations cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). PC2 belongs to the superfamily of TRP (Transient Receptor Potential) proteins that generally function as Ca2+-permeable nonselective cation channels implicated in Ca2+ signaling. PC2 localizes to various cell domains with distinct functions that likely depend on interactions with specific channel partners. Functions include receptor-operated, nonselective cation channel activity in the plasma membrane, intracellular Ca2+ release channel activity in the endoplasmic reticulum (ER), and mechanosensitive channel activity in the primary cilium of renal epithelial cells. Here we summarize our current understanding of the properties of PC2 and how other transmembrane and cytosolic proteins modulate this activity, providing functional diversity and selective regulatory mechanisms to its role in the control of cellular Ca2+ homeostasis.

The Chloride Conductance Inhibitor NS3623 Enhances the Activity of a Non-selective Cation Channel in Hyperpolarizing Conditions

Handbooks of physiology state that the strategy adopted by red blood cells (RBCs) to preserve cell volume is to maintain membrane permeability for cations at its minimum. However, enhanced cation permeability can be measured and observed in specific physiological and pathophysiological situations such as in vivo senescence, storage at low temperature, sickle cell anemia and many other genetic defects affecting transporters, membrane or cytoskeletal proteins. Among cation pathways, cation channels are able to dissipate rapidly the gradients that are built and maintained by the sodium and calcium pumps. These situations are very well-documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. In addition, non-selective cation (NSC) channels present in the RBC membrane have proven difficult to molecular identification and functional characterization. For instance, NSC channel activity can be elicited by Low Ionic Strength conditions (LIS): the associated change in membrane potential triggers its opening in a voltage dependent manner. But, whereas this depolarizing media produces a spectacular activation of NSC channel, Gárdos channel-evoked hyperpolarization's have been shown to induce sodium entry through a pathway thought to be conductive and termed Pcat. Using the CCCP method, which allows to follow fast changes in membrane potential, we show here (i) that hyperpolarization elicited by Gárdos channel activation triggers sodium entry through a conductive pathway, (ii) that chloride conductance inhibition unveils such conductive cationic conductance, (iii) that the use of the specific chloride conductance inhibitor NS3623 (a derivative of Neurosearch compound NS1652), at concentrations above what is needed for full anion channel block, potentiates the non-selective cation conductance. These results indicate that a non-selective cation channel is likely activated by the changes in the driving force for cations rather than a voltage dependence mechanism per se.

Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs.

SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-Å resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca2+-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases.

Histamine depolarizes rat intracardiac ganglion neurons through the activation of TRPC non-selective cation channels

The cardiac plexus, which contains parasympathetic ganglia, plays an important role in regulating cardiac function. Histamine is known to excite intracardiac ganglion neurons, but the underlying mechanism is obscure. In the present study, therefore, the effect of histamine on rat intracardiac ganglion neurons was investigated using perforated patch-clamp recordings. Histamine depolarized acutely isolated neurons with a half-maximal effective concentration of 4.5 μM. This depolarization was markedly inhibited by the H1 receptor antagonist triprolidine and mimicked by the H1 receptor agonist 2-pyridylethylamine, thus implicating histamine H1 receptors. Consistently, reverse transcription-PCR (RT-PCR) and Western blot analyses confirmed H1 receptor expression in the intracardiac ganglia. Under voltage-clamp conditions, histamine evoked an inward current that was potentiated by extracellular Ca2+ removal and attenuated by extracellular Na+ replacement with N-methyl-D-glucamine. This implicated the involvement of non-selective cation channels, which given the link between H1 receptors and Gq/11-protein-phospholipase C signalling, were suspected to be transient receptor potential canonical (TRPC) channels. This was confirmed by the marked inhibition of the inward current through the pharmacological disruption of either Gq/11 signalling or intracellular Ca2+ release and by the application of the TRPC blockers Pyr3, Gd3+ and ML204. Consistently, RT-PCR analysis revealed the expression of several TRPC subtypes in the intracardiac ganglia. Whilst histamine was also separately found to inhibit the M-current, the histamine-induced depolarization was only significantly inhibited by the TRPC blockers Gd3+ and ML204, and not by the M-current blocker XE991. These results suggest that TRPC channels serve as the predominant mediator of neuronal excitation by histamine.

