Postsynaptic Kainate Receptor Recycling and Surface Expression Are Regulated by Metabotropic Autoreceptor Signalling

Kainate receptors (KARs) play fundamentally important roles in controlling synaptic function and regulating neuronal excitability. Postsynaptic KARs contribute to excitatory neurotransmission but the molecular mechanisms underlying their activity‐dependent surface expression are not well understood. Strong activation of KARs in cultured hippocampal neurons leads to the downregulation of postsynaptic KARs via endocytosis and degradation. In contrast, low‐level activation augments postsynaptic KAR surface expression. Here, we show that this increase in KARs is due to enhanced recycling via the recruitment of Rab11‐dependent, transferrin‐positive endosomes into spines. Dominant‐negative Rab11 or the recycling inhibitor primaquine prevents the kainate‐evoked increase in surface KARs. Moreover, we show that the increase in surface expression is mediated via a metabotropic KAR signalling pathway, which is blocked by the protein kinase C inhibitor chelerythrine, the calcium chelator BAPTA and the G‐protein inhibitor pertussis toxin. Thus, we report a previously uncharacterized positive feedback system that increases postsynaptic KARs in response to low‐ or moderate‐level agonist activation and can provide additional flexibility to synaptic regulation.      

Drosophila Bestrophin-1 Currents Are Regulated by Phosphorylation via a CaMKII Dependent Mechanism

Cell swelling induced by hypo-osmotic stress results in activation of volume-regulated anion channels (VRAC) that drive a compensatory regulatory volume decrease. We have previously shown that the Best1 gene in Drosophila encodes a VRAC that is also activated by increases in intracellular Ca²⁺. The role of Best1 as a VRAC has recently been independently confirmed by the Clapham lab in an unbiased RNAi screen. Although dBest1 is clearly a volume-regulated channel, its mechanisms of regulation remain unknown. Here we investigate Drosophila Best1 (dBest1) regulation using the Drosophila S2 cell model system. Because dBest1 activates slowly after establishing whole-cell recording, we tested the hypothesis that the channel is activated by phosphorylation. Two experiments indicate that phosphorylation is required for dBest1 activation: nonspecific protein kinase inhibitors or intracellular perfusion with the non-hydrolyzable ATP analog AMP-PNP dramatically reduce the amplitude of dBest1 currents. Furthermore, intracellular perfusion with ATP-γ-S augments channel activation. The kinase responsible for dBest1 activation is likely Ca²⁺/calmodulin dependent kinase II (CaMKII), because specific inhibitors of this kinase dramatically inhibit dBest1 current activation. Neither specific PKA inhibitors nor inactive control inhibitors have effects on dBest1currents. Our results demonstrate that dBest1 currents are regulated by phosphorylation via a CaMKII dependent mechanism.     

Structure of Excitatory Synapses and GABAA Receptor Localization at Inhibitory Synapses Are Regulated by Neuroplastin-65

Formation, maintenance, and activity of excitatory and inhibitory synapses are essential for neuronal network function. Cell adhesion molecules (CAMs) are crucially involved in these processes. The CAM neuroplastin-65 (Np65) highly expressed during periods of synapse formation and stabilization is present at the pre- and postsynaptic membranes. Np65 can translocate into synapses in response to electrical stimulation and it interacts with subtypes of GABA_A receptors in inhibitory synapses. Here, we report that in the murine hippocampus and in hippocampal primary culture, neurons of the CA1 region and the dentate gyrus (DG) express high Np65 levels, whereas expression in CA3 neurons is lower. In neuroplastin-deficient (Np^−/−) mice the number of excitatory synapses in CA1 and DG, but not CA3 regions is reduced. Notably this picture is mirrored in mature Np^−/− hippocampal cultures or in mature CA1 and DG wild-type (Np^+/+) neurons treated with a function-blocking recombinant Np65-Fc extracellular fragment. Although the number of GABAergic synapses was unchanged in Np^−/− neurons or in mature Np65-Fc-treated Np^+/+ neurons, the ratio of excitatory to inhibitory synapses was significantly lower in Np^−/− cultures. Furthermore, GABA_A receptor composition was altered at inhibitory synapses in Np^−/− neurons as the α1 to α2 GABA_A receptor subunit ratio was increased. Changes of excitatory and inhibitory synaptic function in Np^−/− neurons were confirmed evaluating the presynaptic release function and using patch clamp recording. These data demonstrate that Np65 is an important regulator of the number and function of synapses in the hippocampus.     

Cu,Zn Superoxide Dismutase Maturation and Activity Are Regulated by COMMD1

The maturation and activation of the anti-oxidant Cu,Zn superoxide dismutase (SOD1) are highly regulated processes that require several post-translational modifications. The maturation of SOD1 is initiated by incorporation of zinc and copper ions followed by disulfide oxidation leading to the formation of enzymatically active homodimers. Our present data indicate that homodimer formation is a regulated final step in SOD1 maturation and implicate the recently characterized copper homeostasis protein COMMD1 in this process. COMMD1 interacts with SOD1, and this interaction requires CCS-mediated copper incorporation into SOD1. COMMD1 does not regulate disulfide oxidation of SOD1 but reduces the level of SOD1 homodimers. RNAi-mediated knockdown of COMMD1 expression results in a significant induction of SOD1 activity and a consequent decrease in superoxide anion concentrations, whereas overexpression of COMMD1 exerts exactly the opposite effects. Here, we identify COMMD1 as a novel protein regulating SOD1 activation and associate COMMD1 function with the production of free radicals.     

Dopamine Receptor 1 Modulates the Discharge Activities of Inspiratory and Biphasic Expiratory Neurons via cAMP-Dependent Pathways

Dopamine receptor 1 (D₁R) plays an essential role in regulating respiratory activity in mammals, however, little is known about how this receptor acts to modulate the basic respiratory rhythmogenesis. Here, by simultaneously recording the discharge activities of biphasic expiratory (biphasic E) neurons/inspiratory (I) neurons and the XII nerve rootlets from brainstem slices, we found that the application of D₁R agonist cis-(±)-1-(aminomethyl)-3,4-dihydro-3-phenyl-1H-2-benzopyran-5,6-diolhydrochloride (A68930, 5 μM), or forskolin, an intracellular cAMP-increasing agent, substantially decreased respiratory cycle and expiratory time of both types of neurons, and elevated the integral amplitude and frequency of XII nerve rootlets discharge. These changes were reversed by subsequent application of their antagonists SCH-23390 and Rp-Adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt hydrate (Rp-cAMPS), respectively. Importantly, after pretreatment with Rp-cAMPS, the effects of A68930 in both types of neurons were blocked, suggestive of a cAMP-dependent action of A68930. Thus, the current study indicates that D₁R may modulate basic breathing rhythmogenesis via cAMP-dependent mechanisms.     

The Arrestin-selective Angiotensin AT1 Receptor Agonist [Sar1,Ile4,Ile8]-AngII Negatively Regulates Bradykinin B2 Receptor Signaling via AT1-B2 Receptor Heterodimers

The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT₁ receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B₁ and B₂ bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT₁-B₂ receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT₁ agonist, [Sar¹,Ile⁴,Ile⁸]-AngII, but not the neutral AT₁ antagonist, losartan, inhibits endogenous B₂ receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar¹,Ile⁴, Ile⁸]-AngII require the AT₁ receptor and result from arrestin-dependent co-internalization of AT₁-B₂ heterodimers. BRET₅₀ measurements indicate that AT₁ and B₂ receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar¹,Ile⁴,Ile⁸]-AngII blunts B₂ receptor activation of G_q/11-dependent intracellular calcium influx and G_i/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar¹,Ile⁴,Ile⁸]-AngII has no effect on B₂ receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar¹,Ile⁴,Ile⁸]-AngII promotes internalization of AT₁-B₂ heterodimers. Thus, [Sar¹,Ile⁴,Ile⁸]-AngII exerts lateral allosteric modulation of B₂ receptor signaling by binding to the orthosteric ligand binding site of the AT₁ receptor and promoting co-sequestration of AT₁-B₂ heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT₁ receptor ligands may translate into different pharmacologic actions.     

Multiple Functions of Nm23-H1 Are Regulated by Oxido-Reduction System

Nucleoside diphosphate kinase (NDPK, Nm23), a housekeeping enzyme, is known to be a multifunctional protein, acting as a metastasis suppressor, transactivation activity on c-myc, and regulating endocytosis. The cellular mechanisms regulating Nm23 functions are poorly understood. In this study, we identified the modifications and interacting proteins of Nm23-H1 in response to oxidative stress. We found that Cys109 in Nm23-H1 is oxidized to various oxidation states including intra- and inter-disulfide crosslinks, glutathionylation, and sulfonic acid formation in response to H₂O₂ treatment both in vivo and in vitro. The cross-linking sites and modifications of oxidized Nm23-H1 were identified by peptide sequencing using UPLC-ESI-q-TOF tandem MS. Glutathionylation and oxidation of Cys109 inhibited the NDPK enzymatic activity of Nm23-H1. We also found that thioredoxin reductase 1 (TrxR1) is an interacting protein of Nm23-H1, and it binds specifically to oxidized Nm23-H1. Oxidized Nm23 is a substrate of NADPH-TrxR1-thioredoxin shuttle system, and the disulfide crosslinking is reversibly reduced and the enzymatic activity is recovered by this system. Oxidation of Cys109 in Nm23-H1 inhibited its metastatic suppressor activity as well as the enzymatic activities. The mutant, Nm23-H1 C109A, retained both the enzymatic and metastasis suppressor activities under oxidative stress. This suggests that key enzymatic and metastasis suppressor functions of Nm23-H1 are regulated by oxido-reduction of its Cys109.     

Cholinergic Dilatation and Constriction of Feline Cerebral Blood Vessels Are Mediated by Stimulation of Phosphoinositide Metabolism via Two Different Muscarinic Receptor Subtypes

Abstract: The muscarinic receptors involved in phosphoinositide (PI) hydrolysis have been pharmacologically characterized in cat cerebral blood vessels. Carbachol elicited a concentration-dependent increase in inositol phosphate accumulation [inositol monophosphate, bisphosphate, trisphosphate (IP₃) and tetrakisphosphate] in both major cerebral arteries and small pial vessels, which reached 140–280% of baseline at 10^?3M carbachol (referred to as maximal effect). However, the inositol phosphate accumulation response was found to be biphasic with a submaximal effect (30–50% of the maximal stimulation) obtained at low carbachol concentrations (<10^?5M). Endothelial denudation induced a virtual disappearance of the submaximal PI response without affecting that elicited by high concentrations of carbachol. The pharmacology of the two carbachol-induced PI responses was investigated by comparing the potency of selected muscarinic antagonists to block the IP₃ accumulation induced by 10^?7M (endothelium-dependent submaximal effect) and 10^?4M (endothelium-independent near-maximal effect) carbachol. In both major arteries and pial vessels, the activation of IP₃ production by 10^?4M carbachol was similarly inhibited by muscarinic antagonists with the following averaged rank order of potency (in -log IC₅₀): 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 8.65) > pirenzepine (8.28) > 6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo[b,e][1,4]diazepine-11-one (UH-AH 371; 7.87) > 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,-11-dihydro-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116; 6.62), a pharmacological profile compatible with an M₁ receptor subtype. In contrast, the submaximal stimulation of the PI metabolism elicited by 10^?7M carbachol in major arteries was blocked by the same antagonists with the following order of potency (in -log IC₅₀): 4-DAMP (8.38) > pirenzepine (7.25) > UH-AH 371 (6.25) > AF-DX 116 (5.72), which was reminiscent of an M₃ pharmacological profile. These findings indicate that stimulation of cerebrovascular muscarinic receptors is accompanied by PI hydrolysis via two distinct receptors, most probably the M₁ and M₃ subtypes that have been associated with constriction and dilatation, respectively, of cat cerebral arteries. Furthermore, these results provide strong evidence for an endothelial localization of the M₃ dilatatory receptors within the vessel wall.     

HIV-1 Replication and APOBEC3 Antiviral Activity Are Not Regulated by P Bodies

The APOBEC3 cytidine deaminases play a critical role in host-mediated defense against exogenous viruses, most notably, human immunodeficiency virus type-1 (HIV-1) and endogenous transposable elements. APOBEC3G and APOBEC3F interact with numerous proteins that regulate cellular RNA metabolism, including components of the RNA-induced silencing complex (RISC), and colocalize with a subset of these proteins to mRNA processing bodies (P bodies), which are sites of mRNA translational repression and decay. We sought to determine the role of P bodies and associated proteins in HIV-1 replication and APOBEC3 antiviral activity. While we established a positive correlation between APOBEC3 protein incorporation into virions and localization to P bodies, depletion of the P-body components DDX6 or Lsm1 did not affect HIV-1 replication, APOBEC3 packaging into virions or APOBEC3 protein mediated inhibition of HIV-1 infectivity. In addition, neither HIV-1 genomic RNA nor Gag colocalized with P-body proteins. However, simultaneous depletion of multiple Argonaute family members, the effector proteins of RISC, could modestly increase viral infectivity. Because some APOBEC3 proteins interact with several Argonaute proteins, we also tested whether they could modulate microRNA (miRNA) activity. We found no evidence for the specific regulation of miRNA function by the APOBEC3 proteins, though more general effects on transfected gene expression were observed. In sum, our results indicate that P bodies and certain associated proteins do not regulate HIV-1 replication or APOBEC3 protein antiviral activity. Localization to P bodies may therefore provide a means of sequestering APOBEC3 enzymatic activity away from cellular DNA or may be linked to as yet unidentified cellular functions.     

Fleshy Fruit Expansion and Ripening Are Regulated by the Tomato SHATTERPROOF Gene TAGL1

The maturation and ripening of fleshy fruits is a developmental program that synchronizes seed maturation with metabolism, rendering fruit tissues desirable to seed dispersing organisms. Through RNA interference repression, we show that Tomato AGAMOUS-LIKE1 (TAGL1), the tomato (Solanum lycopersicum) ortholog of the duplicated SHATTERPROOF (SHP) MADS box genes of Arabidopsis thaliana, is necessary for fruit ripening. Tomato plants with reduced TAGL1 mRNA produced yellow-orange fruit with reduced carotenoids and thin pericarps. These fruit are also decreased in ethylene, indicating a comprehensive inhibition of maturation mediated through reduced ACC Synthase 2 expression. Furthermore, ectopic expression of TAGL1 in tomato resulted in expansion of sepals and accumulation of lycopene, supporting the role of TAGL1 in ripening. In Arabidopsis, the duplicate SHP1 and SHP2 MADS box genes regulate the development of separation layers essential for pod shatter. Expression of TAGL1 in Arabidopsis failed to completely rescue the shp1 shp2 mutant phenotypes, indicating that TAGL1 has evolved distinct molecular functions compared with its Arabidopsis counterparts. These analyses demonstrate that TAGL1 plays an important role in regulating both fleshy fruit expansion and the ripening process that together are necessary to promote seed dispersal of fleshy fruit. From this broad perspective, SHP1/2 and TAGL1, while distinct in molecular function, regulate similar activities via their necessity for seed dispersal in Arabidopsis and tomato, respectively.