Biology subjects

Latorre, R.

Publications and source records attributed to Latorre, R..

13 recordsLinked to original sources

bioRxiv2026

Programming cell behavior with synthetic protease-activated receptors

Extracellular proteases are important signaling molecules in coagulation, inflammation, cell migration, and pain. Dysregulation of extracellular protease activity is common in diseases that perturb these critical functions. Engineering cells to sense and respond programmatically to protease activity has applications in biosensing, cell-based screening for protease activity, and therapeutics. Here we report synthetic protease-activated receptors (SynPARs) based on engineered, auto-inhibited G protein-coupled receptors (GPCRs). Relief of autoinhibition by proteolysis enables receptor activation by an exogenous or tethered agonist to generate transgene expression, real-time fluorescence, or endogenous G-protein signaling. We demonstrate SynPAR modularity with diverse secreted proteases, establish a cell-based SynPAR library selection to optimize protease recognition sequences, and control neuronal activity in response to protease activity. Finally, we use SynPARs in the dorsal root ganglion of mice to counteract hyperalgesia produced by trypsin activity, rewiring neurons to produce an analgesic response to a pain-inducing stimulus. Our study establishes SynPAR as a versatile and modular platform for recording, sensing, and responding to pericellular proteolysis. This fills a critical gap in protease-sensing tools and lays the groundwork for protease-activated genetic and cell-based medicines.

bioengineering↗

bioRxiv2026

Inducible activation of PKA in osteoblasts causes a profound high bone turnover phenotype similar to human diseases

Protein kinase A (PKA) is involved in bone biology and is a key mediator of parathyroid hormone signaling in the osteoblast. However, the consequences of sustained PKA activation in bone are unclear. In this study, we inducibly activated PKA in osteoblasts by deleting its major regulatory subunit, Prkar1a, using a Col11-driven Cre system. Prkar1aob-/-mice demonstrated rapid and profound bone pathologies in their femurs, lumbar and caudal vertebrae with cortical bone breakdown and cortical trabecularization. This phenotype was characterized by increased bone turnover and elevated osteoblastic and osteoclastic activities. Transcriptomic and qPCR analyses showed an impairment of osteoblast differentiation with a defect in ossification, expansion of stromal cells, and numbers of both osteoblastic and osteoclastic precursors. Moreover, there were alterations in gene expression of chemokines and Wnt members with enhanced osteoclastogenesis. Altogether, activation of PKA in osteoblasts by inducible deletion of Prkar1a causes a profound high bone turnover phenotype resembling several human bone diseases.

pathology↗

bioRxiv2026

Targeting of subcellular metabotropic glutamate receptor 5 signaling to modulate pain transmission

Metabotropic glutamate receptor 5 (mGlu5) is a class C GPCR crucial for neuronal development and synaptic transmission. mGlu5 is a potential therapeutic target in pain management and modulates pain-associated gene expression and signaling pathways. Although mGlu5 inhibitors have shown promise in treating pain, none have translated to the clinic. Up to 90% of neuronal mGlu5 expression is intracellular, although the precise locations and function of different mGlu5 intracellular pools remains unclear. Building on recent evidence showing the importance of endosome-mediated nociceptive signaling by other GPCRs, we hypothesized that endosomal pools of mGlu5 contribute to pain transmission, and that targeted inhibition of intracellular mGlu5 signaling results in superior analgesia. Using calcium mobilization assays and genetically encoded resonance energy transfer biosensors, we report that upon its activation mGlu5 recruits Gq/11 and Gs to the plasma membrane. Conversely, internalized mGlu5 in endosomes recruits only Gq/11 proteins. mGlu5 signaling is highly dependent on receptor trafficking to endosomes, with sustained nuclear ERK1/2 signaling requiring both receptor internalization and active glutamate transport into the cell. We generated pH responsive nanoparticles loaded with the mGlu5 negative allosteric modulator VU0366058 (DIPMA-VU058), enabling endosome-targeted inhibition of mGlu5. Nanoparticle encapsulation of VU0366058 enhanced inhibition of both acute and sustained nuclear ERK1/2 signaling, and significantly reduced neuronal excitability in nociceptive circuits in spinal cord slices from rats with neuropathic pain. Intrathecal administration of DIPMA-VU058 achieved superior analgesia in both inflammatory and neuropathic models of pain in mice compared to free VU0366058 and the reference compound fenobam. These studies demonstrate the importance of endosome-associated receptors for the complete mGlu5 signaling response. Furthermore, we show that manipulating the cellular distribution of an allosteric modulator can engender location-biased pharmacological effects. Together, we have revealed new and unappreciated roles for endosome-specific mGlu5 signaling and demonstrate that endosome-selective targeting may offer an alternative therapeutic approach for modulating mGlu5 activity.

