Investigating genotype-phenotype relationship of extreme neuropathic pain disorders in a UK national cohort.

The aims of our study were to use whole genome sequencing in a cross-sectional cohort of patients to identify new variants in genes implicated in neuropathic pain, to determine the prevalence of known pathogenic variants and to understand the relationship between pathogenic variants and clinical presentation. Patients with extreme neuropathic pain phenotypes (both sensory loss and gain) were recruited from secondary care clinics in the UK and underwent whole genome sequencing as part of the National Institute for Health and Care Research Bioresource Rare Diseases project. A multidisciplinary team assessed the pathogenicity of rare variants in genes previously known to cause neuropathic pain disorders and exploratory analysis of research candidate genes was completed. Association testing for genes carrying rare variants was completed using the gene-wise approach of the combined burden and variance-component test SKAT-O. Patch clamp analysis was performed on transfected HEK293T cells for research candidate variants of genes encoding ion channels. The results include the following: (i) Medically actionable variants were found in 12% of study participants (205 recruited), including known pathogenic variants: SCN9A(ENST00000409672.1): c.2544T>C, p.Ile848Thr that causes inherited erythromelalgia, and SPTLC1(ENST00000262554.2):c.340T>G, p.Cys133Tr variant that causes hereditary sensory neuropathy type-1. (ii) Clinically relevant variants were most common in voltage-gated sodium channels (Nav). (iii) SCN9A(ENST00000409672.1):c.554G>A, pArg185His variant was more common in non-freezing cold injury participants than controls and causes a gain of function of NaV1.7 after cooling (the environmental trigger for non-freezing cold injury). (iv) Rare variant association testing showed a significant difference in distribution for genes NGF, KIF1A, SCN8A, TRPM8, KIF1A, TRPA1 and the regulatory regions of genes SCN11A, FLVCR1, KIF1A and SCN9A between European participants with neuropathic pain and controls. (v) The TRPA1(ENST00000262209.4):c.515C>T, p.Ala172Val variant identified in participants with episodic somatic pain disorder demonstrated gain-of-channel function to agonist stimulation. Whole genome sequencing identified clinically relevant variants in over 10% of participants with extreme neuropathic pain phenotypes. The majority of these variants were found in ion channels. Combining genetic analysis with functional validation can lead to a better understanding as to how rare variants in ion channels lead to sensory neuron hyper-excitability, and how cold, as an environmental trigger, interacts with the gain-of-function NaV1.7 p.Arg185His variant. Our findings highlight the role of ion channel variants in the pathogenesis of extreme neuropathic pain disorders, likely mediated through changes in sensory neuron excitability and interaction with environmental triggers.

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.

A novel phospho-modulatory mechanism contributes to the calcium-dependent regulation of T-type Ca2+ channels.

Cav3 / T-type Ca2+ channels are dynamically regulated by intracellular Ca2+ ions, which inhibit Cav3 availability. Here, we demonstrate that this inhibition becomes irreversible in the presence of non-hydrolysable ATP analogs, resulting in a strong hyperpolarizing shift in the steady-state inactivation of the residual Cav3 current. Importantly, the effect of these ATP analogs was prevented in the presence of intracellular BAPTA. Additional findings obtained using intracellular dialysis of inorganic phosphate and alkaline phosphatase or NaN3 treatment further support the involvement of a phosphorylation mechanism. Contrasting with Cav1 and Cav2 Ca2+ channels, the Ca2+-dependent modulation of Cav3 channels appears to be independent of calmodulin, calcineurin and endocytic pathways. Similar findings were obtained for the native T-type Ca2+ current recorded in rat thalamic neurons of the central medial nucleus. Overall, our data reveal a new Ca2+ sensitive phosphorylation-dependent mechanism regulating Cav3 channels, with potentially important physiological implications for the multiple cell functions controlled by T-type Ca2+ channels.

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. VIDEO ABSTRACT.

The Novel Activity of Carbamazepine as an Activation Modulator Extends from NaV1.7 Mutations to the NaV1.8-S242T Mutant Channel from a Patient with Painful Diabetic Neuropathy.

