Presynaptic kainate receptor-mediated facilitation of glutamate release involves Ca(2+) -calmodulin at mossy fiber-CA3 synapses

J. Neurochem. (2012) 122, 891-899. ABSTRACT: Presynaptic kainate receptors (KARs) modulate the release of glutamate at synapses established between mossy fibers (MF) and CA3 pyramidal cells in the hippocampus. The activation of KAR by low, nanomolar, kainate concentrations facilitates glutamate release. KAR-mediated facilitation of glutamate release involves the activation of an adenylate cyclase/cyclic adenosine monophosphate/protein kinase A cascade at MF-CA3 synapses. Here, we studied the mechanisms by which KAR activation produces this facilitation of glutamate release in slices and synaptosomes. We find that the facilitation of glutamate release mediated by KAR activation requires an increase in Ca(2+) levels in the cytosol and the formation of a Ca(2+) -calmodulin complex to activate adenylate cyclase. The increase in cytosolic Ca(2+) underpinning this modulation is achieved, both, by Ca(2+) entering via Ca(2+) -permeable KARs and, by the mobilization of intraterminal Ca(2+) stores. Finally, we find that, congruent with the Ca(2+) -calmodulin support of KAR-mediated facilitation of glutamate release, induction of long-term potentiation at MF-CA3 synapses has an obligate requirement for Ca(2+) -calmodulin activity.     

Ca2+/calmodulin binding to PSD‐95 mediates homeostatic synaptic scaling down

Postsynaptic density protein‐95 (PSD‐95) localizes AMPA‐type glutamate receptors (AMPARs) to postsynaptic sites of glutamatergic synapses. Its postsynaptic displacement is necessary for loss of AMPARs during homeostatic scaling down of synapses. Here, we demonstrate that upon Ca²⁺ influx, Ca²⁺/calmodulin (Ca²⁺/CaM) binding to the N‐terminus of PSD‐95 mediates postsynaptic loss of PSD‐95 and AMPARs during homeostatic scaling down. Our NMR structural analysis identified E17 within the PSD‐95 N‐terminus as important for binding to Ca²⁺/CaM by interacting with R126 on CaM. Mutating E17 to R prevented homeostatic scaling down in primary hippocampal neurons, which is rescued via charge inversion by ectopic expression of CaM^R126E, as determined by analysis of miniature excitatory postsynaptic currents. Accordingly, increased binding of Ca²⁺/CaM to PSD‐95 induced by a chronic increase in Ca²⁺ influx is a critical molecular event in homeostatic downscaling of glutamatergic synaptic transmission.     

Decreases in plasma membrane Ca2+‐ATPase in brain synaptic membrane rafts from aged rats

Precise regulation of free intracellular Ca²⁺ concentrations [Ca²⁺]_i is critical for normal neuronal function, and alterations in Ca²⁺ homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca²⁺]_i is the plasma membrane Ca²⁺‐ATPase (PMCA), the high‐affinity transporter that fine tunes the cytosolic nanomolar levels of Ca²⁺. We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non‐raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age‐related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.     

Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short‐term synaptic plasticity

Ca²⁺ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca²⁺–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca²⁺ signals. We solved the structure of Ca²⁺₄–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca²⁺₂–CaM complex. The Ca²⁺ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca²⁺–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca²⁺]_i, whereas the N‐module acts as a sensor at micromolar [Ca²⁺]_i. This Ca²⁺/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca²⁺‐dependent modulation of short‐term synaptic plasticity.     

A role for Ca2+ stores in kainate receptor-dependent synaptic facilitation and LTP at mossy fiber synapses in the hippocampus

Compared with NMDA receptor-dependent LTP, much less is known about the mechanism of induction of NMDA receptor-independent LTP; the most extensively studied form of which is mossy fiber LTP in the hippocampus. In the present study we show that Ca2+-induced Ca2+ release from intracellular stores is involved in the induction of mossy fiber LTP. This release also contributes to the kainate receptor-dependent component of the pronounced synaptic facilitation that occurs during high-frequency stimulation. We also present evidence that the trigger for this Ca2+ release is Ca2+ permeation through kainate receptors. However, these novel synaptic mechanisms can be bypassed when the Ca2+ concentration is raised (from 2 to 4 mM), via a compensatory involvement of L-type Ca2+ channels. These findings suggest that presynaptic kainate receptors at mossy fiber synapses can initiate a cascade involving Ca2+ release from intracellular stores that is important in both short-term and long-term plasticity.     