pharmacology and toxicology↗

bioRxiv2025

Tuning the gate and the gear: The LRRC26 (γ1) subunit modulates intrinsic gating and voltage-sensor coupling of the BK channel

Association of auxiliary subunits ({beta}1-4 and {gamma}1-4) with the pore-forming subunit of the calcium- and voltage-activated potassium (BK) channel provides functional diversity. {gamma}1 promotes a significant leftward shift of the voltage activation curve, ensuring the adequate functioning of secretory glands, allowing the BK channel to release K+ at the cells resting Ca2+ concentration. Given its physiological importance, it is crucial to elucidate the mechanisms of {gamma}1 action. However, structural and functional studies have yielded conflicting conclusions regarding the modulation of BK channels by {gamma}1. Here, using macroscopic, single-channel, and gating current measurements, we demonstrate that at zero mV {gamma}1 increases 92-fold the equilibrium constant that defines the closed- open transition by destabilizing the channels closed configuration and enhancing the coupling between the voltage sensor and the pore domain, without affecting voltage-sensor activation. These results suggest that {gamma}1 not only causes an increase in the energetic coupling between the voltage sensors and the pore but mainly enhances the channel opening reaction. TeaserThe {gamma}1 subunit favors the BK channel pore opening by destabilizing its closed configuration.

biophysics↗

bioRxiv2025

The BK channel-NS1619 agonist complex reveals molecular insights on allosteric activation gating

BK channels play essential roles in a wealth of physiological functions, including regulating smooth muscle tone and neurotransmitter release. Its dysfunction, often caused by loss-of-function mutations, can lead to severe phenotypes, including ataxia and sensory impairment. Despite the therapeutic potential of BK channel agonists, the molecular mechanisms by which they stabilize the pores open conformation remain unclear. Using cryo-electron microscopy and molecular dynamic simulations, we identified that NS1619, a synthetic benzimidazolone agonist, first described as a BK opener, binds within a pocket formed by the S6/RCK1 linker and the S4 transmembrane segment. Agonist binding drives a twisting motion in the S6 segment, enabling critical interactions with residues K330, K331, and F223. Our findings clarify the mechanism of NS1619 and suggest that its binding site can accommodate other agonists, highlighting a promising target for therapeutic development. TeaserBK channel activation by NS1619 reveals key binding interactions, offering insights for designing targeted therapeutic agents.

biophysics↗

bioRxiv2025

Characterization and targeting of the endosomal signaling of the gastrin releasing peptide receptor in pruritus.

Chronic pruritus is a major unmet clinical problem affecting one in four adults. G protein-coupled receptors (GPCRs) are key receptors driving itch signaling and are a therapeutic target for itch relief. The endosomal signaling of GPCRs provides new challenges for understanding how GPCR signaling is regulated, how endosomal signaling of GPCRs contributes to disease states like chronic pruritus and opens new targets for therapeutic development. The Gastrin releasing peptide receptor (GRPR) is a key mediator of pruritus in the spinal cord. Yet, little is known about the molecular mechanisms regulating GRPR signaling in pruritus, if GRPR can signal from endosomes, or the role of endosomal GRPR in the development of pruritus. Here we show the importance of internalization and endosomal signaling of GRPR in pruritus. Agonist induced GRPR internalization and trafficking was quantified using BRET or microscopy while endosomal-mediated ERK signaling was measured using compartmentalized FRET biosensors. Recruitment of G proteins to endosomes was measured with NanoBit BRET. pH sensitive mesoporous silica nanoparticles (MSN) which accumulated in endosomes were used to deliver RC-3095, a GRPR specific antagonist, intracellularly to block endosomal signaling of GRPR. MSN-RC proved more effective than free RC-3095 at inhibiting chloroquine scratching in mice. Our results demonstrate a critical role for GRPR endosomal signaling in itch sensation. These results highlight the ability of endosomally targeted antagonist to inhibit GRPR signaling and provide a new target for developing therapeutics that block GRPR mediated pruritus. Significance StatementGPCRs are dynamic signaling receptors that can continue to signal following internalization and trafficking to endosomes. Using subcellular targeted BRET and FRET based biosensors we can quantify the recruitment of signaling partners like G proteins and arrestins to GRPR from the endosomal compartment. Inhibition of clathrin and dynamin mediated endocytosis allowed to differentiate plasma membrane vs endosomal signaling of GRPR. pH sensitive nanoparticles loaded with the GRPR antagonist RC-3095 are endocytosed to the endosomal network where they specifically target and block endosomal GRPR signaling. Intrathecal injection of RC-3095 loaded nanoparticles blocked chloroquine induced scratching behavior in mice. Thus, intracellular GRPR drives itch sensation and targeted inhibition of intracellular GRPR signaling is a more effective strategy to treat pruritus.

neuroscience↗

bioRxiv2025

Identification of a secreted protease from Bacteroides fragilis that induces intestinal pain and inflammation by cleavage of PAR2

Protease-activated receptor 2 (PAR2) is a central regulator of intestinal barrier function, inflammation and pain. Upregulated intestinal proteolysis and PAR2-signaling are implicated in inflammatory bowel diseases (IBDs) and irritable bowel syndrome (IBS). To identify potential bacterial regulators of PAR2 activity, we developed a functional assay for PAR2 processing and used it to screen conditioned media from a library of diverse gut commensal microbes. We found that multiple bacteria secrete proteases that cleave host PAR2. Using chemoproteomic profiling with a covalent irreversible inhibitor, we identified a previously uncharacterized Bacteroides fragilis serine protease Bfp1, and showed that it cleaves and activates PAR2 in multicellular and murine models. PAR2 cleavage by Bfp1 disrupts the intestinal barrier, sensitizes nociceptors, and triggers colonic inflammation and abdominal pain. Collectively, our findings uncover Bfp1-mediated PAR2-processing as a new axis of host-commensal-interaction in the gut that has the potential to be targeted for therapeutic intervention in IBD or IBS.

microbiology↗

bioRxiv2024

Nanomedicines targeting signaling of protease-activated receptor 2 in organelles provide sustained analgesia

