Development of an oral treatment that rescues gait ataxia and retinal degeneration in a phenotypic mouse model of familial dysautonomia.

Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a splicing mutation in elongator acetyltransferase complex subunit 1 (ELP1). This mutation leads to the skipping of exon 20 and a tissue-specific reduction of ELP1, mainly in the central and peripheral nervous systems. FD is a complex neurological disorder accompanied by severe gait ataxia and retinal degeneration. There is currently no effective treatment to restore ELP1 production in individuals with FD, and the disease is ultimately fatal. After identifying kinetin as a small molecule able to correct the ELP1 splicing defect, we worked on its optimization to generate novel splicing modulator compounds (SMCs) that can be used in individuals with FD. Here, we optimize the potency, efficacy, and bio-distribution of second-generation kinetin derivatives to develop an oral treatment for FD that can efficiently pass the blood-brain barrier and correct the ELP1 splicing defect in the nervous system. We demonstrate that the novel compound PTC258 efficiently restores correct ELP1 splicing in mouse tissues, including brain, and most importantly, prevents the progressive neuronal degeneration that is characteristic of FD. Postnatal oral administration of PTC258 to the phenotypic mouse model TgFD9;Elp1Δ20/flox increases full-length ELP1 transcript in a dose-dependent manner and leads to a 2-fold increase in functional ELP1 in the brain. Remarkably, PTC258 treatment improves survival, gait ataxia, and retinal degeneration in the phenotypic FD mice. Our findings highlight the great therapeutic potential of this novel class of small molecules as an oral treatment for FD.

The nonclassical MHC class I Qa-1 expressed in layer 6 neurons regulates activity-dependent plasticity via microglial CD94/NKG2 in the cortex.

During developmental critical periods, circuits are sculpted by a process of activity-dependent competition. The molecular machinery involved in regulating the complex process of responding to different levels of activity is now beginning to be identified. Here, we show that the nonclassical major histocompatibility class I (MHCI) molecule Qa-1 is expressed in the healthy brain in layer 6 corticothalamic neurons. In the visual cortex, Qa-1 expression begins during the critical period for ocular dominance (OD) plasticity and is regulated by neuronal activity, suggesting a role in regulating activity-dependent competition. Indeed, in mice lacking Qa-1, OD plasticity is perturbed. Moreover, signaling through CD94/NKG2, a known cognate Qa-1 heterodimeric receptor in the immune system, is implicated: selectively targeting this interaction phenocopies the plasticity perturbation observed in Qa-1 knockouts. In the cortex, CD94/NKG2 is expressed by microglial cells, which undergo activity-dependent changes in their morphology in a Qa-1­dependent manner. Our study thus reveals a neuron­microglial interaction dependent upon a nonclassical MHCI molecule expressed in L6 neurons, which regulates plasticity in the visual cortex. These results also point to an unexpected function for the Qa-1/HLA-E (ligand) and CD94/NKG2 (receptor) interaction in the nervous system, in addition to that described in the immune system.

Selective retinal ganglion cell loss and optic neuropathy in a humanized mouse model of familial dysautonomia.

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease caused by a splicing mutation in the gene encoding Elongator complex protein 1 (ELP1, also known as IKBKAP). This mutation results in tissue-specific skipping of exon 20 with a corresponding reduction of ELP1 protein, predominantly in the central and peripheral nervous system. Although FD patients have a complex neurological phenotype caused by continuous depletion of sensory and autonomic neurons, progressive visual decline leading to blindness is one of the most problematic aspects of the disease, as it severely affects their quality of life. To better understand the disease mechanism as well as to test the in vivo efficacy of targeted therapies for FD, we have recently generated a novel phenotypic mouse model, TgFD9; IkbkapΔ20/flox. This mouse exhibits most of the clinical features of the disease and accurately recapitulates the tissue-specific splicing defect observed in FD patients. Driven by the dire need to develop therapies targeting retinal degeneration in FD, herein, we comprehensively characterized the progression of the retinal phenotype in this mouse, and we demonstrated that it is possible to correct ELP1 splicing defect in the retina using the splicing modulator compound (SMC) BPN-15477.

Genetically Stable and Scalable Nanoengineering of Human Primary T Cells via Cell Mechanoporation.

