In-Depth Chemical Analysis of the Brain Extracellular Space Using In Vivo Microdialysis with Liquid Chromatography-Tandem Mass Spectrometry.

Metabolomic analysis of samples acquired in vivo from the brain extracellular space by microdialysis sampling can provide insights into chemical underpinnings of a given brain state and how it changes over time. Small sample volumes and low physiological concentrations have limited the identification of compounds from this compartment, so at present, we have scant knowledge of its composition. As a result, most in vivo measurements have limited depth of analysis. Here, we describe an approach to (1) identify hundreds of compounds in brain dialysate and (2) routinely detect many of these compounds in 5 µL microdialysis samples to enable deep monitoring of brain chemistry in time-resolved studies. Dialysate samples collected over 12 h were concentrated 10-fold and then analyzed using liquid chromatography with iterative tandem mass spectrometry (LC-MS/MS). Using this approach on dialysate from the rat striatum with both reversed-phase and hydrophilic interaction liquid chromatography yielded 479 unique compound identifications. 60% of the identified compounds could be detected in 5 µL of dialysate without further concentration using a single 20 min LC-MS analysis, showing that once identified, most compounds can be detected using small sample volumes and shorter analysis times compatible with routine in vivo monitoring. To detect more neurochemicals, LC-MS analysis of dialysate derivatized with light and isotopically labeled benzoyl chloride was employed. 872 nondegenerate benzoylated features were detected with this approach, including most small-molecule neurotransmitters and several metabolites involved in dopamine metabolism. This strategy allows deeper annotation of the brain extracellular space than previously possible and provides a launching point for defining the chemistry underlying brain states.

Comparative study on physicochemical properties and hypoglycemic activities of intracellular and extracellular polysaccharides from submerged fermentation of Morchella esculenta.

The structural characteristic, physicochemical properties and structure-hypoglycemic activity relationship of intracellular (IPS) and extracellular (EPS) from submerged fermentation of Morchella esculenta were systematically compared and assessed. Both IPS and EPS were neutral, with a triple-helical conformation, and composed of galactose, glucose and mannose monosaccharides in different molar ratios. The molecular weight and particle size of IPS were higher than those of EPS. FTIR and SEM showed that the main functional group absorption peak intensity, glycosidic bond type and surface morphology of the two polysaccharides differed. Analysis of rheological and thermal properties revealed that the viscosity of IPS was higher than that of EPS, while thermal stability of EPS was greater than that of IPS. Hypoglycemic activity analysis in vitro showed that both IPS and EPS were non-competitive inhibitors of α-amylase and α-glucosidase. EPS showed strong digestive enzyme inhibitory activity due to its higher sulphate content and molar ratio of galactose, lower Mw and particle size. Meanwhile, with its higher Mw and apparent viscosity, IPS showed stronger glucose adsorption capacity and glucose diffusion retardation. These results indicate that IPS and EPS differed considerably in structure and physicochemical properties, which ultimately led to differences in hypoglycemic activity. These results not only suggested that IPS and EPS has the potential to be functional foods or hypoglycemic drugs, but also provided a new target for the prevention and treatment of diabetes with natural polysaccharides.

Simultaneous Mapping of the Nanoscale Organization and Redox State of Extracellular Space in Living Brain Tissue.

The spatial organization characteristics and redox status of the extracellular space (ECS) are crucial in the development of brain diseases. However, it remains a challenge to simultaneously capture dynamic changes in microstructural features and redox states at the submicron level within the ECS. Here, we developed a reversible glutathione (GSH)-responsive nanoprobe (RGN) for mapping the spatial organization features and redox status of the ECS in brain tissues with nanoscale resolution. The RGN is composed of polymer nanoparticles modified with GSH-responsive molecules and amino-functionalized methoxypoly(ethylene glycol), which exhibit exceptional single-particle brightness and excellent free diffusion capability in the ECS of brain tissues. Tracking single RGNs in acute brain slices allowed us to dynamically map spatial organizational features and redox levels within the ECS of brain tissues in disease models. This provides a powerful super-resolution imaging method that offers a potential opportunity to study the dynamic changes in the ECS microenvironment and to understand the physiological and pathological roles of the ECS in vivo.

