Defining the functional role of NaV1.7 in human nociception

Loss-of-function mutations in NaV1.7 cause congenital insensitivity to pain (CIP); this voltage-gated sodium channel is therefore a key target for analgesic drug development. Utilizing a multi-modal approach, we investigated how NaV1.7 mutations lead to human pain insensitivity. Skin biopsy and microneurography revealed an absence of C-fiber nociceptors in CIP patients, reflected in a reduced cortical response to capsaicin on fMRI. Epitope tagging of endogenous NaV1.7 revealed the channel to be localized at the soma membrane, axon, axon terminals, and the nodes of Ranvier of induced pluripotent stem cell (iPSC) nociceptors. CIP patient-derived iPSC nociceptors exhibited an inability to properly respond to depolarizing stimuli, demonstrating that NaV1.7 is a key regulator of excitability. Using this iPSC nociceptor platform, we found that some NaV1.7 blockers undergoing clinical trials lack specificity. CIP, therefore, arises due to a profound loss of functional nociceptors, which is more pronounced than that reported in rodent models, or likely achievable following acute pharmacological blockade

Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers

Background: Methods for rapid assessment of interactions of small molecules with membrane proteins in detergent are lacking.  Results: Thermostability measurements of mitochondrial transporters display informative trends about detergent, lipid, substrate, and inhibitor interactions.  Conclusion: Mechanistic insights are obtained by studying the thermostability of mitochondrial transporters.  Significance: Information about the nature of compound interactions with membrane proteins can be obtained rapidly

An iPSC model of hereditary sensory neuropathy-1 reveals L-serine-responsive deficits in neuronal ganglioside composition and axoglial interactions.

Hereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy

The genetics of neuropathic pain from model organisms to clinical application

Neuropathic pain (NeuP) arises due to injury of the somatosensory nervous system and is both common and disabling, rendering an urgent need for non-addictive, effective new therapies. Given the high evolutionary conservation of pain, investigative approaches from Drosophila mutagenesis to human Mendelian genetics have aided our understanding of the maladaptive plasticity underlying NeuP. Successes include the identification of ion channel variants causing hyper-excitability and the importance of neuro-immune signaling. Recent developments encompass improved sensory phenotyping in animal models and patients, brain imaging, and electrophysiology-based pain biomarkers, the collection of large well-phenotyped population cohorts, neurons derived from patient stem cells, and high-precision CRISPR generated genetic editing. We will discuss how to harness these resources to understand the pathophysiological drivers of NeuP, define its relationship with comorbidities such as anxiety, depression, and sleep disorders, and explore how to apply these findings to the prediction, diagnosis, and treatment of NeuP in the clinic

Relations structure - fonction des transporteurs mitochondriaux

The Mitochondrial Carrier Family (MCF) groups integral membrane proteins that transport various metabolites across the inner mitochondrial membrane. This transport is needed in major metabolic pathways occurring in mitochondria and is essential in eukaryotic metabolism. The mis-function of several identified human mitochondrial carriers has been associated with severe diseases. In order to achieve a better understanding of mitochondrial carriers two different families of mitochondrial carriers where chosen to be studied: the AACs (ADP/ATP Carriers) and the UCPs (UnCoupling Proteins). Two heterologous expression systems of these carriers were developed: the cell free and the expression in E. coli as fusion proteins. The cell free system allowed the production and purification of about 0,6 mg of protein per milliliter of reaction mixture and the expression in E.coli proved to be very efficient for the functional characterization of ADP/ATP carriers. A functional test was developed for the uncoupling protein also. This test, based on the measure of electrical currents generated by the transport activity of UCP, allowed the functional characterization of native UCP1.Le passage sélectif d'ions et de métabolites à travers les membranes biologiques est essentiel à de nombreux processus cellulaires fondamentaux. Au niveau de la membrane interne de la mitochondrie, la communication cellulaire et les processus d'échanges sont principalement assurés par les transporteurs mitochondriaux. Ces protéines membranaires jouent un rôle clef dans les fonctions métaboliques des cellules eucaryotes et leur dysfonctionnement est à l'origine d'un certain nombre de maladies graves chez l'homme Parmi les transporteurs mitochondriaux, deux familles ont été étudiées au cours de ce travail : les AACs (ADP/ATP Carriers) et les UCPs (UnCoupling Proteins). Deux systèmes de production hétérologue de ces transporteurs ont été mis en place : la synthèse in vitro et l'expression chez E. coli de protéines de fusion. Le premier a permis la production et la purification d'environ 0,6 mg de protéine par mL de réaction et le deuxième a été exploité afin de réaliser des caractérisations fonctionnelles des transporteurs ADP/ATP. Un test fonctionnel pour la protéine découplante a également été mis au point. Ce test, basé sur la mesure directe des courants électriques associés à l'activité de transport de l'UCP, à permis la caractérisation fonctionnelle de la protéine UCP1 native

