Biology subjects

Zhu, M. X.

Publications and source records attributed to Zhu, M. X..

13 recordsLinked to original sources

bioRxiv2026

Cryo-EM elucidation of stoichiometric plasticity, asymmetric ligand recognition and allosteric coupling in human P2X2/3 heterotrimeric channels

P2X receptors are trimeric ATP-gated ion channels that assemble as homo- or heterotrimers, with heteromeric forms exhibiting intrinsic asymmetry that influences function. Here, we report four high-resolution cryo-EM structures of human P2X2/3 heterotrimers representing distinct functional states, including ATP-bound assemblies (P2X332 and P2X223), the apo form, and a ligand/ATP-bound closed conformation. The three ATP-binding sites show asymmetric recognition of MgATP{superscript 2}- and ATP-, and channel activation requires occupancy of only two MgATP{superscript 2}- molecules. Gefapixant binds a single allosteric site and selectively inhibits MgATP{superscript 2}-, but not ATP-, binding, indicating orthosteric-allosteric coupling within the heterotrimer. Structural features of the transmembrane domain define ion permeation, particularly for Ca{superscript 2}. Despite asymmetric ligand interactions, gating remains largely symmetric, with minor differences in desensitization. These findings provide a structural framework linking asymmetry to coordinated channel function and open avenues for subtype-selective therapeutic intervention.

biophysics↗

bioRxiv2025

DHHC21 is a STIM1 protein S-acyltransferase that modulates immune function in vivo

Depletion of calcium from ER stores leads to the activation of calcium channels on the plasma membrane. This process is termed store-operated calcium entry (SOCE). The proteins STIM1 and STIM2 function as ER calcium sensors, and upon store depletion, they undergo a conformational change that allows them to bind to and gate Orai calcium channels on the plasma membrane. We have shown that both Orai1 and STIM1 are dynamically S-acylated after store depletion, which is required for SOCE. These results suggest the requirement of a calcium-activated protein S-acyltransferase such as DHHC21. Here, we show that DHHC21 is essential for SOCE in vitro and in vivo. Using the depilated mouse model that expresses DHHC21 but can no longer be activated by calcium, we show that DHHC21 activation is required for STIM1 S-acylation and subsequent calcium entry. Plasma membrane-localized DHHC21 is dynamically recruited into Orai1/STIM1 puncta upon store depletion, where it physically binds to STIM1. Finally, we show that depilated mice phenocopy many aspects of autoimmune lymphoproliferative syndrome (ALPS), including defective Fas-mediated calcium release, T cell death, neutropenia, and increased serum vitamin B12 levels. These results suggest that dynamic S-acylation has underappreciated and expansive roles in second messenger signaling and immune system function. Targeting DHHC21 may be therapeutically beneficial for ALPS and diseases associated with deregulated activation of STIM1, such as tubular aggregate myopathy and Stormorken syndrome.

immunology↗

bioRxiv2025

ATP-sensitive isoforms of P2X5: A reevaluation of its function across species

The P2X receptor family comprises ATP-gated non-selective cation channels central to physiological processes across the nervous, immune, cardiovascular, respiratory, and reproductive systems. While P2X1, P2X2, P2X3, P2X4, and P2X7 are well-established as ATP-sensitive, P2X5 isoforms and transcript variants (TVs) have traditionally been considered ATP-insensitive, limiting their functional exploration. This study identifies previously overlooked ATP-sensitive P2X5 isoforms across diverse species. Through gene profiling and next-generation sequencing (NGS), we found that the zfP2X5TV2 isoform dominates ATP-sensitive forms in zebrafish. In mice, ATP-sensitive mP2X5G317 (mP2X5.1) comprises [~]90% of transcripts, while in rats, exon 3-containing rP2X5F191 accounts for over 70%. In human cell lines, ATP-sensitive isoforms retaining exons 3, 7, and 10 represent [~]15-30% of P2X5 transcripts. RNA-seq from human tissues confirms frequent retention of exons 3 and 7 and polymorphic exon 10 expression. ATP-sensitive P2X5 variants were also identified in chickens, bullfrogs, dogs, cows, and naked mole-rats. These findings challenge the prevailing assumption of ATP-insensitivity, highlighting the need to reassess P2X5s roles in physiological and pathological contexts.

physiology↗

bioRxiv2024

Hijacking a dedicated entorhinal-hippocampal extinction circuit to remove traumatic memory