Interference of extracellular soluble algal organic matter on flocculation-sedimentation harvesting of Chlorella sp.

Extracellular soluble algal organic matter (AOM) significantly interferes with microalgae flocculation. This study investigated the effects of various AOM fractions on Chlorella sp. flocculation using ferric chloride (FeCl3), sodium hydroxide (NaOH), and chitosan. All flocculants achieved high separation efficiency (87-99 %), but higher dosages were required in the presence of AOM. High molecular weight (>50 kDa) AOM fraction was identified as the primary inhibitor of flocculation across different pH levels, whereas low/medium molecular weight (<3 and <50 kDa) AOM had minimal impact. Compositional analysis revealed that the inhibitory AOM fraction is a glycoprotein rich in carbohydrates, including neutral, amino, and acidic sugars. The significance of this study is in identifying carboxyl groups (-COOH) from acidic monomers in >50 kDa AOM that inhibit flocculation. Understanding AOM composition and the interaction dynamics between AOM, cells, and flocculants is crucial for enhancing the techno-economics and sustainability of flocculation-based microalgae harvesting.

Reductive carbon dots for reduction, ratiometric fluorescence determination, and intracellular imaging of Au3.

Many studies show that ortho-phenylenediamine (OPD) produces an oxidized fluorescent product when exposed to an oxidizing agent that enables the direct or indirect fluorescence detection of a range chemical and biochemical analytes. However, there is no report on this unique optical behavior for other two isomers of phenylenediamine. This study demonstrates that a simple hydrothermal treatment of para-phenylenediamine (PPD) in the presence of sulfuric acid results in the formation of fluorescent N, S-doped carbon dots (CDs) with triple functionalities including the reduction of Au3+ into gold nanoparticles (AuNPs), the stabilization of the produced AuNPs, and the determination of Au3+ concentration through an intrinsic ratiometric fluorescence signal. In the presence of Au3+, the blue emission of CDs at 437 nm quenched, and a green emission at 540 nm emerged. The linear concentration range for the determination of Au3+ was 20 nM-16 µM with a detection limit of 16 nM. Additionally, the dual emissive CDs-AuNPs hybrid probe showed potential for the indirect fluorescence ratiometric determination of cysteine and sulfide ions. The linear concentration range for cysteine and sulfide ions were 0.25-8 µM and 0.1-6 µΜ, with detection limits of 0.095 µM and 0.041 µM, respectively. Accordingly, CDs were applied to detect Au3+ and S2- in real water samples. Moreover, the synthesized CDs showed no cytotoxicity for HeLa cells up to 300 µg mL-1, as determined by the MTT assay. Therefore, their potential for intracellular imaging of Au3+ in living cells was also investigated.

Delineating cathodic extracellular electron transfer pathways in microbial electrosynthesis: Modulation of polarized potential and Pt@C addition.

Microbial electrosynthesis (MES) is an innovative technology that employs microbes to synthesize chemicals by reducing CO2. A comprehensive understanding of cathodic extracellular electron transfer (CEET) is essential for the advancement of this technology. This study explores the impact of different cathodic potentials on CEET and its response to introduction of hydrogen evolution materials (Pt@C). Without the addition of Pt@C, H2-mediated CEET contributed up to 94.4 % at -1.05 V. With the addition of Pt@C, H2-mediated CEET contributions were 76.6 % (-1.05 V) and 19.9 % (-0.85 V), respectively. BRH-c20a was enriched as the dominated microbe (>80 %), and its relative abundance was largely affected by the addition of Pt@C NPs. This study highlights the tunability of MES performance through cathodic potential control and the addition of metal nanoparticles.