The Cation Channel TMEM63B Is an Osmosensor Required for Hearing

Hypotonic stress causes the activation of swelling-activated nonselective cation channels (NSCCs), which leads to Ca2+-dependent regulatory volume decrease (RVD) and adaptive maintenance of the cell volume; however, the molecular identities of the osmosensitive NSCCs remain unclear. Here, we identified TMEM63B as an osmosensitive NSCC activated by hypotonic stress. TMEM63B is enriched in the inner ear sensory hair cells. Genetic deletion of TMEM63B results in necroptosis of outer hair cells (OHCs) and progressive hearing loss. Mechanistically, the TMEM63B channel mediates hypo-osmolarity-induced Ca2+ influx, which activates Ca2+-dependent K+ channels required for the maintenance of OHC morphology. These findings demonstrate that TMEM63B is an osmosensor of the mammalian inner ear and the long-sought cation channel mediating Ca2+-dependent RVD.

Contractile Behavior of Mouse Aorta Depends on SERCA2 Isoform Distribution: Effects of Replacing SERCA2a by SERCA2b.

The Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) actively pumps Ca2+ into the sarco/endoplasmic reticulum, thereby regulating intracellular Ca2+ concentrations and associated physiological processes. Different SERCA isoforms have been described (SERCA1, 2, and 3) with SERCA2 playing a pivotal role in Ca2+ homeostasis in cardiovascular tissues. In the heart, SERCA2a is the dominant isoform and has been proposed as therapeutic target in patients with heart failure. In the vasculature, both SERCA2a and SERCA2b are expressed with SERCA2b being the predominant isoform. The physiological role of SERCA2a in the vasculature, however, remains incompletely understood. In the present study, we used gene-modified mice in which the alternative splicing of the SERCA2-encoding gene (Atp2a2), underlying the expression of SERCA2a, is prevented and SERCA2a is replaced by SERCA2b. The resulting SERCA2b/b mice provide a unique opportunity to investigate the specific contribution of SERCA2a versus SERCA2b to vascular physiology. Aortic segments of SERCA2b/b (SERCA2a-deficient) and SERCA2a/b (control) mice were mounted in organ baths to evaluate vascular reactivity. SERCA2b/b aortic rings displayed higher contractions induced by phenylephrine (1 μM). Surprisingly, the initial inositol-3-phosphate mediated phasic contraction showed a faster decay of force in SERCA2b/b mice, while the subsequent tonic contraction was larger in SERCA2b/b segments. Moreover, in the presence of the calcium channel blocker diltiazem (35 μM) SERCA2b/b aortic rings showed higher contractions compared to SERCA2a/b, suggesting that SERCA2a (deficiency) modulates the activity of non-selective cation channels. Additionally, in endothelial cell (EC)-denuded aortic segments, the SERCA-inhibitor cyclopiazonic acid (CPA) caused markedly larger contractions in SERCA2b/b mice, while the increases of cytosolic Ca2+ were similar in both strains. Hence, aortas of SERCA2b/b mice appear to have a stronger coupling of intracellular Ca2+ to contraction, which may be in agreement with the reported difference in intracellular localization of SERCA2a versus SERCA2b. Finally, EC-mediated relaxation by acetylcholine and ATP was assessed. Concentration-response-curves for ATP showed a higher sensitivity of aortic segments of SERCA2b/b mice, while no difference in potency between strains were observed for acetylcholine. In summary, despite the relative low expression of SERCA2a in the murine aorta, our results point toward a distinct role in vascular physiology.

The Cation Channel TMEM63B is an Osmosensor Required for Hearing

Life has evolved appropriate adaptive responses to maintain cell shape and volume when facing osmotic stress. Hypotonic stress causes the activation of swelling-activated nonselective cation channels (NSCCs), which leads to a Ca2+-dependent regulatory volume decrease (RVD) and the adaptive maintenance of cell volume; however, the molecular identities of the osmosensitive NSCCs remain unclear. Here, we identified TMEM63B as an osmosensitive NSCC activated by hypotonic stress. TMEM63B is enriched in the inner ear sensory hair cells. Genetic deletion of TMEM63B results in necroptosis of outer hair cells (OHCs) and progressive hearing loss. Mechanistically, the TMEM63B channel mediates hypoosmolarity-induced Ca2+ influx, which activates Ca2+-dependent K+ channels required for the maintenance of OHC morphology. These findings demonstrate that TMEM63B is the osmosensor of mammalian ear and the long-sought cation channel mediating Ca2+-dependent RVD.