Relations structure - fonction des transporteurs mitochondriaux

The Mitochondrial Carrier Family (MCF) groups integral membrane proteins that transport various metabolites across the inner mitochondrial membrane. This transport is needed in major metabolic pathways occurring in mitochondria and is essential in eukaryotic metabolism. The mis-function of several identified human mitochondrial carriers has been associated with severe diseases. In order to achieve a better understanding of mitochondrial carriers two different families of mitochondrial carriers where chosen to be studied: the AACs (ADP/ATP Carriers) and the UCPs (UnCoupling Proteins). Two heterologous expression systems of these carriers were developed: the cell free and the expression in E. coli as fusion proteins. The cell free system allowed the production and purification of about 0,6 mg of protein per milliliter of reaction mixture and the expression in E.coli proved to be very efficient for the functional characterization of ADP/ATP carriers. A functional test was developed for the uncoupling protein also. This test, based on the measure of electrical currents generated by the transport activity of UCP, allowed the functional characterization of native UCP1.Le passage sélectif d'ions et de métabolites à travers les membranes biologiques est essentiel à de nombreux processus cellulaires fondamentaux. Au niveau de la membrane interne de la mitochondrie, la communication cellulaire et les processus d'échanges sont principalement assurés par les transporteurs mitochondriaux. Ces protéines membranaires jouent un rôle clef dans les fonctions métaboliques des cellules eucaryotes et leur dysfonctionnement est à l'origine d'un certain nombre de maladies graves chez l'homme Parmi les transporteurs mitochondriaux, deux familles ont été étudiées au cours de ce travail : les AACs (ADP/ATP Carriers) et les UCPs (UnCoupling Proteins). Deux systèmes de production hétérologue de ces transporteurs ont été mis en place : la synthèse in vitro et l'expression chez E. coli de protéines de fusion. Le premier a permis la production et la purification d'environ 0,6 mg de protéine par mL de réaction et le deuxième a été exploité afin de réaliser des caractérisations fonctionnelles des transporteurs ADP/ATP. Un test fonctionnel pour la protéine découplante a également été mis au point. Ce test, basé sur la mesure directe des courants électriques associés à l'activité de transport de l'UCP, à permis la caractérisation fonctionnelle de la protéine UCP1 native

Relations structure - fonction des transporteurs mitochondriaux

Le passage sélectif d'ions et de métabolites à travers les membranes biologiques est essentiel à de nombreux processus cellulaires fondamentaux. Au niveau de la membrane interne de la mitochondrie, la communication cellulaire et les processus d'échanges sont principalement assurés par les transporteurs mitochondriaux. Ces protéines membranaires jouent un rôle clef dans les fonctions métaboliques des cellules eucaryotes et leur dysfonctionnement est à l'origine d'un certain nombre de maladies graves chez l'homme Parmi les transporteurs mitochondriaux, deux familles ont été étudiées au cours de ce travail: les AACs (ADP/A TI Carriers) et les UCPs (UnCoupling Proteins). Deux systèmes de production hétérologue de ces transporteurs ont été mil en place: la synthèse in vitro et l'expression chez E.coli de protéines de fusion. Le premier a permis la production et 1 purification d'environ 0,6 mg de protéine par mL de réaction et le deuxième a été exploité afin de réaliser dl caractérisations fonctionnelles des transporteurs ADP/ATP. Un test fonctionnel pour la protéine découplante a égalemel été mis au point. Ce test, basé sur la mesure directe des courants électriques associés à l'activité de transport de l'UCP, permis la caractérisation fonctionnelle de la protéine UCPI native.The Mitochondrial Carrier Family (MCF) groups integral membrane proteins that transport various metabolites across, the inner mitochondrial membrane. This transport is needed in major metabolic pathways occurring in mitochondria and is essential in eukaryotic metabolism. The mis-function of several identified human mitochondrial carriers has been associated with severe diseases . ln order to achieve a better understanding of mitochondrial carriers two different families of mitochondrial carriers where chosen to be studied: the AACs (ADP/ATP Carriers) and the UCPs (UnCoupling Proteins). Two heterologous expression systems of these carriers were developed: the cell free and the expression in E. coli as fusion proteins. The cell free system allowed the production and purification of about 0,6 mg of protein per milliliter of reaction mixture and the expression in E.coli proved to be very efficient for the functional characterization of ADPI A TP carriers. A functional test was developed for the uncoupling protein also. This test, based on the measure of electrical currents generated by the transport activity ofUCP, allowed the functional characterization of native UCPI.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF