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1.
Curr Protoc ; 4(2): e959, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38334240

RESUMO

In vitro electrophysiological safety studies have become an integral part of the drug development process because, in many instances, compound-induced QT prolongation has been associated with a direct block of human ether-a-go-go-related gene (hERG) potassium channels or their native current, the rapidly activating delayed rectifier potassium current (IKr ). Therefore, according to the ICH S7B guideline, the in vitro hERG channel patch-clamp assay is commonly used as an early screen to predict the ability of a compound to prolong the QT interval prior to first-in-human testing. The protocols described in this article are designed to assess the effects of acute or long-term exposure to new chemical entities on the amplitude of IKr in HEK293 cells stably transfected with the hERG channel (whole-cell configuration of the patch-clamp technique). Examples of results obtained with moxifloxacin, terfenadine, arsenic, pentamidine, erythromycin, and sotalol are provided for illustrative purposes. © 2024 Wiley Periodicals LLC. Basic Protocol: Measurement of the acute effects of test items in the hERG channel test Alternate Protocol: Measurement of the long-term effects of test items in the hERG channel test.


Assuntos
Canais de Potássio Éter-A-Go-Go , Sotalol , Humanos , Canais de Potássio Éter-A-Go-Go/genética , Técnicas de Patch-Clamp , Células HEK293 , Eritromicina
2.
STAR Protoc ; 5(1): 102917, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38421863

RESUMO

Multiple patch-clamp recordings and morphological reconstruction are powerful approaches for neuronal microcircuitry dissection and cell type classification but are challenging due to the sophisticated expertise needed. Here, we present a protocol for applying these techniques to neurons in the medial entorhinal cortex (MEC) of mice. We detail steps to prepare brain slices containing MEC and perform simultaneous multiple whole-cell recordings, followed by procedures of histological staining and neuronal reconstruction. We then describe how we analyze morphological and electrophysiological features. For complete details on the use and execution of this protocol, please refer to Shi et al.1.


Assuntos
Córtex Entorrinal , Neurônios , Camundongos , Animais , Córtex Entorrinal/fisiologia , Neurônios/fisiologia , Citoplasma , Técnicas de Patch-Clamp , Encéfalo
3.
Sci Rep ; 14(1): 1660, 2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38238544

RESUMO

The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic profile. However, the throughput in recordings is limited because of the manual replacement of patch-pipettes after each attempt which are often also unsuccessful. This has been overcome by automated cleaning the tips in detergent solutions, allowing to reuse the pipette for further recordings. Here, we developed a novel method of automated cleaning by sonicating the tips within the bath solution wherein the cells are placed, reducing the risk of contaminating the bath solution or internal solution of the recording pipette by any detergent and avoiding the necessity of a separate chamber for cleaning. We showed that the patch-pipettes can be used consecutively at least ten times and that the cleaning process does not negatively impact neither the brain slices nor other patched neurons. This method, combined with automated patch-clamp, highly improves the throughput for single and especially multiple recordings.


Assuntos
Detergentes , Ultrassom , Neurônios/fisiologia , Neurofisiologia , Técnicas de Patch-Clamp
4.
Methods Mol Biol ; 2752: 227-243, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38194038

RESUMO

Cells exhibit diverse morphologic phenotypes, biophysical and functional properties, and gene expression patterns. Understanding how these features are interrelated at the level of single cells has been challenging due to the lack of techniques for multimodal profiling of individual cells. We recently developed Patch-seq, a technique that combines whole-cell patch clamp recording, immunohistochemistry, and single-cell RNA-sequencing (scRNA-seq) to comprehensively profile single cells. Here we present a detailed step-by-step protocol for obtaining high-quality morphological, electrophysiological, and transcriptomic data from single cells. Patch-seq enables researchers to explore the rich, multidimensional phenotypic variability among cells and to directly correlate gene expression with phenotype at the level of single cells.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Biofísica , Técnicas de Patch-Clamp , Eletrofisiologia
5.
Methods Mol Biol ; 2766: 191-198, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38270880

RESUMO

To achieve the most accurate assessment of functional Ca2+ channel or modulator properties and their regulation, a patch-clamp technique to record membrane currents is required. This technique has wide applications ranging from recording the activity of native channels in their natural environment to that of recombinant channels expressed in heterologous cells. This chapter introduces the methods that have been used for the detection of calcium release-activated calcium (CRAC) currents, one of the store-operated calcium entry pathways, in human primary T cells. This standard protocol is for laboratories already equipped with a full patch-clamp set-up or for investigators collaborating with laboratories experienced in patch clamp.


