Cell Calcium

Cav1.2 is an L-type voltage-gated Ca2+ channel (VGCC) that supports Ca2+ influx in response to membrane depolarization. Ca2+ entering via Cav1.2 alters gene expression, activates Ca2+-dependent enzymes and has been implicated in synaptic plasticity. ORL-1 is a Gi/o-coupled G protein-coupled receptor (GPCR) that is expressed in the peripheral and central nervous systems. Both Cav1.2 and ORL-1 are expressed in the hippocampus, where they have been implicated in learning and memory. It is well-documented that ORL-1 interacts with another VGCC, Cav2.2. However, less is known about potential interactions between Cav1.2 and ORL-1. Here, we examine the interplay between Cav1.2 (Cav1c, Cav2{delta}-1, Cav{beta}1) and ORL-1 co-expressed in tsA-201 cells by using biochemical, electrophysiological and confocal imaging analysis. Co-immunoprecipitations revealed that ORL-1 independently interacts with Cav1c and Cav2{delta}-1 subunits of the Cav1.2 channel complex. Electrophysiological recordings revealed that co-expression with ORL-1 reduced Cav1.2 peak current density without altering its biophysical properties. Acute perfusion with the ORL-1 receptor agonist nociceptin (1 M) did not alter Cav1.2 current density. Confocal imaging experiments revealed that ORL-1 significantly decreases Cav1.2 plasma membrane expression by disrupting forward trafficking. Interestingly, ORL-1 did not affect Cav1.2 endocytosis. Overall, our results demonstrate a previously unrecognized interaction between ORL-1 and Cav1.2 that alters Cav1.2 membrane expression without affecting biophysical properties.

CO2 opens a subset of connexin hemichannels by binding to a site in the cytoplasmic domain of the channel. From outside the cell, CO2 must cross at least one membrane to reach this site. We have used Neuro-2A cells, which exhibit very low expression of CO2 permeable aquaporins (AQPs) and do not express any of the connexins (Cxs) known to be CO2 sensitive, to evaluate the minimal complement of molecular components required to recapitulate whole cell CO2 sensitivity mediated by connexins (assayed by either whole cell patch clamp recordings or real time recordings of ATP release via a co-expressed genetically encoded ATP sensor). Neuro-2A cells that expressed either Cx26, Cx32 or Cx43 on their own did not exhibit CO2-dependent connexin hemichannel gating. Expression of AQP1 or AQP5 either with or without carbonic anhydrase 2 (CA2) did not reveal any endogenous CO2 sensitivity of Neuro-2A cells. Only by expressing one of Cx26, Cx32 or Cx43 with either AQP1 or AQP5, plus CA2 were we able to reconstitute whole cell CO2 sensitivity. We found that expression of Cx26 with either AQP1 or AQP5 resulted in high levels of cell death. This was prevented by co-expression of CA2. Simulations of the influx and diffusion of CO2 show that CA2 prevents accumulation of intracellular CO2 and excessive activation of Cx26, thus protecting the cells from death. Surveying the transcriptome of cells that express CO2 sensitive connexins shows that many also express CO2 permeable aquaporins and CA2. We suggest that connexins, aquaporins and carbonic anhydrases represent the minimal trifecta of components required for cellular CO2 sensing.

Following ER Ca2+ depletion, Ca2+ release-activated Ca2+ (CRAC) channels are activated by STIM1 at ER-plasma membrane junctions. The restricted localization and low conductance of the CRAC channel (<40 fS) precludes single-channel recordings, limiting studies of CRAC channel gating. Here we describe an optical approach to characterize the gating of HaloTag-fused Orai1 channels labeled with JF646-BAPTA, a Ca2+-sensitive fluorescent dye. While Ca2+ influx through single channels generates fluorescence fluctuations, identifying true gating events is complicated by stochastic transitions of JF646-BAPTA to a non-fluorescent state. To overcome this, we combine TIRF microscopy with whole-cell voltage clamp to control the driving force for Ca2+ entry. We show the open channel intensity at -100 mV reflects Ca2+ saturation of the dyes on each channel, while the closed-channel intensity is defined by the fluorescence at +30 mV, where influx is absent. True gating events can be identified from transitions between the open- and closed-channel levels, distinguishing them from transitions to a non-fluorescent state. We describe the gating behavior of CRAC channels activated by STIM1 after store depletion. Dwell time distributions indicate at least two open and closed states with durations of 0.1 to several seconds, with most channels having an open probability of [&ge;]0.7. We also detect silent channels that colocalize with STIM1 but show no activity over tens of seconds, a population that would be undetectable by whole-cell electrophysiology alone. This method offers an approach to explore CRAC channel gating mechanisms and may be applicable to other Ca2+- permeable channels not amenable to patch-clamp techniques.