Neuropathic pain in patients carrying sodium channel gain-of-function mutations is generally refractory to pharmacotherapy. However, we have shown that pretreatment of cells with clinically achievable concentration of carbamazepine (CBZ; 30 µM) depolarizes the voltage dependence of activation in some NaV1.7 mutations such as S241T, a novel CBZ mode of action of this drug. CBZ reduces the excitability of dorsal root ganglion (DRG) neurons expressing NaV1.7-S241T mutant channels, and individuals carrying the S241T mutation respond to treatment with CBZ. Whether the novel activation-modulating activity of CBZ is specific to NaV1.7, and whether this pharmacogenomic approach can be extended to other sodium channel subtypes, are not known. We report here the novel NaV1.8-S242T mutation, which corresponds to the NaV1.7-S241T mutation, in a patient with neuropathic pain and diabetic peripheral neuropathy. Voltage-clamp recordings demonstrated hyperpolarized and accelerated activation of NaV1.8-S242T. Current-clamp recordings showed that NaV1.8-S242T channels render DRG neurons hyperexcitable. Structural modeling shows that despite a substantial difference in the primary amino acid sequence of NaV1.7 and NaV1.8, the S242 (NaV1.8) and S241 (NaV1.7) residues have similar position and orientation in the domain I S4-S5 linker of the channel. Pretreatment with a clinically achievable concentration of CBZ corrected the voltage dependence of activation of NaV1.8-S242T channels and reduced DRG neuron excitability as predicted from our pharmacogenomic model. These findings extend the novel activation modulation mode of action of CBZ to a second sodium channel subtype, NaV1.8.

Rare NaV1.7 variants associated with painful diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is a common disabling complication of diabetes. Almost half of the patients with DPN develop neuropathic pain (NeuP) for which current analgesic treatments are inadequate. Understanding the role of genetic variability in the development of painful DPN is needed for improved understanding of pain pathogenesis for better patient stratification in clinical trials and to target therapy more appropriately. Here, we examined the relationship between variants in the voltage-gated sodium channel NaV1.7 and NeuP in a deeply phenotyped cohort of patients with DPN. Although no rare variants were found in 78 participants with painless DPN, we identified 12 rare NaV1.7 variants in 10 (out of 111) study participants with painful DPN. Five of these variants had previously been described in the context of other NeuP disorders and 7 have not previously been linked to NeuP. Those patients with rare variants reported more severe pain and greater sensitivity to pressure stimuli on quantitative sensory testing. Electrophysiological characterization of 2 of the novel variants (M1852T and T1596I) demonstrated that gain of function changes as a consequence of markedly impaired channel fast inactivation. Using a structural model of NaV1.7, we were also able to provide further insight into the structural mechanisms underlying fast inactivation and the role of the C-terminal domain in this process. Our observations suggest that rare NaV1.7 variants contribute to the development NeuP in patients with DPN. Their identification should aid understanding of sensory phenotype, patient stratification, and help target treatments effectively.

CACNA1H Mutations Are Associated With Different Forms of Primary Aldosteronism.

Primary aldosteronism (PA) is the most common form of secondary hypertension. Mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D are found in aldosterone producing adenoma (APA) and familial hyperaldosteronism (FH). A recurrent mutation in CACNA1H (coding for Cav3.2) was identified in a familial form of early onset PA. Here we performed whole exome sequencing (WES) in patients with different types of PA to identify new susceptibility genes. Four different heterozygous germline CACNA1H variants were identified. A de novo Cav3.2 p.Met1549Ile variant was found in early onset PA and multiplex developmental disorder. Cav3.2 p.Ser196Leu and p.Pro2083Leu were found in two patients with FH, and p.Val1951Glu was identified in one patient with APA. Electrophysiological analysis of mutant Cav3.2 channels revealed significant changes in the Ca2+ current properties for all mutants, suggesting a gain of function phenotype. Transfections of mutant Cav3.2 in H295R-S2 cells led to increased aldosterone production and/or expression of genes coding for steroidogenic enzymes after K+ stimulation. Identification of CACNA1H mutations associated with early onset PA, FH, and APA suggests that CACNA1H might be a susceptibility gene predisposing to PA with different phenotypic presentations, opening new perspectives for genetic diagnosis and management of patients with PA.

CACNA1H missense mutations associated with amyotrophic lateral sclerosis alter Cav3.2 T-type calcium channel activity and reticular thalamic neuron firing.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. In a recent study by Steinberg and colleagues, 2 recessive missense mutations were identified in the Cav3.2 T-type calcium channel gene (CACNA1H), in a family with an affected proband (early onset, long duration ALS) and 2 unaffected parents. We have introduced and functionally characterized these mutations using transiently expressed human Cav3.2 channels in tsA-201 cells. Both of these mutations produced mild but significant changes on T-type channel activity that are consistent with a loss of channel function. Computer modeling in thalamic reticular neurons suggested that these mutations result in decreased neuronal excitability of thalamic structures. Taken together, these findings implicate CACNA1H as a susceptibility gene in amyotrophic lateral sclerosis.

Phosphorylation of the Cav3.2 T-type calcium channel directly regulates its gating properties.

Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.

A Recurrent Mutation in CACNA1G Alters Cav3.1 T-Type Calcium-Channel Conduction and Causes Autosomal-Dominant Cerebellar Ataxia.

