Biology subjects

Gonzalez, W.

Publications and source records attributed to Gonzalez, W..

11 recordsLinked to original sources

bioRxiv2026

Mechanosensitive channels dominate the minimal ion channelrepertoire in prokaryotes

The eukaryotic genomes encode hundreds of proteins that function as ion channels and transporters. Essential for sustaining life, these proteins mediate the movement of inorganic ions (e.g., K+, Na+, Cl-, and Ca2+) across the plasma membrane according to their electrochemical gradients. In multicellular organisms, a diverse array of ion channels contributes to the maintenance of the resting membrane potential, the regulation of pH, osmolarity, and cell volume, and the control of secretion, electrical excitability, and synaptic activity, among many other fundamental physiological processes. Although independent evolutionary origins have been proposed for several ion channel families, their relative hierarchical importance for cellular viability remains poorly understood. To advance our knowledge of ion channel evolutionary history, we focused on determining the minimal combination of permeabilities that allows cellular viability. To this end, we conducted a survey of representative prokaryotes with small genomes across bacterial and archaeal phyla. By focusing on the smallest genomes, our approach enabled the identification of five ion channel architectures shared among prokaryotes. Among these, non-selective mechanosensitive channels (MscS and MscL) are the most abundant, followed by CLC-type channels, RCK-containing 2TM potassium channels, and proton channels of the MotA/TolQ/ExbB family. The conservation of the mechanosensitive protein architecture across archaeal and bacterial membranes suggests that the capacity to monitor physical membrane integrity predates the requirements for electrical communication.

biophysics↗

bioRxiv2025

SHORT DISORDERED PEPTIDES ARE SUFFICIENT TO CONVERT PROTEINS INTO MECHANOSENSORS THAT RESPOND TO PHYSIOLOGICAL CELLULAR FORCES

Mechanical forces regulate many biological processes and a handful of mechanosensor domains have been identified in proteins that respond to cellular forces. The Notch signaling pathway, a key regulator of cell communication processes ranging from animal development to cancer, is activated by mechanical forces generated by cell-cell interactions. The mechanosensing element of Notch is thought to be a 300 amino acid extracellular domain called negative regulatory region (NRR). Ligand binding exerts mechanical forces that are believed to partially unfold NRR, rendering it susceptible to cleavage by extracellular metalloproteases that leads to activation of signaling. However, some engineered notch-like receptors lacking the NRR can be activated upon ligand binding, although the mechanisms underlying this activation are not known. Here we observe that Notch molecules without the NRR, but with a 12 amino acid sequence present in the extracellular juxta-transmembrane domain (eJTMD) are also cleaved and activated upon interaction with their ligands in cultured cells and in transgenic animals. Furthermore, the [~]12 aa eJTMD from notch genes from other species (chicken, xenopus, zebrafish and Drosophila), from other unrelated transmembrane proteins (erbB4, N-CAM, and E-Cadherin), or multiple artificial sequences of [~]12 amino acids without any apparent structure or specific sequence can also convert proteins into ligand-dependent mechanosensors. These results indicate that short, disordered amino acid sequences that are commonly found in many proteins are capable of imparting mechanosensing capabilities into proteins, suggesting that mechanical force may regulate many more cellular processes than previously suspected.

cell biology↗

bioRxiv2024

A KCNC1 Variant Linked to Rett Syndrome Disrupts ER to Golgi Trafficking of Kv3.1 Channel