Muscle satellite cells (MuSCs) are muscle-resident stem cells that are responsible for myofiber regeneration. Although the importance of calcium ions (Ca2+) in muscle physiology has been well established, the mechanism by which Ca2+ mobilization governs MuSC function remains poorly understood. In this study, we aimed to systematically characterize Ca2+ dynamics in MuSCs and to define the mechanisms regulating these signals during muscle regeneration. By employing modified protocols for mouse MuSC isolation and Ca2+ measurement, we observed spontaneous Ca2+ fluctuations in MuSCs isolated from regenerating muscle after cardiotoxin-induced myofiber injury. Our detailed analysis using chemical Ca2+ indicators and a genetically encoded Ca2+ indicator revealed that the frequency and amplitude of Ca2+ fluctuations increased significantly during the activated and proliferative stages of MuSCs in muscle regeneration. This effect was more pronounced in MuSCs isolated from dystrophic and aged mice. Mechanistically, these Ca2+ fluctuations were at least partially mediated by mechanosensitive ion channels, including PIEZO1 and TRPM7, which promote MuSC migration. Collectively, our findings demonstrate that Ca2+ fluctuations through mechanosensitive ion channels act as a key regulator of MuSC activation during muscle regeneration and may provide new insights into the role of Ca2+ influx in muscle biology and the pathogenesis of muscle diseases.

Amyloid {beta} peptides (A{beta}) trigger Alzheimers disease (AD) but how has remained elusive. A{beta} stimulates the {beta}2 adrenergic receptor ({beta}2AR), which forms a unique signaling complex with the L-type Ca2+ channel (LTCC) CaV1.2. LTCCs have been implicated in the etiology of dementia and AD. We show that A{beta} acutely potentiates CaV1.2 via the {beta}2AR, which triggers postsynaptic recruitment of Ca2+ permeable (CP) AMPARs in hippocampal cultures and impairs LTP in hippocampal slices within minutes. The long-term consequence is a loss of postsynaptic structure of glutamatergic synapses and neurotoxicity. Disrupting this signaling cascade with highly specific tools prevented all of these effects, unifying a number of currently divergent findings on A{beta} synaptotoxicity including dysregulation of AMPARs and synaptic plasticity. TEASERAmyloid {beta} peptide is the primary pathological agent in Alzheimers disease. It affects the nanoscale structure and function of glutamatergic synapses. The molecular mechanisms are largely unknown except for identification of several binding proteins including the {beta}2 adrenergic receptor. We show that this binding potently (EC50<100 nM) augments Ca2+ influx through the L-type Ca channel CaV1.2. This effect leads to improper recruitment of Ca2+-permeable glutamate receptors to postsynaptic sites (EC50<100 nM), synaptic dysfunction and ultimately neuronal death. This work identifies an essential mechanism in amyloid {beta} neurotoxicity and explains many of the observed postsynaptic alterations. HighlightsImmediate effects of A{beta}-induced stimulation of {beta}2AR on Cav1.2: O_LIA{beta} induces phosphorylation of Cav1.2 on S1928 by PKA C_LIO_LIA{beta} augments Cav1.2 activity via {beta}2AR-induced S1928 phosphorylation within seconds C_LI A{beta}-induced {beta}2AR - Cav1.2 signaling has the following synaptotoxic effects. O_LIA{beta} induces postsynaptic accumulation of Ca-permeable AMPARs via {beta}2AR - Cav1.2 signaling within 20 min C_LIO_LIA{beta} impairs long-term potentiation (LTP) via {beta}2AR - Cav1.2 signaling C_LIO_LIA{beta} impairs postsynaptic structure and neuronal viability over 24 h C_LIO_LIPotency of A{beta} in all the above effects is very high (100 nM A{beta} is saturating!) C_LIO_LIAll effects are prevented in S1928A KI mice and acute displaces {beta}2AR from Cav1.2 with tat-Pep1923 C_LI