Although many internalized G protein-coupled receptors (GPCRs) continue to signal, the mechanisms and outcomes of GPCR signaling in organelles are uncertain due to the challenges of measuring organelle-specific signals and of selectively antagonizing receptors in intracellular compartments. Herein, genetically-encoded biosensors targeted to subcellular compartments were used to analyze organelle-specific signaling of protease-activated receptor 2 (PAR2); the propensity of nanoparticles (NPs) to accumulate in endosomes was leveraged to selectively antagonize intracellular PAR2 signaling of pain. PAR2 agonists evoked sustained activation of PAR2, Gq and {beta}-arrestin-1 in early, late and recycling endosomes and the cis- and trans-Golgi apparatus, and activated extracellular signal regulated kinase (ERK) in the cytosol and nucleus, measured with organelle-targeted biosensors. Dendrimer and core-shell polymeric NPs accumulated in early and late endosomes of HEK293 cells, colonic epithelial cells and nociceptors, detected by confocal imaging of fluorescent NPs. NPs efficiently encapsulated and slowly released AZ3451, a negative allosteric PAR2 antagonist. NP-encapsulated AZ3451, but not unencapsulated AZ3451, rapidly and completely reversed PAR2, Gq and {beta}-arrestin-1 activation in endosomes and the Golgi apparatus and ERK activation in the cytosol and nucleus. When administered into the mouse colon lumen, dendrimer NPs accumulated in endosomes of colonocytes and polymeric NPs targeted neurons, sites of PAR2 expression. Both NP-AZ3451 formulations, but not unencapsulated AZ3451, caused long-lasting analgesia and normalized aberrant behavior in preclinical models of inflammatory bowel disease. Thus, organelle-specific PAR2 signals in colonocytes and nociceptors mediate pain. Antagonism of PAR2 in organelles, rather than at the plasma membrane, provides effective pain relief. Significance StatementOnce activated at the cell surface, many GPCRs internalize and continue to signal. The mechanisms and physiological relevance of intracellular GPCR signaling are uncertain. By using organelle-targeted biosensors, we detected sustained activation of the GPCR, PAR2, and its effectors in early, late and recycling endosomes, the cis- and trans-Golgi apparatus, and the cytosol and nucleus. NPs that delivered AZ3451, a PAR2 antagonist, to endosomes disrupted these intracellular signals, whereas unencapsulated AZ3451 was minimally effective. After intracolonic administration to mice, NPs accumulated in colonocytes and neurons. NP-encapsulated AZ3451, but not unencapsulated AZ3451, reversed pain in preclinical models of inflammatory bowel disease. Thus, intracellular PAR2 signaling mediates pain and antagonism of intracellular rather than plasma membrane PAR2 provides effective therapy.

physiology↗

bioRxiv2024

Turning a Kv channel into hot and cold receptor by perturbing its electromechanical coupling.

Voltage-dependent potassium channels (Kv) are extremely sensitive to membrane voltage and play a crucial role in membrane repolarization during action potentials. Kv channels undergo voltage-dependent transitions between closed states before opening. Despite all we have learned using electrophysiological methods and structural studies, we still lack a detailed picture of the energetics of the activation process. We show here that even a single mutation can drastically modify the temperature response of the Shaker Kv channel. Using rapid cell membrane temperature steps (Tsteps), we explored the effects of temperature on the ILT mutant (V369I, I372L, and S376T) and the I384N mutant. The ILT mutant produces a significant separation between the transitions of the voltage sensor domain (VSD) activation and the I384N uncouples its movement from the opening of the domain (PD). ILT and I384N respond to temperature in drastically different ways. In ILT, temperature facilitates the opening of the channel akin to a "hot" receptor, reflecting the temperature dependence of the voltage sensors last transition and facilitating VSD to PD coupling (electromechanical coupling). In I384N, temperature stabilizes the channel closed configuration analogous to a "cold" receptor. Since I384N drastically uncouples the VSD from the pore opening, we reveal the intrinsic temperature dependence of the PD itself. Here, we propose that the electromechanical coupling has either a "loose" or "tight" conformation. In the loose conformation, the movement of the VSD is necessary but not sufficient to efficiently propagate the electromechanical energy to the S6 gate. In the tight conformation the VSD activation is more effectively translated into the opening of the PD. This conformational switch can be tuned by temperature and modifications of the S4 and S4-S5 linker. Our results show that we can modulate the temperature dependence of Kv channels by affecting its electromechanical coupling.

biophysics↗

bioRxiv2023

Structural Basis of Voltage-Dependent Gating in BK Channels and Its Coupling to the Calcium Sensor

The allosteric communication between pore domain, voltage sensors, and Ca2+ binding sites in the Ca2+-activated K+ channel (BK) shapes its multiple physiological roles as the preeminent signal integrator in excitable systems. BK displays shallow voltage sensitivity with very fast gating charge kinetics, yet little is known about the molecular underpinnings of this distinctive behavior. Here, we explore the mechanistic basis of coupling between voltage-sensing domains (VSDs) and calcium sensors in Aplysia BK by locking the VSDs in their resting (R196Q and R199Q) and activated (R202Q) states, with or without calcium. Cryo-EM structures of these mutants reveal unique tilts at the S4 C-terminal end, together with large side-chain rotameric excursions of the gating charges. Importantly, the VSD resting structure (R202Q) also revealed BK in its elusive fully closed state, highlighting the reciprocal relation between calcium and voltage sensors. These structures provide a plausible mechanism where voltage and Ca2+ binding converge physically and couple energetically to define the conformation of the pore domain and thus, BK full functional range.

biophysics↗

bioRxiv2022

ION CHANNEL THERMODYNAMICS STUDIED WITH TEMPERATURE JUMPS MEASURED AT THE MEMBRANE

Perturbing the temperature of a system modifies its energy landscape thus providing a ubiquitous tool to understand biological processes. Here, we developed a framework to generate sudden temperature jumps (Tjumps) and sustained temperature steps (Tsteps) to study the temperature dependence of membrane proteins under voltage-clamp, while measuring the membrane temperature. Utilizing the melanin under the Xenopus laevis oocytes membrane as a photothermal transducer, we achieved short Tjumps up to 10 {o}C in less than 1.5 ms and constant Tsteps for durations up to 150 ms. We followed the temperature at the membrane with submillisecond time resolution by measuring the time-course of membrane capacitance, which is linearly related to temperature. We applied Tjumps in Kir 1.1b, which reveals a highly temperature-sensitive blockage relief and characterized the effects of Tsteps on the temperature-sensitive channels TRPM8 and TRPV1. These newly developed approaches provide a general tool to study membrane proteins thermodynamics.

biophysics↗

bioRxiv2019

Temporal discrimination from the interaction between dynamic synapses and intrinsic subthreshold oscillations

The interaction between synaptic and intrinsic dynamics can efficiently shape neuronal input-output relationships in response to temporally structured spike trains. We use a neuron model with subthreshold oscillations receiving inputs through a synapse with short-term depression and facilitation to show that the combination of intrinsic subthreshold and synaptic dynamics leads to channel-specific nontrivial responses and recognition of specific temporal structures. We employ the Generalized Integrate-and-Fire (GIF) model, which can be subjected to analytical characterization. We map the temporal structure of spike input trains to the type of spike response, and show how the emergence of nontrivial input-output preferences is modulated by intrinsic and synaptic parameters in a synergistic manner. We demonstrate that these temporal input discrimination properties are robust to noise and to variations in synaptic strength, suggesting that they likely contribute to neuronal computation in biological circuits. Furthermore, we also illustrate the presence of these input-output relationships in conductance-based models.\n\nAuthor summaryNeuronal subthreshold oscillations underlie key aspects of information processing in single neuron and network dynamics. Dynamic synapses provide a channel-specific temporal modulation of input information. We combine a neuron model that displays subthreshold oscillations and a dynamic synapse to analytically assess their interplay in processing trains of spike-mediated synaptic currents. Our results show that the co-action of intrinsic and synaptic dynamics builds nontrivial input-output relationships, which are resistant to noise and to changes in synaptic strength. The discrimination of a precise temporal structure of the input signal is shaped as a function of the joint interaction of intrinsic oscillations and synaptic dynamics. This interaction can result in channel-specific recognition of precise temporal patterns, hence greatly expanding the flexibility and complexity in information processing achievable by individual neurons with respect to temporal discrimination mechanisms based on intrinsic neuronal dynamics alone.

neuroscience↗

bioRxiv2019

Calcium-driven regulation of voltage-sensing domains in BK channels

Allosteric interplays between voltage-sensing domains (VSD), Ca2+-binding sites, and the pore domain govern the Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the Ca2+- and voltage-sensing mechanisms crosstalk on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation measuring and analyzing the effects of internal Ca2+ on BK channels gating currents. Our results indicate that the Ca2+ sensors occupancy has a strong impact on the VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single -subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, appear to contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca2+ sensors and VSDs.

biophysics↗