Effective psychotherapy of post-traumatic stress disorder (PTSD) remains challenging due to the fragile nature of fear extinction, for which ventral hippocampal CA1 (vCA1) region is considered as a central hub. However, neither the core pathway nor the cellular mechanisms involved in implementing extinction are known. Here, we unveil a direct pathway, where layer 2a fan cells in the lateral entorhinal cortex (LEC) target parvalbumin-expressing interneurons (PV-INs) in the vCA1 region to propel low gamma-band synchronization of the LEC-vCA1 activity during extinction learning. Bidirectional manipulations of either hippocampal PV-INs or LEC fan cells suffice fear extinction. Gamma entrainment of vCA1 by deep brain stimulation (DBS) or noninvasive transcranial alternating current stimulation (tACS) of LEC persistently enhances the PV-IN activity in vCA1, thereby promoting fear extinction. These results demonstrate that the LEC-vCA1 pathway forms a top-down motif to empower low gamma-band oscillations that facilitate fear extinction. Finally, application of low gamma DBS and tACS to a mouse model with persistent PTSD shows potent efficacy, suggesting that the dedicated LEC-vCA1 pathway can be hijacked for therapy to remove traumatic memory trace. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/593830v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@ca6768org.highwire.dtl.DTLVardef@4ae574org.highwire.dtl.DTLVardef@11e4ff5org.highwire.dtl.DTLVardef@1136cc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Neuropeptide Y co-opts neuronal ensembles for memory lability and stability

Memory engrams are formed by activity-dependent recruitment of distinct subsets of excitatory principal neurons (or neuronal ensembles) whereas inhibitory neurons pivot memory lability and stability1-5. However, the molecular logic for memory engrams to preferentially recruit specific type of interneurons over other subtypes remains enigmatic. Using activity-dependent single-cell transcriptomic profiling6-8 in mice with training of cued fear memory and extinction, we discovered that neuropeptide Y (NPY)-expressing (NPY+) GABAergic interneurons in the ventral hippocampal CA1 (vCA1) region exert fast GABAergic inhibition to facilitate the acquisition of memory, but bifurcate NPY-mediated slow peptidergic inhibition onto distinct sub-ensembles underlying the extinction of single memory trace. Genetically encoded calcium and NPY sensors revealed that both calcium dynamics of NPY+ neurons and their NPY release in vCA1 ramp up as extinction learning progresses while behavioral state switches from "fear-on" to "fear-off". Bidirectional manipulations of NPY+ neurons or NPY itself demonstrated NPY is both necessary and sufficient to control the rate and degree of memory extinction by acting on two physically non-overlapping sub-ensembles composed of NPY1R- and NPY2R-expressing neurons. CRISPR/Cas9-mediated knockout of NPY2R or NPY1R further unravels that NPY co-opts its actions on these two sub-ensembles to gate early fast and late slow stages of extinction. These findings exemplify the intricate spatiotemporal orchestration of slow peptidergic inhibitions from single subtype of GABAergic interneurons to fine-tune engram lability verse stability of memory.

neuroscience↗

bioRxiv2024

Widespread latent hyperactivity of nociceptors outlasts enhanced avoidance behavior following incision injury

Nociceptors with somata in dorsal root ganglia (DRGs) exhibit an unusual readiness to switch from an electrically silent state to a hyperactive state of tonic, nonaccommodating, low-frequency, irregular discharge of action potentials (APs). Ongoing activity (OA) during this state is present in vivo in rats months after spinal cord injury (SCI), and has been causally linked to SCI pain. OA induced by various neuropathic conditions in rats, mice, and humans is retained in nociceptor somata after dissociation and culturing, providing a powerful tool for investigating its mechanisms and functions. An important question is whether similar nociceptor OA is induced by painful conditions other than neuropathy. The present study shows that probable nociceptors dissociated from DRGs of rats subjected to postsurgical pain (induced by plantar incision) exhibit OA. The OA was most apparent when the soma was artificially depolarized to a level within the normal range of membrane potentials where large, transient depolarizing spontaneous fluctuations (DSFs) can approach AP threshold. This latent hyperactivity persisted for at least 3 weeks, whereas behavioral indicators of affective pain - hindpaw guarding and increased avoidance of a noxious substrate in an operant conflict test - persisted for 1 week or less. An unexpected discovery was latent OA in neurons from thoracic DRGs that innervate dermatomes distant from the injured tissue. The most consistent electrophysiological alteration associated with OA was enhancement of DSFs. Potential in vivo functions of widespread, low-frequency nociceptor OA consistent with these and other findings are to amplify hyperalgesic priming and to drive anxiety-related hypervigilance.

neuroscience↗

bioRxiv2023

Readiness of nociceptor cell bodies to generate spontaneous activity results from background activity of diverse ion channels and high input resistance