P/Q-type calcium channel (Cav2.1) is the major channel that mediates Ca2+ influx during action potentials (APs) and evokes neurotransmitter release from presynaptic terminals. Repetitive activity induces its Ca2+-dependent facilitation (CDF) via binding of calmodulin (CaM) superfamily proteins to the IQ-like motif, specifically isoleucine (I) and methionine (M) sites, on the cytoplasmic c-terminus of Cav2.1. However, whether and how CDF contributes to short-term synaptic plasticity remains elusive. By recordings from the calyx of Held terminal in IQ-like motif point mutation knock-in mice (Cav2.1 IM-AA KI), we found that activity-dependent CDF is completely abolished, resulting in lower quantal output and shorter release time course as well as profound reductions in the magnitute of short-term facilitation and depression (STF and STD) in different Ca2+ concentrations. Prolonging deactivation of Ca2+ channels by broadening spike width normalizes quantal output and release time course in Cav2.1 IM-AA synapses, but does not fully rescue STF/STD. These results indicate that CDF of Cav2.1 channels governs the polarity and magnitude of short-term synaptic plasticity in fast-spiking central synapses.

Mitochondrial calcium uniporter (MCU) uptakes calcium into mitochondria to maintain intracellular calcium homeostasis, malfunction of which has been implicated in altered neuronal signaling and disease. Its role in synaptic transmission remains understudied, especially in intact neural circuits. We investigated MCU function at the auditory nerve endbulb of Held synapse and postsynaptic bushy neurons in the cochlear nucleus, using age-matched control and MCU knockout (KO) mice of either sex. Whole-cell voltage- and current-clamp recordings were acquired from acute brain slices to examine synaptic transmission and postsynaptic responses. We found that basal synaptic properties at the endbulb of Held were unchanged in MCU KO mice, whereas synaptic transmission during sustained high-rate activity was significantly altered with a shift toward increased asynchronous release. Similarly, MCU deficiency did not change the intrinsic membrane properties of postsynaptic bushy neurons, but significantly reduced the temporal precision of auditory nerve evoked spikes trains at high rates. These results demonstrate that MCU is largely dispensable under low-rate activity, presumably because its activation requires relatively high calcium concentrations. In contrast, during sustained high-rate activity, MCU becomes an important regulator of synaptic function by reducing asynchronous neurotransmitter release under elevated intracellular calcium. Particularly in the auditory system, where neurons routinely fire at high rates, MCU promotes temporal processing and thereby plays a key role in supporting auditory function. It suggests that impaired MCU function under pathological conditions may be an important mechanism underlying central auditory processing deficits, and consequently contributes to hearing loss

Along with the membrane potential and respiration, mitochondrial matrix volume is a critical parameter that determines mitochondrial function. Mitochondria undergo constant changes in matrix volume and cristae dynamics, and in processes that are critical for normal metabolic rates and pathophysiological responses. Changes in matrix volume cannot be easily measured by conventional fluorescence imaging techniques due to the size of the sub-organellar structures, which are below resolution. This challenge was successfully resolved in studies of isolated mitochondria with the use of scattered light. Here we use dark-field imaging, which relies on scattered light contrast, to measure matrix volume dynamics in living cells. We demonstrate that mitochondrial volume changes can be easily detected as changes in intensity of the scattered light following matrix volume modulation with K+ ionophores or by onset of the permeability transition. Specifically, we found that stimulation of K+ influx leads to increase of mitochondrial matrix volume while stimulation of K+ efflux leads to matrix shrinkage, and that activation of the permeability transition leads to high-amplitude mitochondrial swelling in wild-type but not in cells lacking subunit c of ATP synthase. These results directly demonstrate the dynamic nature of mitochondrial matrix volume and its link to physiological and pathological ion transport.