Thermodynamic controls on rates of iron oxide reduction by extracellular electron shuttles.

Anaerobic microbial respiration in suboxic and anoxic environments often involves particulate ferric iron (oxyhydr-)oxides as terminal electron acceptors. To ensure efficient respiration, a widespread strategy among iron-reducing microorganisms is the use of extracellular electron shuttles (EES) that transfer two electrons from the microbial cell to the iron oxide surface. Yet, a fundamental understanding of how EES-oxide redox thermodynamics affect rates of iron oxide reduction remains elusive. Attempts to rationalize these rates for different EES, solution pH, and iron oxides on the basis of the underlying reaction free energy of the two-electron transfer were unsuccessful. Here, we demonstrate that broadly varying reduction rates determined in this work for different iron oxides and EES at varying solution chemistry as well as previously published data can be reconciled when these rates are instead related to the free energy of the less exergonic (or even endergonic) first of the two electron transfers from the fully, two-electron reduced EES to ferric iron oxide. We show how free energy relationships aid in identifying controls on microbial iron oxide reduction by EES, thereby advancing a more fundamental understanding of anaerobic respiration using iron oxides.

Activation of the plant mevalonate pathway by extracellular ATP.

The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca2+-imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism.

Extracellular HSP90α Induces MyD88-IRAK Complex-Associated IKKα/ß-NF-κB/IRF3 and JAK2/TYK2-STAT-3 Signaling in Macrophages for Tumor-Promoting M2-Polarization.

M2-polarization and the tumoricidal to tumor-promoting transition are commonly observed with tumor-infiltrating macrophages after interplay with cancer cells or/and other stroma cells. Our previous study indicated that macrophage M2-polarization can be induced by extracellular HSP90α (eHSP90α) secreted from endothelial-to-mesenchymal transition-derived cancer-associated fibroblasts. To extend the finding, we herein validated that eHSP90α-induced M2-polarized macrophages exhibited a tumor-promoting activity and the promoted tumor tissues had significant increases in microvascular density but decreases in CD4+ T-cell level. We further investigated the signaling pathways occurring in eHSP90α-stimulated macrophages. When macrophages were exposed to eHSP90α, CD91 and toll-like receptor 4 (TLR4) functioned as the receptor/co-receptor for eHSP90α binding to recruit interleukin (IL)-1 receptor-associated kinases (IRAKs) and myeloid differentiation factor 88 (MyD88), and next elicited a canonical CD91/MyD88-IRAK1/4-IκB kinase α/ß (IKKα/ß)-nuclear factor-κB (NF-κB)/interferon regulatory factor 3 (IRF3) signaling pathway. Despite TLR4-MyD88 complex-associated activations of IKKα/ß, NF-κB and IRF3 being well-known as involved in macrophage M1-activation, our results demonstrated that the CD91-TLR4-MyD88 complex-associated IRAK1/4-IKKα/ß-NF-κB/IRF3 pathway was not only directly involved in M2-associated CD163, CD204, and IL-10 gene expressions but also required for downregulation of M1 inflammatory cytokines. Additionally, Janus kinase 2 (JAK2) and tyrosine kinase 2 (TYK2) were recruited onto MyD88 to induce the phosphorylation and activation of the transcription factor signal transducer and activator of transcription-3 (STAT-3). The JAK2/TYK2-STAT-3 signaling is known to associate with tumor promotion. In this study, the MyD88-JAK2/TYK2-STAT-3 pathway was demonstrated to contribute to eHSP90α-induced macrophage M2-polarization by regulating the expressions of M1- and M2-related genes, proangiogenic protein vascular endothelial growth factor, and phagocytosis-interfering factor Sec22b.

Sodium Bicarbonate Prescription and Extracellular Volume Increase: Real-world Data Results from the AlcalUN Study.

