Domain assembly of NAADP-gated two-pore channels

TPCs (two-pore channels) have recently been identified as targets for the Ca2+-mobilizing messenger NAADP (nicotinic acid–adenine dinucleotide phosphate). TPCs have a unique structure consisting of cytosolic termini, two hydrophobic domains (I and II) each comprising six transmembrane regions and a pore, and a connecting cytosolic loop; however, little is known concerning how these channels are assembled. In the present paper, we report that both domain I and II of human TPCs are capable of independent insertion into membranes, whereas the loop linking the domains fails to insert. Pairs of transmembrane regions within domain I of TPC1 are also capable of insertion, consistent with sequential translational integration of hydrophobic regions. Insertion of the first two transmembrane regions, however, was inefficient, indicating possible interaction between transmembrane regions during translation. Both domains, and each pair of transmembrane regions within domain I, were capable of forming oligomers, highlighting marked redundancy in the molecular determinants driving oligomer formation. Each hydrophobic domain formed dimers upon cross-linking. The first four transmembrane regions of TPC1 also formed dimers, whereas transmembrane regions 5 and 6, encompassing the pore loop, formed both dimers and tetramers. TPCs thus probably assemble as dimers through differential interactions between transmembrane regions. The present study provides new molecular insight into the membrane insertion and oligomerization of TPCs

Predominant Functional Expression of Kv1.3 by Activated Microglia of the Hippocampus after Status epilepticus

BACKGROUND:Growing evidence indicates that the functional state of microglial cells differs according to the pathological conditions that trigger their activation. In particular, activated microglial cells can express sets of Kv subunits which sustain delayed rectifying potassium currents (Kdr) and modulate differently microglia proliferation and ability to release mediators. We recently reported that hippocampal microglia is in a particular activation state after a status epilepticus (SE) and the present study aimed at identifying which of the Kv channels are functionally expressed by microglia in this model. METHODOLOGY/PRINCIPAL FINDINGS:SE was induced by systemic injection of kainate in CX3CR1(eGFP/+) mice and whole cell recordings of fluorescent microglia were performed in acute hippocampal slices prepared 48 h after SE. Microglia expressed Kdr currents which were characterized by a potential of half-maximal activation near -25 mV, prominent steady-state and cumulative inactivations. Kdr currents were almost abolished by the broad spectrum antagonist 4-Aminopyridine (1 mM). In contrast, tetraethylammonium (TEA) at a concentration of 1 mM, known to block Kv3.1, Kv1.1 and 1.2 subunits, only weakly reduced Kdr currents. However, at a concentration of 5 mM which should also affect Kv1.3 and 1.6, TEA inhibited about 30% of the Kdr conductance. Alpha-dendrotoxin, which selectively inhibits Kv1.1, 1.2 and 1.6, reduced only weakly Kdr currents, indicating that channels formed by homomeric assemblies of these subunits are not important contributors of Kdr currents. Finally, agitoxin-2 and margatoxin strongly inhibited the current. CONCLUSIONS/SIGNIFICANCE:These results indicate that Kv1.3 containing channels predominantly determined Kdr currents in activated microglia after SE

TPCs: Endolysosomal channels for Ca2+ mobilization from acidic organelles triggered by NAADP

Two-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltage-gated Ca(2+) and Na(+) channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca(2+) release channels that respond to activation by the Ca(2+) mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled

Defective microglial development in the hippocampus of Cx3cr1 deficient mice

Microglial cells participate in brain development and influence neuronal loss and synaptic maturation. Fractalkine is an important neuronal chemokine whose expression increases during development and that can influence microglia function via the fractalkine receptor, CX3CR1. Mice lacking Cx3cr1 show a variety of neuronal defects thought to be the result of deficient microglia function. Activation of CX3CR1 is important for the proper migration of microglia to sites of injury and into the brain during development. However, little is known about how fractalkine modulates microglial properties during development. Here we examined microglial morphology, response to ATP, and K(+) current properties in acute brain slices from Cx3cr1 knockout mice across postnatal hippocampal development. We found that fractalkine signaling is necessary for the development of several morphological and physiological features of microglia. Specifically, we found that the occurrence of an outward rectifying K(+) current, typical of activated microglia, that peaked during the second and third postnatal week, was reduced in Cx3cr1 knockout mice. Fractalkine signaling also influenced microglial morphology and ability to extend processes in response to ATP following its focal application to the slice. Our results reveal the developmental profile of several morphological and physiological properties of microglia and demonstrate that these processes are modulated by fractalkine signaling

The acid test: the discovery of two-pore channels (TPCs) as NAADP-gated endolysosomal Ca2+ release channels

In this review, we describe the background and implications of our recent discovery that two-pore channels (TPCs) comprise a novel class of calcium release channels gated by the intracellular messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Their localisation to the endolysosomal system highlights a new function for these organelles as targets for NAADP-mediated Ca(2+) mobilisation. In addition, we describe how TPCs may also trigger further Ca(2+) release by coupling to the endoplasmic reticular stores through activation of IP(3) receptors and ryanodine receptors

LE NAADP, UN MESSAGER LIBERANT DU CALCIUM:<br />ETUDE DE L'IMPLICATION DES RESERVES ACIDES ET ROLE DE CD38 DANS LA VOIE DE SYNTHESE