The hippocampal CA3 subfield is central to associative learning and memory consolidation. The principal cells of the CA3, pyramidal neurons, execute these functions by generating hypersynchronous bursts that feed forward to the CA1. Extensive recurrent collateral connections within the CA3 neuron population are crucial for the generation of this burst activity. Double C2 domain-containing protein (Doc2) and synaptotagmin 7 (Syt7) are high-affinity calcium sensors implicated in asynchronous synaptic vesicle (SV) release and in the exocytosis of dense-core vesicles (DCVs). Additionally, Doc2 is a sensor for miniature neurotransmission, whereas Syt7 is involved in synaptic facilitation and SV replenishment. Both Doc2 and Syt7 are expressed in the hippocampus, but their potential roles in spontaneous excitatory network activity remain unanswered. Using whole-cell recordings in disinhibited acute hippocampal slices obtained from juvenile Doc2- and Syt7- knockout (KO) mice (P15-21), we report increased CA3 burst generation without changes in spontaneous excitatory postsynaptic current (sEPSC) frequency or amplitude. Moreover, the intrinsic properties of CA3 pyramidal neurons, such as the resting membrane potential, firing rate and input resistance, are unchanged. We propose that this novel burst phenotype in Doc2- and Syt7- KO mice is unrelated to changes in SV release but might be mediated by changes in neuropeptide release from DCVs. Regardless of the underlying mechanisms, this work reveals that both proteins act to regulate network activity. The hippocampal CA3 subfield is central to associative learning and memory consolidation. The principal cells of the CA3, pyramidal neurons, execute these functions by generating hypersynchronous bursts that feed forward to the CA1. Extensive recurrent collateral connections within the CA3 neuron population are crucial for the generation of this burst activity. Double C2 domain-containing protein (Doc2) and synaptotagmin 7 (Syt7) are high-affinity calcium sensors implicated in asynchronous synaptic vesicle (SV) release and in the exocytosis of dense-core vesicles (DCVs). Additionally, Doc2 is a sensor for miniature neurotransmission, whereas Syt7 is involved in synaptic facilitation and SV replenishment. Both Doc2 and Syt7 are expressed in the hippocampus, but their potential roles in spontaneous excitatory network activity remain unanswered. Using whole-cell recordings in disinhibited acute hippocampal slices obtained from juvenile Doc2- and Syt7- knockout (KO) mice (P15-21), we report increased CA3 burst generation without changes in spontaneous excitatory postsynaptic current (sEPSC) frequency or amplitude. Moreover, the intrinsic properties of CA3 pyramidal neurons, such as the resting membrane potential, firing rate and input resistance, are unchanged. We propose that this novel burst phenotype in Doc2- and Syt7- KO mice is unrelated to changes in SV release but might be mediated by changes in neuropeptide release from DCVs. Regardless of the underlying mechanisms, this work reveals that both proteins act to regulate network activity.

The Ca{superscript 2} release-activated Ca{superscript 2} (CRAC) channel mediates store-operated calcium entry (SOCE), a ubiquitous pathway essential for many cell types, including immune cells. Three Orai (Orai1/2/3) proteins constitute the plasma membrane pore-forming units of CRAC channels that are activated by the endoplasmic reticulum (ER) Ca2+-sensing STIM1/2 proteins when ER Ca2+ stores are depleted. Orai1/2/3 are differentially expressed across primary cells with discernible differences in their structures and biophysical properties. Further, Orai1 has two alternatively translated isoforms: long mammalian-specific Orai1 and the 63-residue shorter Orai1{beta}, which is evolutionarily older and conserved across vertebrates. Whether Orai1/1{beta}/2/3 produce unique cytosolic Ca{superscript 2} signatures that bias transcriptional responses through effectors like NFAT is unclear. Here, we used HEK293 cells engineered to express one native Orai isoform and show that all Orai isoforms couple to NFAT1/4 induction. The magnitude of NFAT1/4 induction for each Orai isoform matches that of SOCE, with the following profile: Orai1{beta}>Orai1>>Orai2>Orai3. Near-native re-expression of either Orai1 or Orai1{beta} in primary murine Orai1-/- CD4 T cells restored SOCE, NFAT activation, cytokine production and promoted near identical transcriptional responses enriched for immune activation pathways. An analysis of genetic and clinical data of human individuals showed that homozygous null mutations selectively abolishing Orai1 are not associated with disease resembling CRAC channelopathy. Primary T cells from individuals homozygous or heterozygous for an Orai1 null mutation showed enhanced, rather than impaired, SOCE and NFAT induction. Our data indicate that NFAT activation and transcriptional outputs are primarily driven by the graded strength of SOCE mediated by each isoform of the Orai quartet.