Membrane Stretch-Induced Activation of a TRPM4-Like Nonselective Cation Channel in Cerebral Artery Myocytes

Membrane Stretch-Induced Activation of a TRPM4-Like Nonselective Cation Channel in Cerebral Artery Myocytes

STIM1-dependent membrane insertion of heteromeric TRPC1-TRPC4 channels in response to muscarinic receptor stimulation.

Muscarinic receptor stimulation results in activation of nonselective cation (NSC) channels in guinea pig adrenal medullary (AM) cells. The biophysical and pharmacological properties of the NSC channel suggest the involvement of heteromeric channels of TRPC1 with TRPC4 or TRPC5. This possibility was explored in PC12 cells and guinea pig AM cells. Proximity ligation assay (PLA) revealed that when exogenously expressed in PC12 cells, TRPC1 forms a heteromeric channel with TRPC4, but not with TRPC5, in a STIM1-dependent manner. The heteromeric TRPC1-TRPC4 channel was also observed in AM cells and trafficked to the cell periphery in response to muscarine stimulation. To explore whether heteromeric channels are inserted into the cell membrane, tags were attached to the extracellular domains of TRPC1 and TRPC4. PLA products developed between the tags in cells stimulated by muscarine, but not in resting cells, indicating that muscarinic stimulation results in the membrane insertion of channels. This membrane insertion required expression of full-length STIM1. We conclude that muscarinic receptor stimulation results in the insertion of heteromeric TRPC1-TRPC4 channels into the cell membrane in PC12 cells and guinea pig AM cells.

Excitatory effect of bradykinin on intrinsic neurons of the rat heart

The heart receives sympathetic and parasympathetic innervation through the intrinsic cardiac nervous system. Although bradykinin (BK) has negative inotropic and chronotropic properties of cardiac contraction, the direct effect of BK on the intrinsic neural network of the heart is still unclear. In the present study, the effect of BK on the intracardiac ganglion neurons isolated from rats was investigated using the perforated patch-clamp technique.Under current-clamp conditions, application of 0.1 μM BK depolarized the membrane, accompanied by repetitive firing of action potentials. When BK was applied repeatedly, the second responses were considerably less intense than the first application. The BK action was fully inhibited by the B2 receptor antagonist Hoe-140, but not by the B1 receptor antagonist des-Arg9-[Leu8]-BK. The BK response was mimicked by the B2 agonist [Hyp3]-BK. The BK-induced depolarization was inhibited by the phospholipase C inhibitor U-73122. BK evoked inward currents under voltage-clamp conditions at a holding potential of −60 mV. Removal of extracellular Ca2+ markedly increased the BK-induced currents, suggesting an involvement of Ca2+-permeable non-selective cation channels. The muscarinic agonist oxotremorine-M (OxoM) also elicited the extracellular Ca2+-sensitive cationic currents. The OxoM response did not exhibit rundown with repeated agonist application. The amplitude of current evoked by 1 μM OxoM was comparable to that induced by 0.1 μM BK. Co-application of 0.1 μM BK and 1 μM OxoM elicited the current whose peak amplitude was almost the same as that elicited by OxoM alone, suggesting that BK and OxoM activate same cation channels. BK also reduced the amplitude of M-current, while the M-current inhibitor XE-991 affected neither resting membrane potential nor the BK-induced depolarization. From these results, we suggest that BK regulates excitability of intrinsic cardiac neurons by both an activation of non-selective cation channels and an inhibition of M-type K+ channels through B2 receptors.

Differences among muscarinic agonists in M1 receptor-mediated nonselective cation channel activation and TASK1 channel inhibition in adrenal medullary cells

