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.

Intracellular calcium waves in cardiac myocytes propagate through a fire-diffuse-fire mechanism in which calcium released from one RyR2 cluster diffuses to neighboring clusters and triggers their activation. Yet propagation faces a fundamental physical difficulty: the calcium signal must cross distances of 1-2 {micro}m between Z-planes while being attenuated by cytosolic buffering and diffusion, and at the same time the release site depletes its local sarcoplasmic reticulum calcium store. How waves propagate efficiently despite these constraints has remained unclear. We developed a three-dimensional computational model of mouse ventricular myocytes at 100 nm resolution to address this question. Our central finding is that co-localization of calsequestrin2 (CASQ2) with RyR2 clusters is required for robust wave propagation. In a physiological model, where CASQ2 is concentrated at release sites as observed experimentally, calcium waves propagate reliably across the cell with velocities that match the experimental range. In contrast, when CASQ2 is distributed uniformly throughout the sarcoplasmic reticulum, keeping total CASQ2 unchanged, the wavefront stalls. These results identify CASQ2-RyR2 co-localization as a key structural requirement for effective calcium wave propagation in ventricular myocytes. Author summaryCalcium waves in cardiomyocytes are thought to underlie the onset of malignant cardiac arrhythmias, such as ventricular tachycardia and fibrillation. Yet, the specific conditions that regulate the transition from local calcium sparks to sustained waves remain poorly understood. Using a newly developed computational model of calcium handling, we demonstrate that the spatial distribution of key regulatory proteins is a critical determinant of arrhythmogenicity. Specifically, we found that calsequestrin2, which buffers Ca2+ within the sarcoplasmic reticulum, must be strictly colocalized with Ca2+ release proteins to facilitate sustained wave propagation. This discovery suggests that cardiac stability depends less on the total quantity of protein and more on its precise architectural organization. The consequences of this finding are significant: it implies that "spatial dysregulation"--where proteins are present but mislocalized--may be a hidden driver of arrhythmias even when protein levels appear normal. This shifts the therapeutic focus from simply altering ion channel conductance to preserving or restoring the structural tethering of the junctional SR. By focusing on the nanodomain architecture, we can better understand how cellular remodeling leads to life-threatening electrical instability.

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.

Every known life form senses and reacts to mechanical forces. These mechanical stimuli can be converted into electrical signals by mechanically gated ion channels, a transduction cascade pivotal to numerous physiological functions including touch, hearing, mechanical pain, circulation, gastrointestinal function, and mechanical loading in various tissues. Despite continuous efforts, numerous mechanically gated ion channels with the mechanotransduction process underlying these physiological functions remain unidentified. Here, we focused on the transmembrane channel-like (TMC) protein family expressed in the cultured cells to identify those with potential mechanosensitive activity. Remarkably, in contrast to human TMC1/2 (HsTMC1/2), human TMC3-8 (HsTMC3-8) proteins are localized to the plasma membrane when heterologously expressed in the cultured cells. Further experiments revealed that mechanical poking stimuli can effectively activate HsTMC3-8. In addition, HsTMC3-8 induced stretch-activated currents and elicited well-resolved single-channel activities in response to negative pressure stimulation. The mutants near the putative pore region altered reversal potentials (Erev) of HsTMC3-8, suggesting that TMC3-8 are likely pore-forming subunits of ion channels. In summary, we proposed that TMC proteins are the largest mammalian mechanically gated ion channel family.

Collective cell migration is a fundamental process driving tissue repair, angiogenesis, and vascular homeostasis. This coordinated movement requires both intercellular communication via gap junctions and precise intracellular Ca{superscript 2} signaling, largely regulated by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump within the endoplasmic reticulum (ER). Historically, carbenoxolone (CBX)--a synthetic derivative of glycyrrhetinic acid--has been widely utilized as a pharmacological tool to inhibit gap junctions and dissect their role in collective cell motility. However, its molecular specificity remains highly controversial. In the present study, using different cellular models, we found that CBX drastically reduces collective cell migration by a previously undescribed function for CBX: a fast, potent, and reversible inhibition of the SERCA pump, which provokes a passive leak of the luminal ER Ca{superscript 2} store. Our findings suggest that the effect of CBX over many cellular responses including cell migration and communication, previously only attributed to gap junction blockade, are indeed the consequence of the disruption of intracellular Ca{superscript 2} homeostasis. One Sentence Summarycarbenoxolone blocks cell migration by inhibiting SERCA