Assuntos
Cálcio , Meio Ambiente , Humanos , Transporte de Íons , Laboratórios , Técnicas de Patch-Clamp
6.
Neuron ; 112(2): 184-200, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-37913772

RESUMO

Layer 1 (L1) of the neocortex acts as a nexus for the collection and processing of widespread information. By integrating ascending inputs with extensive top-down activity, this layer likely provides critical information regulating how the perception of sensory inputs is reconciled with expectation. This is accomplished by sorting, directing, and integrating the complex network of excitatory inputs that converge onto L1. These signals are combined with neuromodulatory afferents and gated by the wealth of inhibitory interneurons that either are embedded within L1 or send axons from other cortical layers. Together, these interactions dynamically calibrate information flow throughout the neocortex. This review will primarily focus on L1 within the primary sensory cortex and will use these insights to understand L1 in other cortical areas.


Assuntos
Neocórtex , Neocórtex/fisiologia , Interneurônios/fisiologia , Axônios , Movimento Celular , Técnicas de Patch-Clamp
7.
STAR Protoc ; 5(1): 102789, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38103195

RESUMO

PIEZO channels sense mechanical forces through conformational rearrangements of a mechanosensory domain called blade. To probe these rearrangements in real time, we have inserted conformational-sensitive cyclic-permuted GFP into several positions of PIEZO1's blade. Here, we describe the step-by-step experimental procedure we developed to simultaneously measure flow-activated ionic currents and fluorometric signals in cells expressing these engineered constructs. We describe steps for performing transfection, seeding cells on coverslips, setting up a perfusion-based fluid shear application system, and performing voltage-clamp fluorometry. For complete details on the use and execution of this protocol, please refer to Ozkan et al. (2023).1.


Assuntos
Técnicas de Patch-Clamp , Conformação Proteica , Fluorometria/métodos
8.
Expert Opin Drug Discov ; 19(3): 331-337, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38108110

RESUMO

INTRODUCTION: Automated Patch Clamp (APC) technology has become an integral element in ion channel research, drug discovery and development pipelines to overcome the use of the highly time-consuming manual patch clamp (MPC) procedures. This automated technology offers increased throughput and promises a new model in obtaining ion channel recordings, which has significant relevance to the development of novel therapies and safety profiling of candidate therapeutic compounds. AREAS COVERED: This article reviews the recent innovations in APC technology, including platforms, and highlights how they have facilitated usage in both industry and academia. The review also provides an overview of the ion channel research endeavors and how APC platforms have contributed to the understanding of ion channel research, pharmacological tools and therapeutics. Furthermore, the authors provide their opinion on the challenges and goals for APC technology going forward to accelerate academic research and drug discovery across a host of therapeutic areas. EXPERT OPINION: It is clear that APC technology has progressed drug discovery programs, specifically in the field of neuroscience and cardiovascular research. The challenge for the future is to keep pace with fundamental research and improve translation of the large datasets obtained.


Assuntos
Ensaios de Triagem em Larga Escala , Canais Iônicos , Humanos , Ensaios de Triagem em Larga Escala/métodos , Descoberta de Drogas/métodos , Tecnologia , Técnicas de Patch-Clamp
9.
Cell Mol Neurobiol ; 44(1): 8, 2023 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-38123823

RESUMO

Multimodal analysis of gene-expression patterns, electrophysiological properties, and morphological phenotypes at the single-cell/single-nucleus level has been arduous because of the diversity and complexity of neurons. The emergence of Patch-sequencing (Patch-seq) directly links transcriptomics, morphology, and electrophysiology, taking neuroscience research to a multimodal era. In this review, we summarized the development of Patch-seq and recent applications in the cortex, hippocampus, and other nervous systems. Through generating multimodal cell type atlases, targeting specific cell populations, and correlating transcriptomic data with phenotypic information, Patch-seq has provided new insight into outstanding questions in neuroscience. We highlight the challenges and opportunities of Patch-seq in neuroscience and hope to shed new light on future neuroscience research.