Phosphatidylinositol (4,5)-bisphosphate (PIP2), the most abundant cellular poly-phosphoinositide (PPI) class of phospholipid, is a central plasma membrane (PM)-associated signaling hub that controls many cellular processes. In this study, we demonstrate that either deletion of the gene encoding actin-binding protein profilin1 (Pfn1) or disruption of Pfn1-actin interaction leads to downregulation of PM PIP2 content in cells. This is also phenocopied when F-actin is depolymerized implying that Pfn1-dependent PIP2 alteration is related to its actin-regulatory function. Phospholipase C (PLC) activity is critical for Pfn1-deficient cells to exhibit the PIP2-related phenotype. These findings, taken together with biochemical signatures of elevated PIP2 hydrolysis (higher baseline PM diacylglycerol-to PIP2 ratio and protein kinase C activity) exhibited by Pfn1-deficient cells, imply that PLC-mediated PIP2 hydrolysis plays a role in Pfn1-dependent regulation of PM PIP2. Furthermore, we unexpectedly found that Pfn1 loss leads to dramatic alterations in several other important forms of lipids, revealing a previously unrecognized role of Pfn1 as a broad regulator of cellular lipid environment that extends beyond PPI control. In conclusion, our study establishes Pfn1 as an important regulator of cellular lipid homeostasis. SUMMARY STATEMENTThis study uncovers a mechanism of how functional loss of Profilin1, a key regulator of actin cytoskeleton, can trigger downregulation of plasma membrane content of PIP2, an important class of phospholipid, in cells.

Neural synchronization is fundamental to brain function and, when it becomes excessive, underlies pathological conditions such as epilepsy. Among brain regions, the temporal lobes, and the hippocampus in particular, exhibit the highest epileptogenic potential, with mesial temporal lobe epilepsy representing the most prevalent form of the condition in humans. Within the hippocampus, extracellular potassium dynamics are central to non-synaptic epileptiform activity, and astrocytic potassium buffering mechanisms have emerged as key regulators of network excitability. Yet the specific contributions of astrocytic gap-junction coupling and potassium spatial buffering to neuronal synchronization across different spatial scales remain poorly understood. To address this gap, we developed a microcircuit biophysical model consisting of two astrocyte-neuron modules, each comprising one astrocyte coupled to five neurons. Astrocyte-neuron interactions are mediated exclusively through shared extracellular potassium dynamics. Using a reduced astrocyte model that captures both local membrane and syncytial potassium buffering, we systematically investigated how astrocytic potassium handling shapes neuronal activity patterns and inter-module synchronization. Our results demonstrate that astrocytes prevent the emergence of pathological states -- such as sustained ictal activity and depolarization block, by stabilizing extracellular potassium levels. Furthermore, we show that astrocytic gap-junction coupling strength critically regulates phase synchronization between neuronal modules: stronger coupling promotes inter-module synchrony under physiological conditions, whereas impaired astrocytic function drives networks toward pathological hypersynchronization when extracellular potassium is elevated. These findings support the hypothesis that astrocytic networks impose modularity on hippocampal neuronal assemblies, and suggest that astrocytic connexins may represent a relevant therapeutic target in epilepsy and other disorders characterized by aberrant neural synchronization. Author summary

This protocol describes manual whole-endolysosome patch-clamp recordings from pharmacologically or genetically enlarged endolysosomes in cultured mammalian cells. Steps include vesicle enlargement, fabrication and fire-polishing of high-resistance pipettes, mechanical dissection and isolation of enlarged vesicles, giga-seal formation, and configuration to whole-endolysosome modes. For complete details on the use and execution of this protocol, please refer to Wang et al1.