Intrinsic neuronal excitability, defined by the balance between input and output signals, is crucial to neural function, and its disruption underlies various neurological diseases. Kv3.1 channels, encoded by KCNC1, are essential for high-frequency action potential firing. Variants in these channels are associated with several subtypes of epilepsy. We report a patient with developmental regression and epilepsy, meeting Rett syndrome criteria, who carries a KCNC1 variant encoding the S474C substitution in Kv3.1 (Kv3.1S474C). Electrophysiological and biochemical assays reveal that Kv3.1S474C reduces channel presence in the plasma membrane and is retained in the endoplasmic reticulum (ER). In murine primary cultures expressing Kv3.1S474C, we observed reduced neuronal firing frequency and exclusion of the channel from the axon initial segment (AIS). Consistently, we found a decreased firing frequency using a conductance-based computational neuronal model. In summary, this study identifies a novel link between a KCNC1 variant and Rett syndrome, highlighting the importance of S474 residue in Kv3.1 channel trafficking and function in neurons. SummaryThis study identifies and characterizes a novel KCNC1 variant associated with classical Rett syndrome. This variant disrupts endoplasmic reticulum (ER) to Golgi trafficking of the Kv3.1 channels, highlighting the variants potential role in altered neuronal excitability and neurodevelopmental disorders.

cell biology↗

bioRxiv2024

Identification of Druggable Binding Sites and Small Molecules as Modulators of TMC1

Our ability to hear and maintain balance relies on the proper functioning of inner ear sensory hair cells, which translate mechanical stimuli into electrical signals via mechano-electrical transducer (MET) channels, composed of TMC1/2 proteins. However, the therapeutic use of ototoxic drugs, such as aminoglycosides and cisplatin, which can enter hair cells through MET channels, often leads to profound auditory and vestibular dysfunction. Despite extensive research on otoprotective compounds targeting MET channels, our understanding of how small-molecule modulators interact with these channels remains limited, hampering the discovery of novel drugs. Here, we propose a structure-based screening approach, integrating 3D-pharmacophore modeling, molecular dynamics simulations of the TMC1+CIB2+TMIE complex, and experimental validation. Our pipeline successfully identified several novel compounds and FDA-approved drugs that reduced dye uptake in cultured cochlear explants, indicating MET-modulation activity. Simulations, molecular docking and free-energy estimations allowed us to identify three potential drug-binding sites within the channel pore, phospholipids, key amino acids involved in modulator interactions, and TMIE as a flexible component of the MET complex. We also identified shared ligand-binding features between TMC and structurally related TMEM16 proteins, providing novel insights into their distinct inhibition. Our pipeline offers a broad application for discovering modulators for mechanosensitive ion channels.

neuroscience↗

bioRxiv2024

Constitutive sodium permeability in a C. elegans two-pore domain potassium channel

Two-pore domain potassium (K2P) channels play a central role in modulating cellular excitability and neuronal function. The unique structure of the selectivity filter in K2P and other potassium channels determines their ability to allow the selective passage of potassium ions across cell membranes. The nematode C. elegans has one of the largest K2P families, with 47 subunit-coding genes. This remarkable expansion has been accompanied by the evolution of atypical selectivity filter sequences that diverge from the canonical TxGYG motif. Whether and how this sequence variation may impact the function of K2P channels has not been investigated so far. Here we show that the UNC-58 K2P channel is constitutively permeable to sodium ions and that a cysteine residue in its selectivity filter is responsible for this atypical behavior. Indeed, by performing in vivo electrophysiological recordings and Ca2+ imaging experiments, we demonstrate that UNC-58 has a depolarizing effect in muscles and sensory neurons. Consistently, unc-58 gain-of-function mutants are hypercontracted, unlike the relaxed phenotype observed in hyperactive mutants of many neuromuscular K2P channels. Finally, by combining molecular dynamics simulations with functional studies in Xenopus laevis oocytes, we show that the atypical cysteine residue plays a key role in the unconventional sodium permeability of UNC-58. As predicting the consequences of selectivity filter sequence variations in silico remains a major challenge, our study illustrates how functional experiments are essential to determine the contribution of such unusual potassium channels to the electrical profile of excitable cells. SIGNIFICANCEPotassium channels play a central role in modulating cellular excitability, particularly of neuronal cells. Their unique structure determines their ability to let ions pass selectively through cell membranes. The impact of pathological or evolutionary variations in this selectivity filter remains difficult to predict. Here, we reveal that UNC-58, a member of the two-pore domain potassium channel family of C. elegans, exhibits an unusual sodium permeability due to a unique cysteine residue in its selectivity filter. Our findings underscore the importance of functional studies to determine how sequence variation in potassium channel selectivity filters can shape the electrical profiles of excitable cells.

physiology↗

bioRxiv2023

Recovery of a learned behavior despite partial restoration of neuronal dynamics after chronic inactivation of inhibitory neurons

Maintaining motor behaviors throughout life is crucial for an individuals survival and reproductive success. The neuronal mechanisms that preserve behavior are poorly understood. To address this question, we focused on the zebra finch, a bird that produces a highly stereotypical song after learning it as a juvenile. Using cell-specific viral vectors, we chronically silenced inhibitory neurons in the pre-motor song nucleus called the high vocal center (HVC), which caused drastic song degradation. However, after producing severely degraded vocalizations for around 2 months, the song rapidly improved, and animals could sing songs that highly resembled the original. In adult birds, single-cell RNA sequencing of HVC revealed that silencing interneurons elevated markers for microglia and increased expression of the Major Histocompatibility Complex I (MHC I), mirroring changes observed in juveniles during song learning. Interestingly, adults could restore their songs despite lesioning the lateral magnocellular nucleus of the anterior neostriatum (LMAN), a brain nucleus crucial for juvenile song learning. This suggests that while molecular mechanisms may overlap, adults utilize different neuronal mechanisms for song recovery. Chronic and acute electrophysiological recordings within HVC and its downstream target, the robust nucleus of the archistriatum (RA), revealed that neuronal activity in the circuit permanently altered with higher spontaneous firing in RA and lower in HVC compared to control even after the song had fully recovered. Together, our findings show that a complex learned behavior can recover despite extended periods of perturbed behavior and permanently altered neuronal dynamics. These results show that loss of inhibitory tone can be compensated for by recovery mechanisms partly local to the perturbed nucleus and do not require circuits necessary for learning.

neuroscience↗

bioRxiv2022

Distribution and frequency of salivary gland tumours: an international multicenter study

BackgroundSalivary gland tumours (SGT) are a relatively rare group of neoplasms with a wide range of histopathological appearance and clinical features. To date, most of the epidemiological studies on salivary gland tumours are limited for a variety of reason including being out of date, extrapolated from either a single centre or country studies, or investigating either major or minor glands only. MethodsThis study aimed to mitigate these shortcomings by analysing epidemiological data including demographic, anatomical location and histological diagnoses of SGT from multiple centres across the world. The analysed data included age, gender, location and histological diagnosis from fifteen centres covering the majority of the world health organisation (WHO) geographical regions between 2006 and 2019. ResultsA total of 5798 cases were analysed including 65% benign and 35% malignant tumours. A slight female predilection (54%) and peak incidence between the fourth and seventh decade for both benign and malignant tumours was observed. The majority (69%) of the SGT presented in major and 31% in the minor glands. The parotid gland was the most common location (70%) for benign and minor glands (46%) for malignant tumours. Pleomorphic adenoma (70%), and Warthins tumour (17%), were the most common benign tumours whereas mucoepidermoid carcinoma (25%) and adenoid cystic carcinoma (16%) were the most frequent malignant tumours. ConclusionsThis multicentre investigation presents the largest cohort study to date analysing salivary gland tumour data from tertiary centres scattered across the globe. These findings should serve as a baseline for future studies evaluating the epidemiological landscape of these tumours.