Effective tumor regression has been observed with chimeric antigen receptor (CAR) T cells; however, the development of an affordable, safe, and effective CAR-T cell treatment remains a challenge. One of the major obstacles is that the suboptimal genetic modification of T cells reduces their yield and antitumor activity, necessitating the development of a next-generation T cell engineering approach. In this study, we developed a nonviral T cell nanoengineering system that allows highly efficient delivery of diverse functional nanomaterials into primary human T cells in a genetically stable and scalable manner. Our platform leverages the unique cell deformation and restoration process induced by the intrinsic inertial flow in a microchannel to create nanopores in the cellular membrane for macromolecule internalization, leading to effective transfection with high scalability and viability. The proposed approach demonstrates considerable potential as a practical alternative technique for improving the current CAR-T cell manufacturing process.

Microfluidic Impedance-Deformability Cytometry for Label-Free Single Neutrophil Mechanophenotyping.

The intrinsic biophysical states of neutrophils are associated with immune dysfunctions in diseases. While advanced image-based biophysical flow cytometers can probe cell deformability at high throughput, it is nontrivial to couple different sensing modalities (e.g., electrical) to measure other critical cell attributes including cell viability and membrane integrity. Herein, an "optics-free" impedance-deformability cytometer for multiparametric single cell mechanophenotyping is reported. The microfluidic platform integrates hydrodynamic cell pinching, and multifrequency impedance quantification of cell size, deformability, and membrane impedance (indicative of cell viability and activation). A newly-defined "electrical deformability index" is validated by numerical simulations, and shows strong correlations with the optical cell deformability index of HL-60 experimentally. Human neutrophils treated with various biochemical stimul are further profiled, and distinct differences in multimodal impedance signatures and UMAP analysis are observed. Overall, the integrated cytometer enables label-free cell profiling at throughput of >1000 cells min-1 without any antibodies labeling to facilitate clinical diagnostics.

Development of a Photonic Switch via Electro-Capillarity-Induced Water Penetration Across a 10-nm Gap.

With narrow and dense nanoarchitectures increasingly adopted to improve optical functionality, achieving the complete wetting of photonic devices is required when aiming at underwater molecule detection over the water-repellent optical materials. Despite continuous advances in photonic applications, real-time monitoring of nanoscale wetting transitions across nanostructures with 10-nm gaps, the distance at which photonic performance is maximized, remains a chronic hurdle when attempting to quantify the water influx and molecules therein. For this reason, the present study develops a photonic switch that transforms the wetting transition into perceivable color changes using a liquid-permeable Fabry-Perot resonator. Electro-capillary-induced Cassie-to-Wenzel transitions produce an optical memory effect in the photonic switch, as confirmed by surface-energy analysis, simulations, and an experimental demonstration. The results show that controlling the wetting behavior using the proposed photonic switch is a promising strategy for the integration of aqueous media with photonic hotspots in plasmonic nanostructures such as biochemical sensors.

Factors associated with myocardial SARS-CoV-2 infection, myocarditis, and cardiac inflammation in patients with COVID-19.

COVID-19 has been associated with cardiac injury and dysfunction. While both myocardial inflammatory cell infiltration and myocarditis with myocyte injury have been reported in patients with fatal COVID-19, clinical-pathologic correlations remain limited. The objective was to determine the relationships between cardiac pathological changes in patients dying from COVID-19 and cardiac infection by SARS-CoV-2, laboratory measurements, clinical features, and treatments. In a retrospective study, 41 consecutive autopsies of patients with fatal COVID-19 were analyzed for the associations between cardiac inflammation, myocarditis, cardiac infection by SARS-CoV-2, clinical features, laboratory measurements, and treatments. Cardiac infection was assessed by in situ hybridization and NanoString transcriptomic profiling. Cardiac infection by SARS-CoV-2 was present in 30/41 cases: virus+ with myocarditis (n = 4), virus+ without myocarditis (n = 26), and virus- without myocarditis (n = 11). In the cases with cardiac infection, SARS-CoV-2+ cells in the myocardium were rare, with a median density of 1 cell/cm2. Virus+ cases showed higher densities of myocardial CD68+ macrophages and CD3+ lymphocytes, as well as more electrocardiographic changes (23/27 vs 4/10; P = 0.01). Myocarditis was more prevalent with IL-6 blockade than with nonbiologic immunosuppression, primarily glucocorticoids (2/3 vs 0/14; P = 0.02). Overall, SARS-CoV-2 cardiac infection was less prevalent in patients treated with nonbiologic immunosuppression (7/14 vs 21/24; P = 0.02). Myocardial macrophage and lymphocyte densities overall were positively correlated with the duration of symptoms but not with underlying comorbidities. In summary, cardiac infection with SARS-CoV-2 is common among patients dying from COVID-19 but often with only rare infected cells. Cardiac infection by SARS-CoV-2 is associated with more cardiac inflammation and electrocardiographic changes. Nonbiologic immunosuppression is associated with lower incidences of myocarditis and cardiac infection by SARS-CoV-2.