Nociceptor cell bodies generate "spontaneous" discharge that can promote ongoing pain in persistent pain conditions. Little is known about the underlying mechanisms. Recordings from nociceptor cell bodies (somata) dissociated from rodent and human dorsal root ganglia (DRGs) have shown that prior pain in vivo is associated with low-frequency discharge controlled by irregular depolarizing spontaneous fluctuations of membrane potential (DSFs), likely produced by transient inward currents across the somal input resistance. Here we show that DSFs are associated with high somal input resistance over a wide range of membrane potentials, including depolarized levels where DSFs approach action potential (AP) threshold. Input resistance and both the amplitude and frequency of DSFs were increased in neurons exhibiting spontaneous activity. Ion substitution experiments indicated that the depolarizing phase of DSFs is generated by spontaneous opening of channels permeable to Na+ and/or Ca2+, and that Ca2+-permeable channels are especially important for larger DSFs. Partial reduction of the amplitude and/or frequency of DSFs by perfusion of pharmacological inhibitors indicated small but significant contributions from Nav1.7, Nav1.8, TRPV1, TRPA1, TRPM4, and N-type Ca2+ channels. Less specific blockers suggested a contribution from NALCN channels, and global knockout suggested a role for Nav1.9. The combination of high somal input resistance plus background activity of diverse ion channels permeable to Na+ and/or Ca2+ produces DSFs that are poised to reach AP threshold if resting membrane potential (RMP) depolarizes, AP threshold decreases, and/or DSFs become enhanced -- all of which have been reported under painful neuropathic and inflammatory conditions.

neuroscience↗

bioRxiv2022

Phosphatidylserine controls synaptic targeting and membrane stability of ASIC1a

Phospholipid-protein interaction is highly specialized at the membranous nanodomains and critical for membrane receptor signaling. Calcium-permeable acid-sensing ion channel isoform 1a (ASIC1a) is a major neuronal proton sensor that contributes to synaptic plasticity. The functional outcome of ASIC1a is dependent on its surface targeting in synaptic subdomains; however, the lipid environment for ASIC1a and its role in channel targeting remain poorly understood. Here, we report that anionic phosphatidylserine (PS) is enriched in dendritic spines during neurodevelopment and it directly binds to ASIC1a through an electrostatic interaction with a di-arginine motif at ASIC1a C-terminus. PS regulates the membrane targeting and function of ASIC1a, which are both strongly suppressed by inhibition of PS synthesis. In cortical neuron dendrites, both PS and ASIC1a are predominately localized to peri-synaptic sites of spine heads, surrounding instead of overlapping with postsynaptic markers, PSD-95 and GluN1. Uncoupling the interaction between PS and ASIC1a by changing the charges to neutral or acidic at the di-arginine PS-binding motif, or applying a membrane penetrating competing peptide, caused mistargeting of ASIC1a at the synaptic sites, an overall increase in internalization and/or cytoplasmic accumulation of ASIC1a, and a decrease in its channel function. Together, our results provide novel insights on lipid microenvironment that governs ASIC1a expression and function at the membrane surface, especially peri-synaptic regions of dendritic spines, through an electrostatic interaction with anionic phospholipids.

neuroscience↗

bioRxiv2022

Dynamic tripartite construct of interregional engram circuits underlies forgetting of extinction memory

Fear extinction allows for adaptive control of learned fear responses but often fails, resulting in a renewal or spontaneous recovery of the extinguished fear, i.e., forgetting of the extinction memory readily occurs. Using an activity-dependent neuronal labeling strategy, we demonstrate that engram neurons for fear extinction memory are dynamically positioned in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and ventral hippocampus (vHPC), which constitute an engram construct in the term of directional engram synaptic connectivity from the BLA or vHPC to mPFC, but not that in the opposite direction, for retrieval of extinction memory. Fear renewal or spontaneous recovery switches the extinction engram construct from an accessible to inaccessible state, whereas additional extinction learning or optogenetic induction of long-term potentiation restores the directional engram connectivity and prevents the return of fear. Thus, the plasticity of engram construct underlies forgetting of extinction memory.

neuroscience↗

bioRxiv2022

Dynamic S-acylation of STIM1 is required for store-operated Ca 2+ entry

Many cell surface stimuli cause calcium release from endoplasmic reticulum (ER) stores to regulate cellular physiology. Upon ER calcium store depletion, the ER-resident protein STIM1 physically interacts with plasma membrane protein Orai1 to induce calcium release-activated calcium (CRAC) currents that conduct calcium influx from the extracellular milieu. Although the physiological relevance of this process is well established, the mechanism supporting the assembly of these proteins is incompletely understood. Earlier we demonstrated a previously unknown post-translational modification of Orai1 with long chain fatty acids, known as S-acylation. We found that S-acylation of Orai1 is dynamically regulated in a stimulus-dependent manner and essential for its function as a calcium channel. Here we show that STIM1 is also rapidly and transiently S-acylated at cysteine 437 upon ER calcium store depletion. S-acylation of STIM1 is required for the assembly of STIM1 into puncta with Orai1 and full CRAC channel function. Together with the S-acylation of Orai1, our data suggest that stimulus-dependent S-acylation of CRAC channel components Orai1 and STIM1 is a critical mechanism facilitating CRAC channel assembly and function.