Multiple lines of evidence point to the volume regulated anion channel (VRAC) as being instrumental for cellular volume regulation in many cell types, including astrocytes. VRAC are thought to open during periods of astrocyte swelling, releasing anions and osmolytes to drive water out of the cell, allowing it to return to baseline volume even under sustained osmotic or ionic challenge, a process called regulatory volume decrease, or RVD. However, the occurrence of RVD and VRACs role in this process has remained controversial, with clear evidence in cultured cells but mixed reports from work in intact brain tissue. In the present study, we aimed to address this gap by generating a transgenic mouse line in which VRAC is conditionally ablated in astrocytes (VRAC cKO) and recording the volume responses of astrocytes in VRAC cKO and control tissue using real-time volume imaging. We found that the effect of VRAC cKO on astrocyte swelling was dependent on whether swelling was evoked by elevated extracellular potassium, or by reduced extracellular osmolarity. We also found that both VRAC and the presence of sufficient intracellular taurine concentration were required to elicit RVD in astrocytes, but only in hypoosmolar conditions. Our findings provide new information on the conditions needed to elicit RVD in intact brain tissue, and that VRAC is required for RVD to occur. Our findings further suggest that reduction of intracellular ion concentration is essential for VRAC to be activated, rather than simply membrane expansion. Future experiments will examine the solute release aspect of VRAC activation upon astrocyte swelling, as well as the contributions of VRAC to pathological volume dysregulation.

Repetitive cytosolic Ca2+ spikes in pancreatic acinar cells, elicited by low (physiological) concentrations of acetylcholine (ACh), cholecystokinin (CCK) and gastrin releasing peptide (GRP), control secretion of digestive enzymes, whereas high-intensity stimulation induces sustained Ca2+ elevation initiating acute pancreatitis. Since inositol trisphosphate (IP3) was discovered as an intracellular Ca2+ releasing messenger, it has been assumed that a major class of G-protein coupled receptors relies on this pathway. We have now compared the mechanisms of action of the three physiological stimulants, all acting on different receptors, but each coupled to the IP3 pathway. Low concentrations of CCK and GRP cannot elicit Ca2+ signals without co-operation of an additional intracellular mechanism. CCK-elicited Ca2+ signalling requires activation of intracellular receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), whereas this is not the case for the action of GRP that nevertheless relies on the operation of CD38, the enzyme involved in the synthesis of both cyclic ADP ribose and NAADP. Even Ca2+ signals elicited by ACh are partially dependent on CD38. It is engagement of these additional non-IP3 pathways that allows low concentrations of secretagogues to elicit safe Ca2+ spiking and therefore secretion, obviating the need for potentially toxic high levels of secretagogues.

Reaction diffusion (RD) systems play a fundamental role in numerous biochemical and biophysical processes. Here, we present a novel analytical framework for solving RD equations by applying the Wentzel Kramers Brillouin Jeffreys (WKBJ) formalism to Ca nanodomains generated by individual membrane channels, a widely used paradigm for intracellular Ca signaling. Previous models have primarily focused on stationary Ca nanodomains while neglecting diffusion and saturation of intracellular Ca buffers and sensors. In contrast, we derive analytical solutions without these simplifying assumptions. Our analysis demonstrates that sustained Ca influx generates continuously expanding distributions of free Ca, whereas Ca bound buffers and sensors propagate as traveling waves. These predictions are supported experimentally by measurements of one-dimensional fluorescence profiles produced by single-channel activity and two-dimensional profiles generated by whole cell Ca currents. The analytical framework developed here readily extends Michaelis Menten type kinetics to reaction diffusion systems and may therefore be broadly applicable to biochemical and biophysical processes in which diffusion cannot be neglected.

Arginine vasopressin (AVP) increases excitability of myometrial smooth muscle cells (MSMCs) through Gq-coupled AVP receptors. Although excitability requires membrane depolarization, the mechanisms linking AVP receptor activation to membrane depolarization and Ca{superscript 2} signaling are incompletely understood. Here, we show that AVPR1 is the predominant AVP receptor in primary MSMCs. In Xenopus oocytes, AVP signals through AVPR1 to inhibit SLO2.1-mediated potassium currents, reducing current amplitude to approximately 60% of control currents. Consistent with suppression of a hyperpolarizing conductance, AVP depolarized a myometrial cell line (hTERT-HM) and increased intracellular Ca{superscript 2} signaling. Analysis of Ca{superscript 2} dynamics revealed that the initial Ca{superscript 2} peak was largely preserved under conditions limiting extracellular Ca{superscript 2} entry, consistent with intracellular store release. Conversely, the oscillatory phase depended on extracellular Ca{superscript 2} influx and was reduced by SLO2.1 knockdown. Together, these findings support a model in which AVP preferentially signals through AVPR1A to inhibit SLO2.1, depolarize myometrial cells, enhance VDCC-dependent Ca{superscript 2} entry, and promote excitability, enhancing conditions for uterine contraction.