Assuntos
Perfilação da Expressão Gênica , Análise de Célula Única , Análise de Sequência de RNA , Técnicas de Patch-Clamp , Transcriptoma
10.
Science ; 382(6667): eadf6484, 2023 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-37824669

RESUMO

Human cortex transcriptomic studies have revealed a hierarchical organization of γ-aminobutyric acid-producing (GABAergic) neurons from subclasses to a high diversity of more granular types. Rapid GABAergic neuron viral genetic labeling plus Patch-seq (patch-clamp electrophysiology plus single-cell RNA sequencing) sampling in human brain slices was used to reliably target and analyze GABAergic neuron subclasses and individual transcriptomic types. This characterization elucidated transitions between PVALB and SST subclasses, revealed morphological heterogeneity within an abundant transcriptomic type, identified multiple spatially distinct types of the primate-specialized double bouquet cells (DBCs), and shed light on cellular differences between homologous mouse and human neocortical GABAergic neuron types. These results highlight the importance of multimodal phenotypic characterization for refinement of emerging transcriptomic cell type taxonomies and for understanding conserved and specialized cellular properties of human brain cell types.


Assuntos
Neurônios GABAérgicos , Interneurônios , Neocórtex , Animais , Humanos , Camundongos , Fenômenos Eletrofisiológicos , Neurônios GABAérgicos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Interneurônios/metabolismo , Neocórtex/citologia , Neocórtex/metabolismo , Técnicas de Patch-Clamp
11.
PLoS Comput Biol ; 19(8): e1011342, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37603559

RESUMO

Bayesian Active Learning (BAL) is an efficient framework for learning the parameters of a model, in which input stimuli are selected to maximize the mutual information between the observations and the unknown parameters. However, the applicability of BAL to experiments is limited as it requires performing high-dimensional integrations and optimizations in real time. Current methods are either too time consuming, or only applicable to specific models. Here, we propose an Efficient Sampling-Based Bayesian Active Learning (ESB-BAL) framework, which is efficient enough to be used in real-time biological experiments. We apply our method to the problem of estimating the parameters of a chemical synapse from the postsynaptic responses to evoked presynaptic action potentials. Using synthetic data and synaptic whole-cell patch-clamp recordings, we show that our method can improve the precision of model-based inferences, thereby paving the way towards more systematic and efficient experimental designs in physiology.


Assuntos
Aprendizagem Baseada em Problemas , Projetos de Pesquisa , Teorema de Bayes , Potenciais de Ação , Técnicas de Patch-Clamp
12.
Cell Rep ; 42(8): 112904, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37531251

RESUMO

Acquisition of neuronal circuit architectures, central to understanding brain function and dysfunction, remains prohibitively challenging. Here I report the development of a simultaneous and sequential octuple-sexdecuple whole-cell patch-clamp recording system that enables architectural reconstruction of complex cortical circuits. The method unveils the canonical layer 1 single bouquet cell (SBC)-led disinhibitory neuronal circuits across the mouse somatosensory, motor, prefrontal, and medial entorhinal cortices. The ∼1,500-neuron modular circuits feature the translaminar, unidirectional, minicolumnar, and independent disinhibition and optimize cortical complexity, subtlety, plasticity, variation, and redundancy. Moreover, architectural reconstruction uncovers age-dependent deficits at SBC-disinhibited synapses in the senescence-accelerated mouse prone 8, an animal model of Alzheimer's disease. The deficits exhibit the characteristic Alzheimer's-like cortical spread and correlation with cognitive impairments. These findings decrypt operations of the elementary processing units in healthy and Alzheimer's mouse cortices and validate the efficacy of octuple-sexdecuple patch-clamp recordings for architectural reconstruction of complex neuronal circuits.


Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Camundongos , Animais , Neurônios/fisiologia , Córtex Entorrinal , Técnicas de Patch-Clamp
13.
Nature ; 620(7973): 366-373, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37468637

RESUMO

Neurons in the posterior parietal cortex contribute to the execution of goal-directed navigation1 and other decision-making tasks2-4. Although molecular studies have catalogued more than 50 cortical cell types5, it remains unclear what distinct functions they have in this area. Here we identified a molecularly defined subset of somatostatin (Sst) inhibitory neurons that, in the mouse posterior parietal cortex, carry a cell-type-specific error-correction signal for navigation. We obtained repeatable experimental access to these cells using an adeno-associated virus in which gene expression is driven by an enhancer that functions specifically in a subset of Sst cells6. We found that during goal-directed navigation in a virtual environment, this subset of Sst neurons activates in a synchronous pattern that is distinct from the activity of surrounding neurons, including other Sst neurons. Using in vivo two-photon photostimulation and ex vivo paired patch-clamp recordings, we show that nearby cells of this Sst subtype excite each other through gap junctions, revealing a self-excitation circuit motif that contributes to the synchronous activity of this cell type. These cells selectively activate as mice execute course corrections for deviations in their virtual heading during navigation towards a reward location, for both self-induced and experimentally induced deviations. We propose that this subtype of Sst neurons provides a self-reinforcing and cell-type-specific error-correction signal in the posterior parietal cortex that may help with the execution and learning of accurate goal-directed navigation trajectories.


Assuntos
Neurônios , Lobo Parietal , Animais , Camundongos , Aprendizagem , Neurônios/metabolismo , Lobo Parietal/citologia , Lobo Parietal/metabolismo , Objetivos , Somatostatina/metabolismo , Inibição Neural , Navegação Espacial , Técnicas de Patch-Clamp , Junções Comunicantes/metabolismo
14.
J Gen Physiol ; 155(8)2023 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-37347216

RESUMO

Mitochondria are double-membrane organelles crucial for oxidative phosphorylation, enabling efficient ATP synthesis by eukaryotic cells. Both of the membranes, the highly selective inner mitochondrial membrane (IMM) and a relatively porous outer membrane (OMM), harbor a number of integral membrane proteins that help in the transport of biological molecules. These transporters are especially enriched in the IMM, where they help maintain transmembrane gradients for H+, K+, Ca2+, PO43-, and metabolites like ADP/ATP, citrate, etc. Impaired activity of these transporters can affect the efficiency of energy-transducing processes and can alter cellular redox state, leading to activation of cell-death pathways or metabolic syndromes in vivo. Although several methodologies are available to study ion flux through membrane proteins, the patch-clamp technique remains the gold standard for quantitatively analyzing electrogenic ion exchange across membranes. Direct patch-clamp recordings of mitoplasts (mitochondria devoid of outer membrane) in different modes, such as whole-mitoplast or excised-patch mode, allow researchers the opportunity to study the biophysics of mitochondrial transporters in the native membrane, in real time, in isolation from other fluxes or confounding factors due to changes in ion gradients, pH, or mitochondrial potential (ΔΨ). Here, we summarize the use of patch clamp to investigate several membrane proteins of mitochondria. We demonstrate how this technique can be reliably applied to record whole-mitoplast Ca2+ currents mediated via mitochondrial calcium uniporter or H+ currents mediated by uncoupling protein 1 and discuss critical considerations while recording currents from these small vesicles of the IMM (mitoplast diameter = 2-5 µm).