Effect of tricyclohexylhydroxytin on synaptosomal Ca2+-dependent ATP hydrolysis and rat brain subcellular calmodulin

Effect of tricyclohexylhydroxytin (plictran) on Ca2+-ATPase activity was studied in rat brain synaptosomes under in vitro and in vivo conditions. Plictran inhibited basal Ca2+-ATPase activity with an IC50 value of 6 nM suggesting its interaction with calcium transport phenomenon. Plictran inhibited calmodulin (CaM) activated Ca2+-ATPase in a concentration-dependent manner. A complete reversal of calmodulin activation of Ca2+-ATPase was observed with 2-3 nM plictran. A 50 per cent decrease of CaM activated Ca2+-ATPase was observed with 0.5 nM plictran, a concentration at which no significant effect was observed on basal enzyme activity. Of all the brain fractions studied, calmodulin levels in P2 fractions alone were reduced significantly to about 75 per cent of control values in plictran treated rats. The synaptosomal Ca2+-ATPase was also decreased by 35 per cent, 42 per cent and 65 per cent in 10, 20 and 40 mg plictran kg-1 day-1 treated rats for 3 days respectively. The activity levels of Ca2+-ATPase in 10 and 20 mg plictran kg-1 day-1 treated rats were restored to normal level by exogenously added calmodulin. These results suggest that plictran may disrupt synaptic function by altering calcium and calmodulin regulated processes in the central nervous system.     

Pre‐synaptic GABAB receptors inhibit glutamate release through GIRK channels in rat cerebral cortex

Neuronal G protein‐gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post‐synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABA_B receptors. In this study, we show for the first time that GABA_B receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABA_B receptors reduces glutamate release and the Ca²⁺ influx mediated by N‐type Ca²⁺ channels in a mode insensitive to the GIRK channel blocker tertiapin‐Q and consistent with direct inhibition of this voltage‐gated Ca²⁺ channel. However, by means of weak stimulation protocols, we reveal that GABA_B receptors also reduce glutamate release mediated by P/Q‐type Ca²⁺ channels, and that these responses are reversed by the GIRK channel blocker tertiapin‐Q. Consistent with the functional interaction between GABA_B receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre‐synaptic boutons of asymmetric synapses co‐express GABA_B receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post‐synaptic level, also occurs at glutamatergic nerve terminals.     

Rab3a interacting molecule (RIM) and the tethering of pre‐synaptic transmitter release site‐associated CaV2.2 calcium channels

Biochemical and physiological evidence suggest that pre‐synaptic calcium channels are attached to the transmitter release site within the active zone by a molecular tether. A recent study has proposed that ‘Rab3a Interacting Molecule’ (RIM) serves as the tether for CaV2.1 channels in mouse brain, based in part on biochemical co‐immunoprecipitation (co‐IP) using a monoclonal antibody, mRIM. We previously argued against this idea for CaV2.2 calcium channel at chick synapses based on experiments using a different anti‐RIM antibody, pRIM1,2: while staining for the two proteins co‐localized and co‐varied at the transmitter release face, consistent with an association, they failed to co‐IP from a synaptosome membrane lysate. RIM is, however, a family of proteins and we tested the possibility that the mRIM antibody used in the more recent study identifies a particular channel‐tethering variant. We find that co‐immunostaining with mRIM and anti‐CaV2.2 antibody neither co‐localized nor co‐varied at the transmitter release face and the two proteins did not co‐IP, arguing against a common protein complex and a key CaV2.2 scaffolding role for RIM at the active zone. The differing results might be reconciled, however, in a model where a RIM family member contributes to a protein bridge that anchors the pre‐fusion secretory vesicle to the calcium channel protein complex.     

Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca2+‐ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation

The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca²⁺‐ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein‐undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l⁻¹, respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca²⁺‐ATPase (PMCA) activity, Ca²⁺/CaM activated EGM Ca²⁺ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca²⁺/CaM to activate EGM Ca²⁺‐ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition. Copyright © 2016 John Wiley & Sons, Ltd.     

Phosphorylation of Cav1.2 on S1928 uncouples the L‐type Ca2+ channel from the β2 adrenergic receptor

Agonist‐triggered downregulation of β‐adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of β₂AR signaling highly specific for Ca_v1.2. We find that β₂‐AR binding to Ca_v1.2 residues 1923–1942 is required for β‐adrenergic regulation of Ca_v1.2. Despite the prominence of PKA‐mediated phosphorylation of Ca_v1.2 S1928 within the newly identified β₂AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the β₂AR from Ca_v1.2 upon β‐adrenergic stimulation rendering Ca_v1.2 refractory for several minutes from further β‐adrenergic stimulation. This effect is lost in S1928A knock‐in mice. Although AMPARs are clustered at postsynaptic sites like Ca_v1.2, β₂AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the β₂AR from Ca_v1.2 is a uniquely specific desensitization mechanism of Ca_v1.2 regulation by highly localized β₂AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT‐LTP) depends on Ca_v1.2 and its regulation by channel‐associated β₂AR.     

Capacitation suppression by mouse seminal vesicle autoantigen involves a decrease in plasma membrane Ca2+‐ATPase (PMCA)‐mediated intracellular calcium

Successful fertilization is tightly regulated by capacitation and decapacitation processes. Without appropriate decapacitation regulation, sperm would undergo a spontaneous acrosome reaction which leads to loss of fertilization ability. Seminal plasma is known to negatively regulate sperm capacitation. However, the suppressive mechanisms still remain unclear. In this study, we demonstrate the decapacitation mechanism of mouse seminal vesicle autoantigen (SVA) might target membrane sphingomyelin (SPM) and regulate plasma membrane Ca²⁺‐ATPase (PMCA) activity. The SVA was shown to suppress sperm capacitation induced by a broad panel of capacitation factors (bovine serum albumin (BSA), PAF, and cyclodextrin (CD)). Furthermore, SVA significantly decreased [Ca²⁺]_i and NaHCO₃‐induced [cAMP]_i. Cyclic AMP agonists bypassed the SVA's suppressive ability. Importantly, the SVA may regulate PMCA activity which was evidenced by the fact that the SVA decreased the [Ca²⁺]_i and intracellular pH (pH_i) of sperm; meanwhile, a PMCA inhibitor (carboxyeosin) could reverse SVA's suppression of [Ca²⁺]_i. The potential target of the SVA on membrane SPM/lipid rafts was highlighted by the high binding affinity of SPM–SVA (with a K_d of ～3 µM) which was close to the IC₅₀ of SVA's suppressive activity. Additionally, treatment of mink lung epithelial cells with the SVA enhanced plasminogen activator inhibitor (PAI)‐1 expression stimulated by tumor growth factor (TGF)‐β and CD. These observations supported the membrane lipid‐raft targeting of SVA. In summary, in this paper, we demonstrate that the decapacitation mechanism of the SVA might target membrane sphingolipid SPM and regulate PMCA activity to lower [Ca²⁺]_i, thereby decreasing the [cAMP]_i level and preventing sperm pre‐capacitation. J. Cell. Biochem. 111: 1188–1198, 2010. © 2010 Wiley‐Liss, Inc.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca2+/calmodulin‐dependent kinase II

Calmodulin (CaM) functions depend on interactions with CaM‐binding proteins, regulated by . Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM‐binding region of /calmodulin‐dependent kinase II (CaMKII_290−309) have been studied using biophysical methods. These proteins have opposite dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that and CaM interact very rapidly, and with moderate affinity ( ). Calmodulin‐CaMKII_290−309 interactions were only detected in the presence of , exhibiting fast kinetics and nanomolar affinity ( ). The CaM–Ng interaction had higher affinity under ‐depleted ( and k ₋₁ = 1.6 × 10⁻¹s⁻¹) than ‐saturated conditions ( ). The IQ motif of Ng (Ng₂₇₋₅₀) had similar affinity for CaM as Ng under ‐saturated conditions ( ), but no interaction was seen under ‐depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng ( ) and CaMKII_290−309( ) interactions. Although CaMKII_290−309 showed expected interaction characteristics, they may be different for full‐length CaMKII. The data for full‐length Ng, but not Ng₂₇₋₅₀, agree with the current model on Ng regulation of /CaM signaling.