Muscarinic receptor stimulation induces depolarizing inward currents and catecholamine secretion in adrenal medullary (AM) cells from various mammals. In guinea-pig AM cells muscarine and oxotremorine at concentrations ≤ 1 μM produce activation of nonselective cation channels with a similar potency and efficacy, whereas muscarine at higher concentrations produces not only nonselective cation channel activation, but also TASK1 channel inhibition. In rat AM cells, the muscarinic M1 receptor is involved in TASK1 channel inhibition in response to muscarinic agonists, and the efficacy of oxotremorine is half that of muscarine. These pharmacological findings might indicate that different muscarinic receptor subtypes are responsible for the regulation of nonselective cation and TASK1 channel activities. The present study aimed to determine the muscarinic receptor subtypes involved in nonselective cation channel activation in guinea-pig and mouse AM cells. The inward current evoked by 1 μM muscarine was completely suppressed by 100 μM quinine, whereas 30 μM muscarine-induced inward currents were comprised of quinine-sensitive and -insensitive components. The electrophysiological and pharmacological properties of the muscarine-induced currents indicated that the quinine-sensitive and insensitive components are due to nonselective cation channel activation and TASK1 channel inhibition, respectively. Muscarine at 30 μM failed to induce any current in AM cells treated with muscarinic toxin 7 or genetically deleted of the M1 receptor. The KD value of VU0255035 against the muscarinic receptor mediating nonselective cation channel activation was 17.5 nM. These results indicate that the M1 receptor mediates nonselective cation channel activation as well as TASK1 channel inhibition.

Maitotoxin Is a Potential Selective Activator of the Endogenous Transient Receptor Potential Canonical Type 1 Channel in Xenopus laevis Oocytes.

Maitotoxin (MTX) is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP). Several reports indicate that MTX is an activator of non-selective cation channels (NSCC) in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP) channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM) concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA) approach and the two-electrode voltage-clamp technique (TEVC). The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1) protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels.

Reactive oxygen species increase neuronal excitability via activation of nonspecific cation channel in rat medullary dorsal horn neurons.

The caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn; MDH) receives direct inputs from small diameter primary afferent fibers that predominantly transmit nociceptive information in the orofacial region. Recent studies indicate that reactive oxygen species (ROS) is involved in persistent pain, primarily through spinal mechanisms. In this study, we aimed to investigate the role of xanthine/xanthine oxidase (X/XO) system, a known generator of superoxide anion (O2·−), on membrane excitability in the rat MDH neurons. For this, we used patch clamp recording and confocal imaging. An application of X/XO (300 µM/30 mU) induced membrane depolarization and inward currents. When slices were pretreated with ROS scavengers, such as phenyl N-tert-butylnitrone (PBN), superoxide dismutase (SOD), and catalase, X/XO-induced responses decreased. Fluorescence intensity in the DCF-DA and DHE-loaded MDH cells increased on the application of X/XO. An anion channel blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), significantly decreased X/XO-induced depolarization. X/XO elicited an inward current associated with a linear current-voltage relationship that reversed near −40 mV. X/XO-induced depolarization reduced in the presence of La3+, a nonselective cation channel (NSCC) blocker, and by lowering the external sodium concentration, indicating that membrane depolarization and inward current are induced by influx of Na+ ions. In conclusion, X/XO-induced ROS modulate the membrane excitability of MDH neurons, which was related to the activation of NSCC.

Clock-driven vasopressin neurotransmission mediates anticipatory thirst prior to sleep.

Circadian rhythms have evolved to anticipate and adapt animals to the constraints of the earth's 24-hour light cycle. Although the molecular processes that establish periodicity in clock neurons of the suprachiasmatic nucleus (SCN) are well understood, the mechanisms by which axonal projections from the central clock drive behavioural rhythms are unknown. Here we show that the sleep period in mice (Zeitgeber time, ZT0-12) is preceded by an increase in water intake promoted entirely by the central clock, and not motivated by physiological need. Mice denied this surge experienced significant dehydration near the end of the sleep period, indicating that this water intake contributes to the maintenance of overnight hydromineral balance. Furthermore, this effect relies specifically on the activity of SCN vasopressin (VP) neurons that project to thirst neurons in the OVLT (organum vasculosum lamina terminalis), where VP is released as a neurotransmitter. SCN VP neurons become electrically active during the anticipatory period (ZT21.5-23.5), and depolarize and excite OVLT neurons through the activation of postsynaptic VP V1a receptors and downstream non-selective cation channels. Optogenetic induction of VP release before the anticipatory period (basal period; ZT19.5-21.5) excited OVLT neurons and prompted a surge in water intake. Conversely, optogenetic inhibition of VP release during the anticipatory period inhibited the firing of OVLT neurons and prevented the corresponding increase in water intake. Our findings reveal the existence of anticipatory thirst, and demonstrate this behaviour to be driven by excitatory peptidergic neurotransmission mediated by VP release from central clock neurons.