A hallmark of dilated cardiomyopathy (DCM) is calcium mishandling, including reduced transport activity of the SERCA calcium pump in cardiac muscle cells. This has focused attention on SERCA as mechanism of disease and potential therapeutic target. Previously, diminished SERCA activity has been attributed to decreased protein expression, but recent studies suggest SERCA levels are unchanged in DCM. Thus, another mechanism must be responsible for the deficit. Since proteolysis is increased and proteosome function is impaired in DCM, we reasoned that accumulation of toxic protein fragments may contribute to SERCA dysfunction. In particular, previous studies showed diverse species of hydrophobic -helices can inhibit SERCA, so we hypothesized that SERCA may become congested with transmembrane peptides that mimic endogenous regulatory partners. We purified cell membranes from non-failing and DCM human ventricles and subjected them to mass spectrometry to identify protein species upregulated in DCM. Select candidates were screened for binding and inhibition of SERCA. Several small membrane proteins and membrane protein fragments bound avidly to SERCA and significantly reduced cellular calcium stores. The data suggest a novel pathophysiological mechanism in which transmembrane protein debris obstructs SERCA function and regulation, contributing to cardiac muscle dysfunction in heart failure.

For years, it was believed that G-protein-coupled receptors (GPCRs) activated or modulated by changes in physiological pH were mechanistically regulated by pH-sensitive histidine residues on the receptors exterior. More recent studies have shown that traditional acid-sensing GPCRs (GPR4, GPR65, GPR68) contain buried acidic amino acids in their 7-transmembrane regions and can be activated by pH-dependent mechanisms. The focus of our research has been the adenosine A2A receptor (A2AR) and its ability to activate at low pH levels without having the typical acidic triad structure found in previously studied acid-sensing GPCRs. We used a combination of bioinformatics and a humanized yeast-based platform, DCyFIR, to identify potential structures involved in the proton-sensing mechanism of A2AR. We also validated some of our A2AR variant yeast phenotypes using mammalian cells. Our data suggest that certain mutations eliminate the pH sensitivity of the A2AR while preserving agonist-induced signaling. Additionally, we demonstrated that one of our mutations (N284D) eliminates pH sensing but increases agonist potency, thereby providing a structural explanation for how pH-sensing and sodium binding occur that differs from previously established models. Overall, these data indicate that G-protein-coupled receptors can sense pH in several ways and demonstrate the potential of a novel, pH-insensitive A2AR in acid-related contexts.

Substantial progress in glial electrophysiology has revealed that astrocytes, which account for half of the cells in the human brain, exhibit membrane potentials that often reflect changes in the extracellular environment. Such responses are mediated by a variety of biochemicals, including potassium and neurotransmitters. Recent advances in voltage imaging have provided new insights into voltage activity in astrocyte peripheries, revealing highly localized depolarization that depends on local presynaptic activity. However, the electrophysiological properties of these isolated peripherals have not been explored due to limitations of spatial and temporal resolution. In this study, we aimed to explore differences in the electrophysiological response between whole-cell stimulation and isolated stimuli at different locations in the cell. Therefore, we constructed an empirical conductance-based NEURON model using a realistic morphology to simultaneously capture both astrocyte processes and soma electrophysiological dynamics. Our results predict a breakdown of the Nernstian behavior of astrocytes when potassium stimuli are localized. Instead, local responses are governed by their conductance ratios. Furthermore, we observe strong capabilities for isolating neurotransmitter responses to specific synaptic inputs, with minimal effect on the astrocyte soma. Our study highlights asymmetrical responses of astrocytic electrophysiology that depend on the spatial scale of stimulation.