In pancreatic acinar cells, the secretagogues Acetylcholine (ACh) and cholecystokinin (CCK) trigger Ca2+ oscillations via the Ca2+ releasing second messengers IP3 and cADPR for ACh, and NAADP and cADPR for CCK. The existence of multiple messengers and multiple intracellular Ca2+ stores suggests a possible cooperation of these messengers and a selective recruitment of different Ca2+ stores.Our work reveals that these Ca2+ oscillations evoked by CCK are initiated by Ca2+ release from the lysosome and endosome by NAADP. In the case of ACh stimulation, Ca2+ oscillations are initiated by Ca2+ release from the zymogen granule by IP3. The Ca2+ signals are then maintained and amplified by Ca2+ release from the endoplasmic reticulum (ER) under control of the cADPR.We propose that the different Ca2+ releasing second messengers determine agonist-specific Ca2+ signatures by controlling the contribution of each acidic store and the ER.Our data on NAADP synthesis show that the surface antigen CD38 is the main NAADP synthesizing enzyme in mice, as well as an other NAADP synthesizing enzyme that exists in brain. We also report first evidence that nitric oxyde is capable of inhibiting NAADP synthesis in skeletal muscle.Dans les cellules acineuses du pancréas exocrine, les agonistes acétylcholine (ACh) et Cholécystokinine (CCK) déclenchent des oscillations calciques via les messagers libérant du calcium IP3 et cADPR pour l'ACh, et NAADP et cADPR pour la CCK. L'existence de plusieurs messagers et de plusieurs réserves intracellulaires de Ca2+ pose la question d'une possible coopération de ces messagers, et du recrutement sélectif des différentes réserves de Ca2+.Notre travail montre que lors de l'initiation des oscillations calciques induites par la CCK, le NAADP recrute le Ca2+ du lysosome et de l'endosome, tandis que dans le cas de l'ACh, l'IP3 recrute le Ca2+ des granules de zymogène. Cette réponse calcique est ensuite maintenue et amplifiée par le réticulum endoplasmique (RE) sous contrôle du cADPR. Nous proposons que les messagers déterminent les signatures calciques des agonistes en contrôlant la contribution de chaque réserve acide et du RE.Notre travail montre que la principale enzyme de synthèse de NAADP chez la souris est l'antigène de surface CD38, bien qu'une autre enzyme de synthèse existe dans le cerveau. Nos résultats montrent aussi pour la première fois que dans les muscles squelettiques, le monoxyde d'azote inhibe la synthèse de NAADP

Le NAADP, un messager libérant du calcium (étude de l'implication des réserves acides et rôle de CD38 dans la voie de synthèse)

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Generation of Specific Ca2+ Signals from Ca2+ Stores and Endocytosis by Differential Coupling to Messengers

It remains unclear how different intracellular stores could interact and be recruited by Ca2+-releasing messengers to generate agonist-specific Ca2+ signatures. In addition, refilling of acidic stores such as lysosomes and secretory granules occurs through endocytosis, but this has never been investigated with regard to specific Ca2+ signatures. In pancreatic acinar cells, acetylcholine (ACh), cholecystokinin (CCK), and the messengers cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and inositol 1,4,5-trisphosphate (IP3) evoke repetitive local Ca2+ spikes in the apical pole. Our work reveals that local Ca2+ spikes evoked by different agonists all require interaction of acid Ca2+ stores and the endoplasmic reticulum (ER), but in different proportions. CCK and ACh recruit Ca2+ from lysosomes and from zymogen granules through different mechanisms; CCK uses NAADP and cADPR, respectively, and ACh uses Ca2+ and IP3, respectively. Here, we provide pharmacological evidence demonstrating that endocytosis is crucial for the generation of repetitive local Ca2+ spikes evoked by the agonists and by NAADP and IP3. We find that cADPR-evoked repetitive local Ca2+ spikes are particularly dependent on the ER. We propose that multiple Ca2+-releasing messengers determine specific agonist-elicited Ca2+ signatures by controlling the balance among different acidic Ca2+ stores, endocytosis, and the ER

Effects of α-dendrotoxin (A, 50 nM), agitoxin-2 (B, 10 and 50 nM) and margatoxin (C, 1 and 10 nM) on the leak subtracted current induced by a voltage step from −70 to +40 mV (black traces recorded in control, red traces after drug application).

<p>The leak conductances of the cells in A, B and C were 338, 862 and 332 pS, respectively. The graphs on the right represent the conductance, normalized to its maximum value, as a function of the membrane potential and its inhibition by α-dendrotoxin (A, n = 4, Dtx), agitoxin-2 (B, AgTx, n = 14 for 10 nM, n = 6 for 50 nM) and margatoxin (C, MgTX, n = 8 for 1 nM, n = 11 for 10 nM).</p

Calcium-activated potassium currents in resting and activated microglia.

<p>A, B Examples of currents induced by voltage steps from 0 to +80 mV before (black traces) and after (red traces) bath application of TEA (1 mM) with 1 µM (left panel) and 0 µM (right panel) estimated intracellular free calcium in microglial cells from control (A) and from epileptic (B) mice. C Summary of the effects of TEA (1 mM, red column) and intracellular calcium on the current densities induced by voltage steps from 0 to +80 mV in microglia of control and epileptic mice (paired <i>t test</i>, *p<0.05).</p