Biology subjects

Alcaino, C.

Publications and source records attributed to Alcaino, C..

4 recordsLinked to original sources

bioRxiv2025

Mechanisms of Activation and Serotonin release from Human Enterochromaffin Cells

Background and AimsGastrointestinal (GI) enterochromaffin (EC) cells are specialised sensors of luminal stimuli. They secrete most of the bodys serotonin (5-HT), and are critical for modulating GI motility, secretion, and sensation, while also signalling satiety and intestinal discomfort. The aim of this study was to investigate mechanisms underlying the regulation of human EC cells, and the relative importance of direct nutrient stimulation compared with neuronal and paracrine regulation. MethodsIntestinal organoids from human duodenal biopsies were modified using CRISPR-Cas9 to specifically label EC cells with either the fluorescent protein Venus or the cAMP sensor Epac1-S. EC cells were purified by fluorescence-activated cell sorting for analysis by bulk RNA sequencing and liquid chromatography mass spectrometry peptidomics. The function of human EC cells was studied using single cell patch clamp, calcium and cAMP imaging and 5-HT ELISA assays. ResultsHuman EC cells showed expression of receptors for nutrients (including GPR142, GPBAR1, GPR119, FFAR2, OR51E1, OR51E2), gut hormones (including SSTR1,2&5, NPY1R, GIPR) and neurotransmitters (ADRA2A, ADRB1). Functional assays revealed EC responses (calcium, cAMP and/or secretion) to a range of stimuli, including bacterial metabolites, aromatic amino acids and adrenergic agonists. Electrophysiological recordings showed that isovalerate increased action potential firing. Conclusions5-HT release from EC cells controls many physiological functions and is currently being targeted to treat disorders of the gut-brain axis. Studying ECs from human organoids enables improved understanding of the molecular mechanisms underlying EC cell activation, which is fundamental for the development of new strategies to target 5-HT-related gut and metabolic disorders. SynopsisHuman duodenal organoids expressing fluorescent proteins in enterochromaffin cells were used to study mechanisms underlying serotonin secretion. Different expression of key sensory receptors was identified by transcriptomic analysis, and validated by live cell second messenger imaging and secretion assays.

cell biology↗

bioRxiv2024

Cav3.1 is a leucine sensor in POMC neurons mediating appetite suppression and weight loss

Hypothalamic leucine sensing promotes satiety and weight loss but an understanding of how leucine regulates neuronal activity is lacking. Here we show that Cacna1g, encoding the T-type voltage-gated calcium channel Cav3.1, is enriched in hypothalamic leucine-sensing neurons and mediates leucine sensing. Pharmacological inhibition of Cav3.1 blunts leucine-induced activation of POMC neurons as well as the anorectic response to leucine in vivo. In addition, genetic deletion of Cacna1g in POMC neurons abolishes the appetite- and weight-suppressive effects of high-protein feeding. Mechanistically, we show that leucine binds to the voltage-sensing segment of Cav3.1, thereby reducing its threshold for voltage-dependent activation. Last, pharmacological activation of hypothalamic Cav3.1 promotes weight loss in diet-induced obese mice and potentiates the weight loss response to GLP-1 receptor agonism. These results reveal that Cav3.1 is a neuronal leucine sensor and a relevant weight loss target.

neuroscience↗

bioRxiv2024

GLP1R agonists activate human POMC neurons

Drugs like semaglutide (a.k.a. Ozempic/Wegovy) that activate the glucagon-like peptide-1 receptor (GLP-1R) are a promising therapy for obesity and type 2 diabetes (T2D). Animal studies suggest that these drugs likely function by stimulating GLP-1R on appetite-suppressing neuron populations in the brain, but it is still unclear how they act to reduce food intake in humans. We therefore generated appetite-regulatory hypothalamic neurons from human pluripotent stem cells (hPSCs) to study their responses to GLP-1R agonists by calcium imaging and electrophysiology. We found that hPSC-derived proopiomelanocortin (POMC) and other hypothalamic neuron subtypes expressed GLP1R mRNA, and many of these neurons robustly responded to GLP-1R agonists by membrane depolarization, increased action potential firing, and extracellular calcium influx that persisted long after agonist withdrawal. The observed GLP-1R-induced response was likely mediated by the activation of PKA and L-type calcium channels, and led to significant changes in gene expression. These findings provide mechanistic insight into how GLP-1R agonists may suppress appetite in humans.

neuroscience↗

bioRxiv2022

Mechanosensitive pore opening of a prokaryotic voltage-gated sodium channel

Voltage-gated ion channels orchestrate electrical activities that drive mechanical functions in contractile tissues such as the heart and gut. In turn, contractions change membrane tension and impact ion channels. Voltage-gated ion channels are mechanosensitive, but the mechanisms of mechanosensitivity remain poorly understood. Here, we leverage the relative simplicity of NaChBac, a prokaryotic sodium channel from Bacillus halodurans, to investigate its mechanosensitivity. In whole-cell experiments on heterologously transfected HEK293 cells, shear stress reversibly altered the kinetic properties of NaChBac and increased its maximum current, comparably to the mechanosensitive eukaryotic sodium channel NaV1.5. In single-channel experiments, patch suction reversibly increased the open probability of a NaChBac mutant with inactivation removed. A simple kinetic mechanism featuring a mechanosensitive pore opening transition explained the overall response to force, whereas an alternative model with mechanosensitive voltage sensor activation diverged from the data. Structural analysis of NaChBac identified a large displacement of the hinged intracellular gate, and mutagenesis at the hinge abolished NaChBac mechanosensitivity, further supporting the proposed mechanism. Overall, our results suggest that NaChBac responds to force because its pore is intrinsically mechanosensitive. This mechanism may apply to other voltage-gated ion channels, including NaV1.5.

physiology↗