pathology↗

bioRxiv2021

Using a Homeogram to Detect Sleep in Free-living Animals

O_LISleep is appreciated as a behavior critical to homeostasis, performance, and fitness. Yet, most of what we know about sleep comes from humans or controlled laboratory experiments. Assessing sleep in wild animals is challenging, as it is often hidden from view, and electrophysiological recordings that define sleep states are difficult to obtain. Accelerometers have offered great insight regarding gross movement, although ambiguous quiescent states like sleep have been largely ignored, limiting our understanding of this ubiquitous behavior. C_LIO_LIWe developed a broadly applicable sleep detection method called a homeogram that can be applied to accelerometer data collected from wild animals. We applied our methodology to detect sleep in free-ranging North American red squirrels (Tamiasciurus hudsonicus) in a region that experiences drastic seasonal shifts in light, temperature, and behavioral demands. C_LIO_LIOur method characterized sleep in a manner consistent with limited existing studies and expanded those observations to provide evidence that red squirrels apply unique sleep strategies to cope with changing environments. C_LIO_LIApplying our analytical strategy to accelerometer data from other species may open new possibilities to investigate sleep patterns for researchers studying wild animals. C_LI

animal behavior and cognition↗

bioRxiv2020

Sex differences in central inflammatory pain sensitization are associated with differential expression of glycine receptors and GLP-1 at the spinal cord

BackgroundFemales have higher inflammatory pain representation. However, sex differences in central pain sensitization and the regulation of nociceptive response to peripheral inflammation remain unclear. The central pain sensitization is mediated by inhibitory neurotransmission and glial cell activity dysregulation where spinal glycine and GLP-1 receptors have described play a critical role. ObjectivesThe aim of this study was to compare the mechanical withdrawal nociceptive threshold with spinal glycine receptor subunits and GLP-1 expression in adult male and female rats after inflammatory hypersensitivity. MethodsSex differences in inflammatory nociception were evaluated before and after intraplantar hindpaw Zymosan A injection in Sprague-Dawley rats. Mechanical paw withdrawal thresholds were tested using von Frey filaments. Western blot was used to measure GlyRs subunits protein levels in the spinal cord. GLP-1 was determined using the Magnetic Luminex Assay. ResultsA reduced nociceptive threshold was observed in males and females rats after 4 hours of inflammatory Zymosan A injection. However, this reduction was significantly major in females. Western blot analysis demonstrated significantly increased 1, 2, 3 and {beta} GlyR subunit levels in male rats. Female rats only increased 3 and {beta} GlyR subunits after Zymosan A injection. GLP-1 was reduced in female spinal tissues after an inflammatory injury. ConclusionsOur study indicates that sex differences in nociceptive threshold after inflammatory Zymosan A rat pain sensitization is related to the sex differences in glycine receptor subunits and GLP-1 expression at the spinal cord.

physiology↗

bioRxiv2019

Evolutionary analyses unveil the molecular mechanism of fast inactivation in calcium-permeable TRP channels

Fundamental for calcium homeostasis in vertebrates, TRPV5 and TRPV6 are calcium-selective ion channels belonging to the Transient Receptor Potential (TRP) gene family. Detailed phylogenetic analysis suggests that duplications from which TRPV5 and TRPV6 originated occurred independently in the ancestors of mammals, sauropsids, amphibians, and chondrichthyans. Coincident with the conquest of land and the physiological changes needed to maintain calcium homeostasis, our expression analyses suggest a change in the expression pattern from gills to kidney. Within amniotes, we identified a traceable sequence signature of three amino acids at the amino-terminal intracellular region that correlates well with both of the duplication events and the phenotype of fast inactivation observed in TRPV6 channels. Electrophysiological recordings and mutagenesis suggest that calcium-induced fast inactivation is an evolutionary innovation that emerged independently from each duplication event. Simulations of molecular dynamics suggest that calcium-induced channel rearrangements are important modulatory regions of TRP channels.

evolutionary biology↗

bioRxiv2018

Extracellular Cation Binding Pocket Is Essential For Ion Conduction Of OsHKT2;2

HKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity, and performed molecular dynamics simulations in the presence of sodium and potassium ions. By analyzing ion-protein interactions, we identified a cation binding pocket on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion binding pocket and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results.

plant biology↗