Oral alkalization with sodium bicarbonate (NaHCO3 ) or citrate is prescribed for conditions ranging from metabolic acidosis to nephrolithiasis. Although most nephrologists/urologists use this method routinely, extracellular volume (ECV) increase is the main feared adverse event reported for NaHCO3 . Thus far, no trial has specifically studied this issue in a real-world setting. AlcalUN (NCT03035812) is a multicentric, prospective, open-label cohort study with nationwide (France) enrollment in 18 (public and private) nephrology/urology units. Participants were adult outpatients requiring chronic (>1 month) oral alkalization by either NaHCO3 -containing or no-NaHCO3 -containing agents. The ECV increase (primary outcome) was judged based on body weight increase (ΔBW), blood pressure increase (ΔBP), and/or new-onset edema at the first follow-up visit (V1). From February 2017 to February 2020, 156 patients were enrolled. After a median 106 days of treatment, 91 (72%) patients reached the primary outcome. They had lower systolic (135 (125, 141) vs. 141 (130, 150), P = 0.02) and diastolic (77 (67, 85) vs. 85 (73, 90), P = 0.03) BP values, a higher plasma chloride (106.0 (105.0, 109.0) vs. 105.0 (102.0, 107.0), P = 0.02) at baseline, and a less frequent history of nephrolithiasis (32 vs. 56%, P = 0.02). Patients experienced mainly slight ΔBP (< 10 mmHg). The primary outcome was not associated (P = 0.79) with the study treatment (129 received NaHCO3 and 27 received citrate). We subsequently developed three different models of propensity score matching; each confirmed our results. Chronic oral alkalization with NaHCO3 is no longer associated with an ECV increase compared to citrate in real-life settings.

Extracellular hyaluronate pressure shaped by cellular tethers drives tissue morphogenesis.

How tissues acquire complex shapes is a fundamental question in biology and regenerative medicine. Zebrafish semicircular canals form from invaginations in the otic epithelium (buds) that extend and fuse to form the hubs of each canal. We find that conventional actomyosin-driven behaviors are not required. Instead, local secretion of hyaluronan, made by the enzymes uridine 5'-diphosphate dehydrogenase (ugdh) and hyaluronan synthase 3 (has3), drives canal morphogenesis. Charged hyaluronate polymers osmotically swell with water and generate isotropic extracellular pressure to deform the overlying epithelium into buds. The mechanical anisotropy needed to shape buds into tubes is conferred by a polarized distribution of actomyosin and E-cadherin-rich membrane tethers, which we term cytocinches. Most work on tissue morphogenesis ascribes actomyosin contractility as the driving force, while the extracellular matrix shapes tissues through differential stiffness. Our work inverts this expectation. Hyaluronate pressure shaped by anisotropic tissue stiffness may be a widespread mechanism for powering morphological change in organogenesis and tissue engineering.

Lauric Acid, a Dietary Saturated Medium-Chain Fatty Acid, Elicits Calcium-Dependent Eryptosis.

Cardiovascular diseases (CVD) are a leading cause of mortality worldwide, and dietary habits represent a major risk factor for dyslipidemia; a hallmark of CVD. Saturated fatty acids contribute to CVD by aggravating dyslipidemia, and, in particular, lauric acid (LA) raises circulating cholesterol levels. The role of red blood cells (RBCs) in CVD is increasingly being appreciated, and eryptosis has recently been identified as a novel mechanism in CVD. However, the effect of LA on RBC physiology has not been thoroughly investigated. RBCs were isolated from heparin-anticoagulated whole blood (WB) and exposed to 50-250 µM of LA for 24 h at 37 °C. Hemoglobin was photometrically examined as an indicator of hemolysis, whereas eryptosis was assessed by Annexin V-FITC for phosphatidylserine (PS) exposure, Fluo4/AM for Ca2+, light scatter for cellular morphology, H2DCFDA for oxidative stress, and BODIPY 581/591 C11 for lipid peroxidation. WB was also examined for RBC, leukocyte, and platelet viability and indices. LA caused dose-responsive hemolysis, and Ca2+-dependent PS exposure, elevated erythrocyte sedimentation rate (ESR), cytosolic Ca2+ overload, cell shrinkage and granularity, oxidative stress, accumulation of lipid peroxides, and stimulation of casein kinase 1α (CK1α). In WB, LA disrupted leukocyte distribution with elevated neutrophil-lymphocyte ratio (NLR) due to selective toxicity to lymphocytes. In conclusion, this report provides the first evidence of the pro-eryptotic potential of LA and associated mechanisms, which informs dietary interventions aimed at CVD prevention and management.