Pentacyclic Triterpene Profile and Its Biosynthetic Pathway in Cecropia telenitida as a Prospective Dietary Supplement.

Promising research over the past decades has shown that some types of pentacyclic triterpenes (PTs) are associated with the prevention of type 2 diabetes (T2D), especially those found in foods. The most abundant edible sources of PTs are those belonging to the ursane and oleanane scaffold. The principal finding is that Cecropia telenitida contains abundant oleanane and ursane PT types with similar oxygenation patterns to those found in food matrices. We studied the compositional profile of a rich PT fraction (DE16-R) and carried out a viability test over different cell lines. The biosynthetic pathway connected to the isolated PTs in C. telenitida offers a specific medicinal benefit related to the modulation of T2D. This current study suggests that this plant can assemble isobaric, positional isomers or epimeric PT. Ursane or oleanane scaffolds with the same oxygenation pattern are always shared by the PTs in C. telenitida, as demonstrated by its biosynthetic pathway. Local communities have long used this plant in traditional medicine, and humans have consumed ursane and oleanane PTs in fruits since ancient times, two key points we believe useful in considering the medicinal benefits of C. telenitida and explaining how a group of molecules sharing a closely related scaffold can express effectiveness.

Localism "Reimagined": Building a Robust Localist Paradigm for Overcoming Emerging Conservation Challenges.

Governance gaps at both the federal and state level increasingly necessitate local action and remain a key driver of community-based solutions. A localist paradigm-encompassing models such as community-based management, citizen science, and cooperative research-offers a promising approach for bridging governance gaps by engaging citizens, co-producing knowledge, fostering trust, and developing innovative solutions to address complex conservation challenges. Yet, despite notable successes, significant barriers constrain widespread implementation of localist approaches. This is particularly evident in natural resource-dependent communities. Rural communities are increasingly faced with a range of conservation challenges related to rapid climate and land-use changes but often they lack the capacity to support locally based initiatives to better anticipate, plan for, and mitigate these changes. We examined four diverse conservation cases based on localist approaches in Maine, USA, to bring to the fore key factors that influence outcomes in different social-ecological contexts. We compared cases along three frequently discussed dimensions-governance systems, social adaptive capacities, and technology and data characteristics and found that localist outcomes vary widely depending on key metrics within each of these dimensions. There is no single way to advance localism, but we offer multiple ways to incorporate a community-based perspective into management. This synthesis of data from our collective participatory research projects provides guidance to maximize the potential of localist conservation approaches in complex social and biophysical arenas.

Immobilization of E. coli expressing Bacillus pumilus CynD in three organic polymer matrices.

Cyanide is toxic to most living organisms. The toxicity of cyanide derives from its ability to inhibit the enzyme cytochrome C oxidase of the electronic transport chain. Despite its high toxicity, several industrial processes rely on the use of cyanide, and considerable amounts of industrial waste must be adequately treated before discharge. Biological treatments for the decontamination of cyanide waste include the use of microorganisms and enzymes. Regarding the use of enzymes, cyanide dihydratase (CynD), which catalyzes the conversion of cyanide into ammonia and formate, is an attractive candidate. Nevertheless, the main impediment to the effective use of this enzyme for the biodegradation of cyanide is the marked intolerance to the alkaline pH at which cyanide waste is kept. In this work, we explore the operational capabilities of whole E. coli cells overexpressing Bacillus pumilus CynD immobilized in three organic polymer matrices: chitosan, polyacrylamide, and agar. Remarkably, the immobilized cells on agar and polyacrylamide retained more than 80% activity even at pH 10 and displayed high reusability. Conversely, the cells immobilized on chitosan were not active. Finally, the suitability of the active complexes for the degradation of free cyanide from a solution derived from the gold processing industry was demonstrated.

Do geographically isolated wetlands influence landscape functions?

Geographically isolated wetlands (GIWs), those surrounded by uplands, exchange materials, energy, and organisms with other elements in hydrological and habitat networks, contributing to landscape functions, such as flow generation, nutrient and sediment retention, and biodiversity support. GIWs constitute most of the wetlands in many North American landscapes, provide a disproportionately large fraction of wetland edges where many functions are enhanced, and form complexes with other water bodies to create spatial and temporal heterogeneity in the timing, flow paths, and magnitude of network connectivity. These attributes signal a critical role for GIWs in sustaining a portfolio of landscape functions, but legal protections remain weak despite preferential loss from many landscapes. GIWs lack persistent surface water connections, but this condition does not imply the absence of hydrological, biogeochemical, and biological exchanges with nearby and downstream waters. Although hydrological and biogeochemical connectivity is often episodic or slow (e.g., via groundwater), hydrologic continuity and limited evaporative solute enrichment suggest both flow generation and solute and sediment retention. Similarly, whereas biological connectivity usually requires overland dispersal, numerous organisms, including many rare or threatened species, use both GIWs and downstream waters at different times or life stages, suggesting that GIWs are critical elements of landscape habitat mosaics. Indeed, weaker hydrologic connectivity with downstream waters and constrained biological connectivity with other landscape elements are precisely what enhances some GIW functions and enables others. Based on analysis of wetland geography and synthesis of wetland functions, we argue that sustaining landscape functions requires conserving the entire continuum of wetland connectivity, including GIWs.

Intracellular Delivery of Nanomaterials via an Inertial Microfluidic Cell Hydroporator.

The introduction of nanomaterials into cells is an indispensable process for studies ranging from basic biology to clinical applications. To deliver foreign nanomaterials into living cells, traditionally endocytosis, viral and lipid nanocarriers or electroporation are mainly employed; however, they critically suffer from toxicity, inconsistent delivery, and low throughput and are time-consuming and labor-intensive processes. Here, we present a novel inertial microfluidic cell hydroporator capable of delivering a wide range of nanomaterials to various cell types in a single-step without the aid of carriers or external apparatus. The platform inertially focuses cells into the channel center and guides cells to collide at a T-junction. Controlled compression and shear forces generate transient membrane discontinuities that facilitate passive diffusion of external nanomaterials into the cell cytoplasm while maintaining high cell viability. This hydroporation method shows superior delivery efficiency, is high-throughput, and has high controllability; moreover, its extremely simple and low-cost operation provides a powerful and practical strategy in the applications of cellular imaging, biomanufacturing, cell-based therapies, regenerative medicine, and disease diagnosis.

Voriconazole and squamous cell carcinoma after lung transplantation: A multicenter study.

This study evaluated the independent contribution of voriconazole to the development of squamous cell carcinoma (SCC) in lung transplant recipients, by attempting to account for important confounding factors, particularly immunosuppression. This international, multicenter, retrospective, cohort study included adult patients who underwent lung transplantation during 2005-2008. Cox regression analysis was used to assess the effects of voriconazole and other azoles, analyzed as time-dependent variables, on the risk of developing biopsy-confirmed SCC. Nine hundred lung transplant recipients were included. Median follow-up time from transplantation to end of follow-up was 3.51 years. In a Cox regression model, exposure to voriconazole alone (adjusted hazard ratio 2.39, 95% confidence interval 1.31-4.37) and exposure to voriconazole and other azole(s) (adjusted hazard ratio 3.45, 95% confidence interval 1.07-11.06) were associated with SCC compared with those unexposed after controlling for important confounders including immunosuppressants. Exposure to voriconazole was associated with increased risk of SCC of the skin in lung transplant recipients. Residual confounding could not be ruled out because of the use of proxy variables to control for some confounders. Benefits of voriconazole use when prescribed to lung transplant recipients should be carefully weighed versus the potential risk of SCC. EU PAS registration number: EUPAS5269.