The cardiac sodium channel NaV1.5 initiates each heartbeat by generating the rapid depolarizing upstroke of the action potential. Dysregulation of NaV1.5 gating can produce cardiac arrhythmias by slowing inactivation, increasing late sodium current (INa,L), and impairing electrical stability. Here, we show that phosphatidylinositol-4,5-bisphosphate (PIP2) is a critical membrane cofactor that stabilizes NaV1.5 gating. Acute PIP2 depletion in human iPSC-derived cardiomyocytes, produced by activation of endogenous AT1 receptors, activation of an engineered M3q-DREADD, or optogenetic recruitment of CRY2-pseudojanin, shifted voltage dependence, slowed fast inactivation, and increased INa,L. These effects were prevented by augmenting intracellular PIP2, required PLC activity when driven by Gq-coupled receptors, and were independent of downstream Ca2+ or PKC signaling. Unlike the skeletal-muscle isoform NaV1.4, NaV1.5 displayed PIP2-dependent shifts in both activation and steady-state inactivation, indicating isoform-specific lipid coupling. Induced-fit docking and molecular dynamics simulations identified a PIP2-interaction interface between the domain IV voltage sensor and pore that contains disease-linked residues. The disease-reported variant R1644C weakened and redistributed the predicted PIP2-contact network, produced elevated basal INa,L, showed enhanced sensitivity to PIP2 depletion, and caused an approximately 30-fold reduction in apparent functional PIP2 sensitivity in excised patches. These findings define a lipid-dependent mechanism that stabilizes NaV1.5 gating and reveal how physiological Gq signaling and inherited channel variants can converge on the channel-PIP2 axis to promote proarrhythmic late sodium current.

The mitochondrial intermembrane space (IMS) is a critical regulatory interface for mitochondrial calcium (mCa2+) flux. Positioned between the outer and inner mitochondrial membranes, the IMS links cytosolic Ca2+ signal to regulated Ca2+ uptake into the matrix. This positioning allows the IMS to influence mCa2+ transport and Ca2+-dependent mitochondrial metabolism. mCa2+ homeostasis is governed mainly by the mitochondrial calcium uniporter complex (mtCU), which mediates mCa2+ uptake, and the Na+/Ca2+ exchanger NCLX, which drives mCa2+ efflux. However, whether IMS regulatory events, particularly proteolytic remodeling by IMS proteases, control this transport machinery remains unclear. Using complementary knockout and overexpression approaches targeting ten IMS proteases (NLN, ATP23, IMMP1L, IMMP2L, YME1L1, OMA1, LACTB2, PARL, and HTRA2), we identified protease-specific remodeling of mtCU components and NCLX abundance. Transcriptomic and proteomic analyses showed that these changes arise largely from protease-specific control of transporter stability rather than transcriptional regulation alone. Proximity-labeling proteomics further revealed spatial associations between IMS proteases and mCa2+ transport components. Functionally, perturbing IMS proteases altered mCa2+ flux and reduced mCa2+ retention capacity, indicating impaired buffering against Ca2+ overload. Together, these findings identify IMS proteases as a proteostatic regulatory network controlling mCa2+ transport and establish a mechanistic link between mitochondrial proteostasis and Ca2+ homeostasis.

Pore forming toxins (PFTs) are cytotoxins secreted in water-soluble form by pathogenic bacteria. They have the ability to form pores in the membrane of host cells, ultimately leading to cell death by lytic activity. Staphylococcus aureus produces a variety of bi-component PFTs, the leukocidins, which target and lyse particular leukocytes, erythrocytes and endothelial cells through specific interactions with membrane receptors. Most of these receptors belong to the family of complement or chemokine receptors that are G protein-coupled receptors (GPCRs). Gamma-hemolysins (Hlgs) are the major leukocidins secreted by S. aureus, and form receptor-dependent hetero-octameric pores through mechanisms that are not fully elucidated. Studying these molecular mechanisms is technically challenging due to the requirement of specific receptors in a lipid bilayer environment. In the present article, we developed a simple and highly sensitive method allowing cell surface expression of a large diversity of target receptors and recording in real-time, currents generated by neo-formed pores. This method is based on the heterologous expression of receptors in Xenopus oocytes and on the two-electrode voltage-clamp technique with electrophysiological robots. Using this approach, we characterized the concentration dependent-kinetics of pore formation, determined the receptor density as a limiting factor, showed specific response to non-cognate pairing of PFTs, observed cell surface binding of F subunits preceding pore formation and propose a hybrid model of subunit oligomerization. This method could be easily implemented for the in vitro characterization of various PFTs on a wide diversity of membrane receptors, to decipher early mechanisms of pore formation or to screen therapeutic agents blocking the cytotoxicity of receptor-dependent PFTs. Author SummaryStaphylococcus aureus is a bacterial species naturally present in our external flora and environment, but it is also one of the main pathogens responsible for nosocomial infections in hospital, with strains having highly problematic multi-resistance to antibiotics. S. aureus is able to secrete various virulence factors, some of which can specifically target and lyse our immune cells, making us more vulnerable to this pathogen. Thus, leukotoxins bind to receptors on the cell surface, drastically change their conformation and form cytotoxic pores in the membrane. Studying the molecular mechanisms underlying the formation of these pores is technically challenging due to their requirement for specific receptors. Here, we tested a simple electrophysiological method enabling the real-time measurement of pore formation on model cells (Xenopus oocytes), which express the receptors of interest. We were thus able to elucidate the kinetics of pore formation, the limiting role of receptors in this process, and propose a complementary model to the standard model. We also demonstrated the ability of this method to detect pore formation of non-cognate pairs of subunits and suggest further applications to characterize pore-forming properties of other toxins, to identify new target receptors, or to screen therapeutic agents inhibiting the formation of pores.

Approximately half of the adult sinoatrial node (SAN) consists of non-myocyte populations, indicating that cardiac pacemaking depends on interactions within a multicellular tissue rather than on pacemaker cardiomyocytes alone. Among these, an S100B-associated cell population has been implicated in pacemaker function, yet its identity and physiological roles remain poorly understood. These cells are rare and dispersed throughout the small, structurally complex SAN, making them difficult to observe repeatedly while preserving the native multicellular environment. Here, we established a dissociated multicellular culture of adult mouse SAN tissue on soft collagen-gelatin hydrogels that retains spontaneous electrical activity and permits longitudinal live imaging of S100B-associated cells. Using an S100B-EGFP+ reporter, we identified at least six reproducible morphological and behavioral phenotypes, including migration, proliferation, phagocytic behavior, and spontaneous self-organization into three-dimensional clusters. Cultures remained spontaneously electrically active for more than 10 days in vitro, with peak activity around day 10. This multicellular culture model bridges the gap between intact SAN preparations and isolated-cell cultures, allowing repeated observation of rare S100B-associated cells within a spontaneously active multicellular environment. HighlightsO_LIThe platform enables longitudinal live imaging of rare S100B-associated cells within an diverse multicellular SAN culture. C_LIO_LILive imaging reveals at least six reproducible morphological and behavioral phenotypes of S100B-associated cells. C_LIO_LIDissociated multicellular SAN cultures remain spontaneously electrically active for more than 10 days in vitro. C_LI

Purinergic signaling coordinates diverse epithelial responses to extracellular nucleotides such as ATP, ADP, and UDP. Although many epithelial cell types co-express multiple P2 receptors, the logic by which these receptors integrate nucleotide signals has remained unclear. Here, using primary human airway epithelial cells as a model, we reveal a hierarchical system in which P2Y2 functions as a central licensing receptor that both enables and constrains downstream activation of P2Y6 and P2Y12. Molecular analysis, calcium assays, and pharmacological profiling show that P2Y6 and P2Y12 exhibit intrinsic activity when co-express to P2Y2 but in turn lose responsiveness to their specific agonists upon upstream activation of P2Y2. This gating mechanism filters background noise by secondary nucleotides and enforces contextual control over downstream signaling. These findings uncover a previously unrecognized principle of purinergic receptor coordination that may apply broadly across epithelial systems, and offer new insight into nucleotide signaling as a therapeutic target.

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.