Riddled with holes: Understanding air space formation in plant leaves.

Plants use energy from sunlight to transform carbon dioxide from the air into complex organic molecules, ultimately producing much of the food we eat. To make this complex chemistry more efficient, plant leaves are intricately constructed in 3 dimensions: They are flat to maximise light capture and contain extensive internal air spaces to increase gas exchange for photosynthesis. Many years of work has built up an understanding of how leaves form flat blades, but the molecular mechanisms that control air space formation are poorly understood. Here, I review our current understanding of air space formation and outline how recent advances can be harnessed to answer key questions and take the field forward. Increasing our understanding of plant air spaces will not only allow us to understand a fundamental aspect of plant development, but also unlock the potential to engineer the internal structure of crops to make them more efficient at photosynthesis with lower water requirements and more resilient in the face of a changing environment.

Investigating the K+ sensitivity of cellular metabolism by extracellular flux analysis.

We have recently demonstrated that the activity of hexokinase 2 is dependent on the intracellular potassium ion (K+) concentration ([K+]). To analyze the K+ dependency of the cell metabolism in cell populations, we used an extracellular flux analyzer to assess oxygen consumption and acidification rates as well-established measures of oxidative- and glycolytic metabolic activities. This protocol describes in detail how a potential K+ sensitivity of the cell metabolism can be elucidated by extracellular flux analysis. For complete details on the use and execution of this protocol, please refer to Bischof et al. (2021).

Z-form extracellular DNA is a structural component of the bacterial biofilm matrix.

Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.

Protocol for intracellular and extracellular metabolite detection in human embryonic stem cells.

Metabolic homeostasis is critical for cell pluripotency and differentiation in human embryonic stem cells (hESCs). It has been reported that metabolic changes specifically regulate cellular signaling during hESC differentiation. This protocol describes procedures for both cell culture and detection of intracellular and extracellular metabolites in hESCs by liquid chromatography-mass spectrometry. Metabolites in glycolysis, citric acid cycle, pentose phosphate pathway, and other metabolic processes can be detected using this approach. For complete details on the use and execution of this protocol, please refer to Song et al., (2019), Yang et al., (2019), Meng et al., (2018), and Chen et al., (2011b).

Development of High Affinity Calcitonin Analog Fragments Targeting Extracellular Domains of Calcitonin Family Receptors.

The calcitonin and amylin receptors (CTR and AMY receptors) are the drug targets for osteoporosis and diabetes treatment, respectively. Salmon calcitonin (sCT) and pramlintide were developed as peptide drugs that activate these receptors. However, next-generation drugs with improved receptor binding profiles are desirable for more effective pharmacotherapy. The extracellular domain (ECD) of CTR was reported as the critical binding site for the C-terminal half of sCT. For the screening of high-affinity sCT analog fragments, purified CTR ECD was used for fluorescence polarization/anisotropy peptide binding assay. When three mutations (N26D, S29P, and P32HYP) were introduced to the sCT(22-32) fragment, sCT(22-32) affinity for the CTR ECD was increased by 21-fold. CTR was reported to form a complex with receptor activity-modifying protein (RAMP), and the CTR:RAMP complexes function as amylin receptors with increased binding for the peptide hormone amylin. All three types of functional AMY receptor ECDs were prepared and tested for the binding of the mutated sCT(22-32). Interestingly, the mutated sCT(22-32) also retained its high affinity for all three types of the AMY receptor ECDs. In summary, the mutated sCT(22-32) showing high affinity for CTR and AMY receptor ECDs could be considered for developing the next-generation peptide agonists.