Pentacyclic Triterpenes from Cecropia telenitida Can Function as Inhibitors of 11ß-Hydroxysteroid Dehydrogenase Type 1.

Plant extracts from the genus Cecropia have been used by Latin-American traditional medicine to treat metabolic disorders and diabetes. Previous results have shown that roots of Cecropia telenitida contain pentacyclic triterpenes and these molecules display a hypoglycemic effect in an insulin-resistant murine model. The pharmacological target of these molecules, however, remains unknown. Several lines of evidence indicate that pentacyclic triterpenes inhibit the 11ß-hydroxysteroid dehydrogenase type 1 enzyme, which highlights the potential use of this type of natural product as phytotherapeutic or botanical dietary supplements. The main goal of the study was the evaluation of the inhibitory effect of Cecropia telenitida molecules on 11ß-hydroxysteroid dehydrogenase type 1 enzyme activity. A pre-fractionated chemical library was obtained from the roots of Cecropia telenitida using several automated chromatography separation steps and a homogeneous time resolved fluorescence assay was used for the bio-guided isolation of inhibiting molecules. The screening of a chemical library consisting of 125 chemical purified fractions obtained from Cecropia telenitida roots identified one fraction displaying 82% inhibition of the formation of cortisol by the 11ß-hydroxysteroid dehydrogenase type 1 enzyme. Furthermore, a molecule displaying IC50 of 0.95 ± 0.09 µM was isolated from this purified fraction and structurally characterized, which confirms that a pentacyclic triterpene scaffold was responsible for the observed inhibition. Our results support the hypothesis that pentacyclic triterpene molecules from Cecropia telenitida can inhibit 11ß-hydroxysteroid dehydrogenase type 1 enzyme activity. These findings highlight the potential ethnopharmacological use of plants from the genus Cecropia for the treatment of metabolic disorders and diabetes.

The Thumb Domain Mediates Acid-sensing Ion Channel Desensitization.

Acid-sensing ion channels (ASICs) are cation-selective proton-gated channels expressed in neurons that participate in diverse physiological processes, including nociception, synaptic plasticity, learning, and memory. ASIC subunits contain intracellular N and C termini, two transmembrane domains that constitute the pore, and a large extracellular loop with defined domains termed the finger, ß-ball, thumb, palm, and knuckle. Here we examined the contribution of the finger, ß-ball, and thumb domains to activation and desensitization through the analysis of chimeras and the assessment of the effect of covalent modification of introduced Cys at the domain-domain interfaces. Our studies with ASIC1a-ASIC2a chimeras showed that swapping the thumb domain between subunits results in faster channel desensitization. Likewise, the covalent modification of Cys residues at selected positions in the ß-ball-thumb interface accelerates the desensitization of the mutant channels. Studies of accessibility with thiol-reactive reagents revealed that the ß-ball and thumb domains reside apart in the resting state but that they become closer to each other in response to extracellular acidification. We propose that the thumb domain moves upon continuous exposure to an acidic extracellular milieu, assisting with the closing of the pore during channel desensitization.

Inertial Microfluidic Cell Stretcher (iMCS): Fully Automated, High-Throughput, and Near Real-Time Cell Mechanotyping.

Mechanical biomarkers associated with cytoskeletal structures have been reported as powerful label-free cell state identifiers. In order to measure cell mechanical properties, traditional biophysical (e.g., atomic force microscopy, micropipette aspiration, optical stretchers) and microfluidic approaches were mainly employed; however, they critically suffer from low-throughput, low-sensitivity, and/or time-consuming and labor-intensive processes, not allowing techniques to be practically used for cell biology research applications. Here, a novel inertial microfluidic cell stretcher (iMCS) capable of characterizing large populations of single-cell deformability near real-time is presented. The platform inertially controls cell positions in microchannels and deforms cells upon collision at a T-junction with large strain. The cell elongation motions are recorded, and thousands of cell deformability information is visualized near real-time similar to traditional flow cytometry. With a full automation, the entire cell mechanotyping process runs without any human intervention, realizing a user friendly and robust operation. Through iMCS, distinct cell stiffness changes in breast cancer progression and epithelial mesenchymal transition are reported, and the use of the platform for rapid cancer drug discovery is shown as well. The platform returns large populations of single-cell quantitative mechanical properties (e.g., shear modulus) on-the-fly with high statistical significances, enabling actual usages in clinical and biophysical studies.