Cerebrospinal fluid (CSF) provides a specialized extracellular environment for the central nervous system, which is predominantly produced by the choroid plexus, a highly vascularized epithelial structure whose ion transport processes are fundamental to CSF secretion, composition, and homeostasis. The mechanisms of Na+ entry into choroid plexus epithelial cells (CPECs) from the interstitial side remain disputed. The slc4a10 gene product encoding the Na+-dependent Cl-/HCO3- exchanger, Ncbe, was suggested as a key transport mechanism based on its impact on the cell's Na+-dependent regulation of intracellular pH and its basolateral membrane expression. The current study was undertaken to directly assess the contribution of Ncbe to the Na+ uptake into CPECs. Intracellular Na+ was recorded by fluorometry using the Na+ probe Sodium Binding Fluorescent Indicator in clusters of CPECs with access to both the luminal and basolateral membranes. Removal of extracellular Na+ reduced the apparent ex vivo intracellular [Na+] to ~5 mM from a baseline of ~43 mM in the absence of CO2/HCO3- and ~54 mM in the presence of CO2/HCO3-. Flame photometry estimated the intracellular [Na+] ex vivo to ~28 mM. The CO2/HCO3--dependent rate of [Na+] recovery amounted to ~53% of the total recovery rate upon re-addition of Na+. Experiments with access to only the luminal membrane show a [Na+] recovery of a similar rate as observed in the absence of CO2/HCO3- in the clusters. The CO2/HCO3--independent [Na+] recovery was inhibited to ~50% by the NKCC1 inhibitor bumetanide and to ~30% by the TRPv4 inhibitor RN1734. NHE contributed to a minor extent to the CO2/HCO3--independent transport. The HCO3- transport inhibitor DIDS, however, inhibited the total [Na+] recovery rate to ~50%, indicating a role for Ncbe rather than NBCn1 in the cellular [Na+] recovery. Indeed, docking of DIDS into Ncbe and NBCn1 indicated that both proteins can accommodate the binding of DIDS. However, the orientation of the DIDS poses in Ncbe suggests a binding mode more similar to that found in the Anion Exchangers (SLC4A1-3), which seems to accommodate the covalent-type docking more than NBCn1. The Ncbe inhibition by DIDS was supported by the rate of [Na+] recovery that was significantly higher in CPECs from Ncbe-wt than Ncbe-ko mice in the presence of CO2/HCO3-. As both NKCC1 and TRPv4 are localized to the luminal membrane, the findings collectively suggest that Ncbe is the most prominent mechanism for Na+ entry into CPECs expressed at the basolateral side. We suggest Ncbe as the rate-limiting mechanism in the vectorial Na+ transport driving CSF secretion.

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.

Osteocytes detect mechanical forces within bone through signaling processes organized at the plasma membrane. Membrane cholesterol regulates membrane organization, dynamics, and mechanical properties, yet its role in osteocyte mechanotransduction in vivo remains unknown. Here, we developed an intravital multiphoton imaging approach to quantify membrane-associated uptake, retention, and clearance alongside load-induced Ca2+ signaling in osteocytes within the intact metatarsal. Using fluorescent nanoparticles and a membrane-labeling probe, we tracked these processes before and after pharmacological cholesterol depletion. Cholesterol depletion reduced osteocyte membrane uptake and retention and altered clearance in both sexes, while affecting load-induced Ca2+ signaling in a sex-dependent manner. In females, cholesterol depletion increased both the proportion of osteocytes responding to mechanical loading and the magnitude of their responses, whereas neither outcome changed in males. These findings identify plasma membrane cholesterol as a regulator of osteocyte membrane dynamics and mechanical responsiveness in vivo. More broadly, this work establishes an approach for directly examining how membrane composition and turnover regulate mechanotransduction in cells embedded within their native tissue environment.

Reliable neurotransmitter release critically depends on the spatial relationship between voltage- gated calcium channels (VGCCs) and presynaptic release sites. Single particle tracking of endogenous CaV2.1 channels at glutamatergic synapses of hippocampal neurons revealed that apart from CaV2.1 channels aggregated in stable nanodomain clusters, a substantial fraction of Cav2.1 channels remained mobile, raising the question of whether these dispersed channels contribute to synaptic function. Mathematical modelling predicted that dispersed Cav2.1 channels cooperatively enhance release reliability. Upon repetitive stimulation, mobile CaV2.1 channels enable alternative use of release sites and thereby reduce the probability of failed presynaptic release. Both optogenetic immobilisation of CaV2.1 channels per se or activation of GABAB receptors (GABABRs) alone increase the failure rate and can lead to synaptic silencing. However, optogenetic clustering CaV2.1 channels prior to GABABR activation increases the fraction of synapses that remain active even in presence of GABABR agonist. The contribution of mobile channels to reliable neurotransmitter release is frequency-dependent and is minor at stimulation frequencies 1 Hz but becomes strong at frequencies over 10 Hz. These results demonstrate that mobile presynaptic CaV2.1 channels increase the frequency range of synaptic transmission but are particularly sensitive to metabotropic GABABR-mediated inhibition in glutamatergic hippocampal synapses.