Assuntos
Cálcio , Membranas Mitocondriais , Membranas Mitocondriais/metabolismo , Técnicas de Patch-Clamp , Cálcio/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo
15.
Adv Sci (Weinh) ; 10(25): e2301854, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37386797

RESUMO

Optoelectronic biointerfaces have gained significant interest for wireless and electrical control of neurons. Three-dimentional (3D) pseudocapacitive nanomaterials with large surface areas and interconnected porous structures have great potential for optoelectronic biointerfaces that can fulfill the requirement of high electrode-electrolyte capacitance to effectively transduce light into stimulating ionic currents. In this study, the integration of 3D manganese dioxide (MnO2 ) nanoflowers into flexible optoelectronic biointerfaces for safe and efficient photostimulation of neurons is demonstrated. MnO2 nanoflowers are grown via chemical bath deposition on the return electrode, which has a MnO2 seed layer deposited via cyclic voltammetry. They facilitate a high interfacial capacitance (larger than 10 mF cm-2 ) and photogenerated charge density (over 20 µC cm-2 ) under low light intensity (1 mW mm-2 ). MnO2 nanoflowers induce safe capacitive currents with reversible Faradaic reactions and do not cause any toxicity on hippocampal neurons in vitro, making them a promising material for biointerfacing with electrogenic cells. Patch-clamp electrophysiology is recorded in the whole-cell configuration of hippocampal neurons, and the optoelectronic biointerfaces trigger repetitive and rapid firing of action potentials in response to light pulse trains. This study points out the potential of electrochemically-deposited 3D pseudocapacitive nanomaterials as a robust building block for optoelectronic control of neurons.


Assuntos
Eletroquímica , Luz , Compostos de Manganês , Nanoestruturas , Neurônios , Óxidos , Potenciais de Ação/efeitos da radiação , Capacitância Elétrica , Eletroquímica/métodos , Eletrodos , Eletrólitos/química , Eletrólitos/efeitos da radiação , Eletrofisiologia , Hipocampo/citologia , Compostos de Manganês/química , Nanoestruturas/efeitos adversos , Nanoestruturas/química , Nanoestruturas/efeitos da radiação , Neurônios/metabolismo , Neurônios/efeitos da radiação , Óxidos/química , Técnicas de Patch-Clamp , Estimulação Luminosa , Tecnologia sem Fio , Humanos , Animais , Ratos
16.
DNA Cell Biol ; 42(8): 481-487, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37311169

RESUMO

The mitochondrial permeability transition pore (mPTP) is a channel in the mitochondrial inner membrane that is activated by excessive calcium uptake. In this study, we used a whole-mitoplast patch-clamp approach to investigate the ionic currents associated with mPTP at the level of the whole single mitochondrion. The whole-mitoplast conductance was at the level of 5 to 7 nS, which is consistent with the presence of three to six single mPTP channels per mitochondrion. We found that mPTP currents are voltage dependent and inactivate at negative potential. The currents were inhibited by cyclosporine A and adenosine diphosphate. When mPTP was induced by oxidative stress, currents were partially blocked by the adenine nucleotide translocase inhibitor bongkrekic acid. Our data suggest that the whole-mitoplast patch-clamp approach is a useful method for investigating the biophysical properties and regulation of the mPTP.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Técnicas de Patch-Clamp , Mitocôndrias , Membranas Mitocondriais , Cálcio/farmacologia
17.
Molecules ; 28(10)2023 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-37241833

RESUMO

Biocytin, a chemical compound that is an amide formed from the vitamin biotin and the amino acid L-lysine, has been used as a histological dye to stain nerve cells. Electrophysiological activity and morphology are two key characteristics of neurons, but revealing both the electrophysiological and morphological properties of the same neuron is challenging. This article introduces a detailed and easy-to-operate procedure for single-cell labeling in combination with whole-cell patch-clamp recording. Using a recording electrode filled with a biocytin-containing internal solution, we demonstrate the electrophysiological and morphological characteristics of pyramidal (PNs), medial spiny (MSNs) and parvalbumin neurons (PVs) in brain slices, where the electrophysiological and morphological properties of the same individual cell are elucidated. We first introduce a protocol for whole-cell patch-clamp recording in various neurons, coupled with the intracellular diffusion of biocytin delivered by the glass capillary of the recording electrode, followed by a post hoc procedure to reveal the architecture and morphology of biocytin-labeled neurons. An analysis of action potentials (APs) and neuronal morphology, including the dendritic length, number of intersections, and spine density of biocytin-labeled neurons, were performed using ClampFit and Fiji Image (ImageJ), respectively. Next, to take advantage of the techniques introduced above, we uncovered defects in the APs and the dendritic spines of PNs in the primary motor cortex (M1) of deubiquitinase cylindromatosis (CYLD) knock-out (Cyld-/-) mice. In summary, this article provides a detailed methodology for revealing the morphology as well as the electrophysiological activity of a single neuron that will have many applications in neurobiology.