Improving management of small natural features on private lands by negotiating the science-policy boundary for Maine vernal pools.

Vernal pools are far more important for providing ecosystem services than one would predict based on their small size. However, prevailing resource-management strategies are not effectively conserving pools and other small natural features on private lands. Solutions are complicated by tensions between private property and societal rights, uncertainties over resource location and function, diverse stakeholders, and fragmented regulatory authority. The development and testing of new conservation approaches that link scientific knowledge, stakeholder decision-making, and conservation outcomes are important responses to this conservation dilemma. Drawing from a 15-y history of vernal pool conservation efforts in Maine, we describe the coevolution of pool conservation and research approaches, focusing on how research-based knowledge was produced and used in support of management decisions. As management shifted from reactive, top-down approaches to proactive and flexible approaches, research shifted from an ecology-focused program to an interdisciplinary program based on social-ecological systems. The most effective strategies for linking scientific knowledge with action changed as the decision-makers, knowledge needs, and context for vernal pool management advanced. Interactions among stakeholders increased the extent to which knowledge was coproduced and shifted the objective of stakeholder engagement from outreach to research collaboration and development of innovative conservation approaches. New conservation strategies were possible because of the flexible, solutions-oriented collaborations and trust between scientists and decision-makers (fostered over 15 y) and interdisciplinary, engaged research. Solutions to the dilemma of conserving small natural features on private lands, and analogous sustainability science challenges, will benefit from repeated negotiations of the science-policy boundary.

The Significant Surface-Water Connectivity of "Geographically Isolated Wetlands".

We evaluated the current literature, coupled with our collective research expertise, on surface-water connectivity of wetlands considered to be "geographically isolated" (sensu Tiner Wetlands 23:494-516, 2003a) to critically assess the scientific foundation of grouping wetlands based on the singular condition of being surrounded by uplands. The most recent research on wetlands considered to be "geographically isolated" shows the difficulties in grouping an ecological resource that does not reliably indicate lack of surface water connectivity in order to meet legal, regulatory, or scientific needs. Additionally, the practice of identifying "geographically isolated wetlands" based on distance from a stream can result in gross overestimates of the number of wetlands lacking ecologically important surface-water connections. Our findings do not support use of the overly simplistic label of "geographically isolated wetlands". Wetlands surrounded by uplands vary in function and surface-water connections based on wetland landscape setting, context, climate, and geographic region and should be evaluated as such. We found that the "geographically isolated" grouping does not reflect our understanding of the hydrologic variability of these wetlands and hence does not benefit conservation of the Nation's diverse wetland resources. Therefore, we strongly discourage use of categorizations that provide overly simplistic views of surface-water connectivity of wetlands fully embedded in upland landscapes.

Conformational Fine-Tuning of Pore-Forming Peptide Potency and Selectivity.

To better understand the sequence-structure-function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding-folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the "negative" peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.

Independent contribution of extracellular proton binding sites to ASIC1a activation.

Acid-sensing ion channels (ASICs) are a group of trimeric cation permeable channels gated by extracellular protons that are mainly expressed in the nervous system. Despite the structural information available for ASIC1, there is limited understanding of the molecular mechanism that allows these channels to sense and respond to drops in extracellular pH. In this report, we employed the substituted cysteine accessibility method and site-directed mutagenesis to examine the mechanism of activation of ASIC1a by extracellular protons. We found that the modification of E238C and D345C channels by MTSET reduced proton apparent affinity for activation. Furthermore, the introduction of positively charged residues at position 345 rendered shifted biphasic proton activation curves. Likewise, channels bearing mutations at positions 79 and 416 in the palm domain of the channel showed reduced proton apparent affinity and biphasic proton activation curves. Of significance, the effect of the mutations at positions 79 and 345 on channel activation was additive. E79K-D345K required a change to a pH lower than 2 for maximal activation. In summary, this study provides direct evidence for the presence of two distinct proton coordination sites in the extracellular region of ASIC1a, which jointly facilitate pore opening in response to extracellular acidification.