Evolutionary insight into the ionotropic glutamate receptor superfamily of photosynthetic organisms

Photosynthetic eukaryotes have a complex evolutionary history shaped by multiple endosymbiosis events that required a tight coordination between the organelles and the rest of the cell. Plant ionotropic glutamate receptors (iGLRs) form a large superfamily of proteins with a predicted or proven non-selective cation channel activity regulated by a broad range of amino acids. They are involved in different physiological processes such as C/N sensing, resistance against fungal infection, root and pollen tube growth and response to wounding and pathogens. Most of the present knowledge is limited to iGLRs located in plasma membranes. However, recent studies localized different iGLR isoforms to mitochondria and/or chloroplasts, suggesting the possibility that they play a specific role in bioenergetic processes.In this work, we performed a comparative analysis of GLR sequences from bacteria and various photosynthetic eukaryotes. In particular, novel types of selectivity filters of bacteria are reported adding new examples of the great diversity of the GLR superfamily. The highest variability in GLR sequences was found among the algal sequences (cryptophytes, diatoms, brown and green algae). GLRs of land plants are not closely related to the GLRs of green algae analyzed in this work. The GLR family underwent a great expansion in vascular plants. Among plant GLRs, Clade III includes sequences from Physcomitrella patens, Marchantia polymorpha and gymnosperms and can be considered the most ancient, while other clades likely emerged later. In silico analysis allowed the identification of sequences with a putative target to organelles. Sequences with a predicted localization to mitochondria and chloroplasts are randomly distributed among different type of GLRs, suggesting that no compartment-related specific function has been maintained across the species.

Potential Treatment for Neuropsychiatry Disorders with TRPC5 Modulators

Patent Application Title: Quinazoline-2,4(1H,3H)-dione Derivatives as TRPC5 Modulators for the Treatment of Neuropsychiatry Disorders Patent Application Number: WO 2016/023826 Al Publication date: 18 February 2016 Priority Application: US 62/035,578 Priority date: 11 August 2014 Inventors: Chenard, B. L.; Gallaschun, R. J. Applicants: Hydra Biosciences, Inc.; 45 Moulton Street, Cambridge, MA, 02138, USA Boehringer Ingelheim International Gmbh; Binger Strasse 173, 55216 Ingelheim am Rhein (DE) Disease Area: Neuropsychiatry Disorders Biological Target: Short transient receptor potential channel 5 (TRPC5) Summary: The invention in this patent application relates to novel quinazoline-2,4(1H,3H)-dione derivatives. These compounds are TRPC5 modulators and may be used as medication for the treatment of conditions such as neuropsychiatric disorder, neurodegenerative disorder, nephropathy, and seizure disorder. Ion channel proteins mediate the ion flux across cellular membranes. Their proper expressions and functions are important for the maintenance of cell function as well as intracellular communications. Ion channels are important in modulating membrane potential and ion flux in cells. The misregulation of membrane potential and aberrant calcium handling are implicated in the pathogenesis of numerous diseases. Therefore, compounds that can either promote or inhibit particular ion channels are of great interest and may potentially provide therapy for different diseases. The short transient receptor potential channel 5 (TRPC5) is one of seven mammalian transient receptor potential channel proteins. TRPC5 is a multipass cation channel membrane protein that modulates the flux of calcium and sodium ions across cellular membranes. The influx of sodium and calcium leads to a depolarization of the cell, which in turn increases the probability that voltage-gated ion channels will reach the threshold required for activation. Thus, activation of nonselective cation channels can increase electrical excitability and the frequency of several voltage-dependent events including, but not limited to, neuronal action potentials, cardiac action potentials, smooth muscle contraction, cardiac muscle contraction, and skeletal muscle contraction. In addition, the activation of nonselective cation channels such as TRPC5 causes calcium influx and alters the intracellular free calcium concentrations. This has profound effects on signal transduction, gene expression, and cellular phenotype. Gene expression events include, but are not limited to, production of mRNAs encoding cell surface receptors, ion channels, and kinases. These changes in gene expression can lead to hyperexcitability in that cell. TRPC5 protein may form homomultimeric species such as a homo-TRPC5 tetramer, or it may form heteromultimeric structures with other transient receptor proteins such as TRPC1, TRPC3, and TRPC4. The term TRPC5 is used generically to include either polymeric form. Compounds that can modulate the function of TRPC5, such as the compounds described in this patent application, can maintain calcium homeostasis, modulate intracellular calcium levels, modulate membrane polarization, and potentially treat or prevent several diseases and disorders associated with calcium and/or sodium homeostasis or dys-homeostasis.