Assuntos
Lisina , Neurônios , Animais , Camundongos , Técnicas de Patch-Clamp , Células Piramidais/fisiologia , Enzima Desubiquitinante CYLD
18.
J Neurosci Methods ; 394: 109898, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37236404

RESUMO

Brain organoids represent a new model system for studying developmental human neurophysiology. Methods for studying the electrophysiology and morphology of single neurons in organoids require acute slices or dissociated cultures. While these methods have advantages (e.g., visual access, ease of experimentation), they risk damaging cells and circuits present in the intact organoid. To access single cells within intact organoid circuits, we have demonstrated a method for fixturing and performing whole cell patch clamp recording from intact brain organoids using both manual and automated tools. We demonstrate applied electrophysiology methods development followed by an integration of electrophysiology with reconstructing the morphology of the neurons within the brain organoid using dye filling and tissue clearing. We found that whole cell patch clamp recordings could be achieved both on the surface and within the interior of intact human brain organoids using both manual and automated methods. Manual experiments were higher yield (53 % whole cell success rate manual, 9 % whole cell success rate automated), but automated experiments were more efficient (30 patch attempts per day automated, 10 patch attempts per day manual). Using these methods, we performed an unbiased survey of cells within human brain organoids between 90 and 120 days in vitro (DIV) and present preliminary data on morphological and electrical diversity in human brain organoids. The further development of intact brain organoid patch clamp methods could be broadly applicable to studies of cellular, synaptic, and circuit-level function in the developing human brain.


Assuntos
Encéfalo , Neurônios , Humanos , Neurônios/fisiologia , Encéfalo/fisiologia , Fenômenos Eletrofisiológicos , Técnicas de Patch-Clamp , Organoides
19.
J Vis Exp ; (193)2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-37010276

RESUMO

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.


Assuntos
Hipotálamo , Kisspeptinas , Humanos , Animais , Kisspeptinas/metabolismo , Técnicas de Patch-Clamp , Hipotálamo/metabolismo , Hormônio Liberador de Gonadotropina , Neurônios/fisiologia
20.
Cell Rep ; 42(4): 112394, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-37058408

RESUMO

The ATP-sensitive K+ (KATP) channel is a key regulator of hormone secretion from pancreatic islet endocrine cells. Using direct measurements of KATP channel activity in pancreatic ß cells and the lesser-studied α cells, from both humans and mice, we provide evidence that a glycolytic metabolon locally controls KATP channels on the plasma membrane. The two ATP-consuming enzymes of upper glycolysis, glucokinase and phosphofructokinase, generate ADP that activates KATP. Substrate channeling of fructose 1,6-bisphosphate through the enzymes of lower glycolysis fuels pyruvate kinase, which directly consumes the ADP made by phosphofructokinase to raise ATP/ADP and close the channel. We further show the presence of a plasma membrane-associated NAD+/NADH cycle whereby lactate dehydrogenase is functionally coupled to glyceraldehyde-3-phosphate dehydrogenase. These studies provide direct electrophysiological evidence of a KATP-controlling glycolytic signaling complex and demonstrate its relevance to islet glucose sensing and excitability.


Assuntos
Membrana Celular , Células Secretoras de Glucagon , Glicólise , Células Secretoras de Insulina , Humanos , Animais , Camundongos , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Canais KATP/metabolismo , Técnicas de Patch-Clamp , Eletrofisiologia , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Lactato Desidrogenases/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Difosfato de Adenosina/metabolismo , Fosfofrutoquinases/metabolismo
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