cell biology↗

bioRxiv2020

Thiamine pyrophosphokinase deficiency induces Alzheimer's pathology

BackgroundThiamine diphosphate (TDP) reduction plays an important role in cerebral glucose hypometabolism, the neurodegenerative indicator, in Alzheimers disease (AD). The mechanism underlying TDP reduction remains elusive. Thus, it is critical to define the mechanism and its effect on neurodegeneration, the pathological basis of the disease occurrence and progression. MethodsThe mRNA levels of all known genes associated with thiamine metabolism, including thiamine pyrophosphokinase (TPK), Solute Carrier Family 19 Member 2 (SLC19A2), SLC19A3, and SLC25A19, in brain samples of patients with AD and other neurodegenerative disorders in multiple independent datasets were analyzed. TPK protein levels were further examined in the brain tissues of AD patients and control subjects. A mouse model with conditional knockout (cKO) of TPK gene in the excitatory neurons of adult brain was established. ResultsThe brain TPK mRNA level was markedly lower in AD patients, but not in other neurodegenerative disorders. The brain TPK protein level was also significantly decreased in AD patients. TPK gene knockout in the mice caused cerebral glucose hypometabolism, {beta}-amyloid deposition, Tau hyperphosphorylation, neuroinflammation, and neuronal loss and brain atrophy. Cross-species correlation analysis revealed the similar changes of gene profiling between the cKO mice and AD patients. ConclusionsThe deficiency of brain TPK, a key enzyme for TDP synthesis, is specific to AD. The cKO mice show AD-associated phenotypes and could serve as a new mouse model for AD studies. Our study provides a novel insight into the critical role of TPK in AD pathogenesis and its potential for the disease treatment.

neuroscience↗

bioRxiv2019

Membrane insertion of chromogranin B for granule maturation in regulated secretion

Regulated secretion serves responses to specific stimuli in eukaryotes. An anion conductance was found essential for maturation and acidification of secretory granules four decades ago, but its genetic identity was unknown. We now demonstrate that chromogranin B (CHGB), an obligate granule protein, constitutes the long-sought anion channel. High-pressure freezing immuno-electron microscopy and biochemical assays showed native CHGB in close proximity to secretory granule membranes, and its membrane-bound and soluble forms both reconstituted Cl- channels. Release of secretory granules delivered CHGB clusters to plasma membranes, which dominate whole-cell anion conductance. Intragranular pH measurements and cargo maturation assays found that CHGB channels supported proinsulin - insulin conversion and dopamine-loading in neuroendocrine cells. {beta}-cells from Chgb-/- mice exhibited significant granule deacidification, accounting for hyperproinsulinemia, altered glucose-tolerance response and lower dopamine concentration in chromaffin granules in these animals. Membrane insertion of well-conserved CHGB is thus indispensable for granule maturation in exocrine, endocrine and neuronal cells. HighlightsO_LINative CHGB is amphipathic and distributes in the lumen and membranes of secretory granules with contrastingly different destinies and functions. C_LIO_LINative CHGB, once delivered to cell surface via granule exocytosis, dominates anion conductance in plasma membranes. C_LIO_LICHGB channels facilitate granule acidification and cargo maturation in cultured and primary neuroendocrine cells. C_LIO_LICHGB channels from bovine, rat and mouse cells all serve the long-missing, intra-organellar anion shunt pathway in the secretory granules for regulated secretion. C_LI

cell biology↗

bioRxiv2018

VPAC1 couples with TRPV4 channel to promote calcium-dependent gastric cancer progression

Although VPAC1 and its ligand vasoactive intestinal peptide (VIP) are important in gastrointestinal physiology, their involvements in progression of gastrointestinal tumor have not been explored. Here, we found that higher expression of VIP/VPAC1 was observed in gastric cancer compared to the adjacent normal tissues. The increased expression of VIP/VPAC1 in gastric cancer correlated positively with invasion, tumor stage, lymph node, distant metastases, and poor survival. Moreover, high expression of VIP and VPAC1, advanced tumor stage and distant metastasis were independent prognostic factors. VPAC1 activation by VIP markedly induced TRPV4-mediated Ca2+ entry, and eventually promoted gastric cancer progression in a Ca2+ signaling-dependent manner. Inhibition of VPAC1 and its signaling pathway could block the progressive responses. VPAC1/TRPV4/Ca2+ signaling in turn enhanced the expression and secretion of VIP in gastric cancer cells, enforcing a positive feedback regulation mechanism. Taken together, our study demonstrate that VPAC1 is significantly overexpressed in gastric cancer and VPAC1/TRPV4/Ca2+ signaling axis could enforce a positive feedback regulation in gastric cancer progression. VIP/VPAC1 may serve as potential prognostic markers and therapeutic targets for gastric cancer.

cancer biology↗