Screening and Characterization of Two Extracellular Polysaccharide-Producing Bacteria from the Biocrust of the Mu Us Desert.

The extracellular polysaccharide (EPS) matrix embedding microbial cells and soil particles plays an important role in the development of biological soil crusts (BSCs), which is widely recognized as beneficial to soil fertility in dryland worldwide. This study examined the EPS-producing bacterial strains YL24-1 and YL24-3 isolated from sandy soil in the Mu Us Desert in Yulin, Shaanxi province, China. The strains YL24-1 and YL24-3 were able to efficiently produce EPS; the levels of EPS were determined to be 257.22 µg/mL and 83.41 µg/mL in cultures grown for 72 h and were identified as Sinorhizobium meliloti and Pedobacter sp., respectively. When the strain YL24-3 was compared to Pedobacter yulinensis YL28-9T using 16S rRNA gene sequencing, the resemblance was 98.6% and the strain was classified as Pedobacter sp. using physiological and biochemical analysis. Furthermore, strain YL24-3 was also identified as a subspecies of Pedobacter yulinensis YL28-9T on the basis of DNA-DNA hybridization and polar lipid analysis compared with YL28-9T. On the basis of the EPS-related genes of relevant strains in the GenBank, several EPS-related genes were cloned and sequenced in the strain YL24-1, including those potentially involved in EPS synthesis, assembly, transport, and secretion. Given the differences of the strains in EPS production, it is possible that the differences in gene sequences result in variations in the enzyme/protein activities for EPS biosynthesis, assembly, transport, and secretion. The results provide preliminary evidence of various contributions of bacterial strains to the formation of EPS matrix in the Mu Us Desert.

A innovative prognostic symbol based on neutrophil extracellular traps (NETs)-related lncRNA signature in non-small-cell lung cancer.

Neutrophil extracellular traps (NETs) are closely related to cancer progression. NETs-related lncRNAs play crucial roles in non-small-cell lung cancer (NSCLC) but there have been no systematic studies regarding NETs-related long noncoding RNA (lncRNA) signatures to forecast the prognosis of NSCLC patients. It's essential to build commensurate NETs-related lncRNA signatures. The expression profiles of prognostic mRNAs and lncRNAs and relevant clinical data of NSCLC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The NETs-related genes came from the results of our transcriptome RNA microarray data. The co-expression network of lncRNAs and NETs-related genes was structured to confirm NETs-related lncRNAs. The 19 lncRNAs correlated with overall survival (OS) were selected by exploiting univariate Cox regression (P < 0.05). Lasso regression and multivariate Cox regression (P < 0.05) were utilized to develop a 12-NETs-related lncRNA signature. We established a risk score based on the signature, which suggested that patients in the high-risk group displayed significantly shorter OS than patients in the low-risk group (P < 0.0001, P = 0.0023 respectively in the two cohorts). The risk score worked as an independent predictive factor for OS in both univariate and multivariate Cox regression analyses (HR> 1, P< 0.001). Additionally, by RT-qPCR, we confirmed that NSCLC cell lines have higher levels of the three adverse prognostic NETs-related lncRNAs than normal lung cells. The expression of lncRNAs significantly increases after NETs stimulation. In short, the 12 NETs-related lncRNAs and their model could play effective roles as molecular markers in predicting survival for NSCLC patients.

PRRT2 modulates presynaptic Ca2+ influx by interacting with P/Q-type channels.

Loss-of-function mutations in proline-rich transmembrane protein-2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We find that either acute or constitutive PRRT2 deletion induces a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that is insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype parallels a decrease in somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation, pull-down assays, and proteomics reveal a specific and direct interaction of PRRT2 with P/Q-type Ca2+ channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca2+ channels reduce their clustering at the active zone, with a corresponding decrease in the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in ensuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.