Aminoglycoside (AG) antibiotic safety is limited by ototoxicity, the mitigation of which is vital considering bacterial resistance mediated erosion of our antibiotic arsenal. Previously, we observed tectorial membrane (TM) sequestration of Ca2+. We hypothesized that the TM sequesters other cations, including the AG gentamicin. We proposed to test the effect of TM genetic ablation on ototoxicity and TM-AG sequestration. After intraperitoneal AG-furosemide, TM-lacking Tecta{Delta}ENT/{Delta}ENT mice showed limited outer hair cell loss, unlike wildtype littermates. Spectroscopy measurements of gentamicin-Texas red (GTTR) were made in isolated wildtype and TectaY1870C TMs and guinea pig cochleae following direct or intraperitoneal GTTR administration. TM-GTTR sequestration was observed in all cases, while negatively correlated with TectaY1870C zygosity. In summary, we discovered a novel TM component in the AG ototoxicity pathway. Intact TM structure is necessary for sequestration, and the TM modulates AG ototoxicity. TM-GTTR sequestration following systemic injection indicates that this phenomenon occurs during AG therapy. Single sentence summaryOtotoxic aminoglycosides collect inside the acellular tectorial membrane of the inner ear, likely due to electrostatic interactions, and the structural status of that membrane modulates the toxic effect of those aminoglycosides on sensory hair cells.

Neurons maintain functionality through homeostatic regulation of spiking activity over extended timescales. Calcium dependent conductance expression is known to regulate mean firing rate, but this is not sufficient to ensure dynamic range in spiking activity and sensitivity to input. This raises the question of whether firing rate variance can be sensed and controlled intracellularly. Using conductance-based models, we demonstrate that time-averaged intracellular calcium dynamics inherently provide a direct readout of both the mean and variance of spiking activity. We show that calcium-based feedback regulation of membrane conductance density can therefore jointly stabilize firing rate mean and variance against input disturbances. Because tuning maximal conductances modulates the underlying relationship between rate statistics, a cells homeostatic response is statedependent rather than fixed. As a consequence, cell-type-specific mixtures of ionic conductances yield distinct homeostatic modalities, implying that cell type dictates homeostatic behaviour as well as spiking and integrative properties.

Regulation of the K+-Cl- cotransporter KCC2 is a critical determinant of the efficacy of inhibition in the central nervous system and KCC2 hypofunction appears at the root of several neurological disorders. Both BDNF-TrkB and NMDAR signaling regulate KCC2, but how they interact remains unknown. Here we show that these two signaling pathways act synergistically to differentially modulate KCC2 function and expression through post-translational regulation, via distinct Ca2+ signalling modes. Blocking ryanodine-dependent intracellular Ca2+ release prevented TrkB-, but not NMDAR-mediated downregulation. TrkB-signalling in absence of NMDAR activation modulated KCC2 function but not expression. In contrast, NMDAR activation induced KCC2 internalization dependent on extracellular Ca2+ influx. In turn, calpain-mediated KCC2 degradation, but not internalization, required Ca2+ influx through voltage-gated Ca2+ channels. While TrkB-activation potentiated the effect of NMDAR on KCC2, the reverse was not true. Yet, strong NMDAR activation was sufficient to cause TrkB-independent KCC2 downregulation. Finally, prolonged, but not short-term inhibition of KCC2 activity caused NMDAR-dependent KCC2 downregulation. These findings reveal, for the first time, that a co-transporter function can be regulated through other means than membrane expression: through a continuum of interwoven synergistic processes, from function to internalization to degradation, scaling with time and stimulus strength.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a crucial role in regulating various microglial functions, including phagocytosis, inflammation, chemotaxis, and proliferation. Recent studies have demonstrated that TREM2 cooperates with DAP12 to mediate intracellular signaling essential for these processes. Despite the importance of the TREM2-DAP12 complex in microglial physiology, the mechanisms controlling its expression and activity remain poorly understood. In this study, we report that the soluble ectodomain of TREM2 (sTREM2) regulates microglial phagocytic activity by attenuating the surface expression of DAP12. We found that stimulation of the microglial cell line BV2 with recombinant sTREM2 reduces the membrane expression of DAP12, but not that of TREM2. In addition, sTREM2 binds to full-length TREM2, leading to the uncoupling of TREM2 from DAP12. Furthermore, pre-treatment of BV2 cells with sTREM2 significantly inhibited amyloid-{beta} incorporation. These findings suggest that sTREM2 negatively regulates TREM2 signaling through the destabilization of the TREM2-DAP12 complex, and act as a novel bioactive molecule that modulates TREM2 signaling under physiological and pathological conditions.