Hereditary cerebellar ataxias (CAs) are neurodegenerative disorders clinically characterized by a cerebellar syndrome, often accompanied by other neurological or non-neurological signs. All transmission modes have been described. In autosomal-dominant CA (ADCA), mutations in more than 30 genes are implicated, but the molecular diagnosis remains unknown in about 40% of cases. Implication of ion channels has long been an ongoing topic in the genetics of CA, and mutations in several channel genes have been recently connected to ADCA. In a large family affected by ADCA and mild pyramidal signs, we searched for the causative variant by combining linkage analysis and whole-exome sequencing. In CACNA1G, we identified a c.5144G>A mutation, causing an arginine-to-histidine (p.Arg1715His) change in the voltage sensor S4 segment of the T-type channel protein Cav3.1. Two out of 479 index subjects screened subsequently harbored the same mutation. We performed electrophysiological experiments in HEK293T cells to compare the properties of the p.Arg1715His and wild-type Cav3.1 channels. The current-voltage and the steady-state activation curves of the p.Arg1715His channel were shifted positively, whereas the inactivation curve had a higher slope factor. Computer modeling in deep cerebellar nuclei (DCN) neurons suggested that the mutation results in decreased neuronal excitability. Taken together, these data establish CACNA1G, which is highly expressed in the cerebellum, as a gene whose mutations can cause ADCA. This is consistent with the neuropathological examination, which showed severe Purkinje cell loss. Our study further extends our knowledge of the link between calcium channelopathies and CAs.

Inhibition of Cav3.2 T-type Calcium Channels by Its Intracellular I-II Loop.

Voltage-dependent calcium channels (Cav) of the T-type family (Cav3.1, Cav3.2, and Cav3.3) are activated by low threshold membrane depolarization and contribute greatly to neuronal network excitability. Enhanced T-type channel activity, especially Cav3.2, contributes to disease states, including absence epilepsy. Interestingly, the intracellular loop connecting domains I and II (I-II loop) of Cav3.2 channels is implicated in the control of both surface expression and channel gating, indicating that this I-II loop plays an important regulatory role in T-type current. Here we describe that co-expression of this I-II loop or its proximal region (Δ1-Cav3.2; Ser(423)-Pro(542)) together with recombinant full-length Cav3.2 channel inhibited T-type current without affecting channel expression and membrane incorporation. Similar T-type current inhibition was obtained in NG 108-15 neuroblastoma cells that constitutively express Cav3.2 channels. Of interest, Δ1-Cav3.2 inhibited both Cav3.2 and Cav3.1 but not Cav3.3 currents. Efficacy of Δ1-Cav3.2 to inhibit native T-type channels was assessed in thalamic neurons using viral transduction. We describe that T-type current was significantly inhibited in the ventrobasal neurons that express Cav3.1, whereas in nucleus reticularis thalami neurons that express Cav3.2 and Cav3.3 channels, only the fast inactivating T-type current (Cav3.2 component) was significantly inhibited. Altogether, these data describe a new strategy to differentially inhibit Cav3 isoforms of the T-type calcium channels.

Assaying the proton transport and regulation of UCP1 using solid supported membranes.

The uncoupling protein 1 (UCP1) is a mitochondrial protein that carries protons across the inner mitochondrial membrane. It has an important role in non-shivering thermogenesis, and recent evidence suggests its role in human adult metabolism. Using rapid solution exchange on solid supported membranes, we succeeded in measuring electrical currents generated by the transport activity of UCP1. The protein was purified from mouse brown adipose tissue, reconstituted in liposomes and absorbed on solid supported membranes. A fast pH jump activated the ion transport, and electrical signals could be recorded. The currents were characterized by a fast rise and a slow decay, were stable over time, inhibited by purine nucleotides and activated by fatty acids. This new assay permits direct observation of UCP1 activity in controlled cell-free conditions, and opens up new possibilities for UCP1 functional characterization and drug screening because of its robustness and its potential for automation.

Impaired transport of nucleotides in a mitochondrial carrier explains severe human genetic diseases.

The mitochondrial ADP/ATP carrier (AAC) is a prominent actor in the energetic regulation of the cell, importing ADP into the mitochondria and exporting ATP toward the cytoplasm. Severe genetic diseases have been ascribed to specific mutations in this membrane protein. How minute, well-localized modifications of the transporter impact the function of the mitochondria remains, however, largely unclear. Here, for the first time, the relationship between all documented pathological mutations of the AAC and its transport properties is established. Activity measurements combined synergistically with molecular-dynamics simulations demonstrate how all documented pathological mutations alter the binding affinity and the translocation kinetics of the nucleotides. Throwing a bridge between the pathologies and their molecular origins, these results reveal two distinct mechanisms responsible for AAC-related genetic disorders, wherein the mutations either modulate the association of the nucleotides to the carrier by modifying its electrostatic signature or reduce its conformational plasticity.

Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant.

Structural studies of membrane protein are still challenging due to several severe bottlenecks, the first being the overproduction of well-folded proteins. Several expression systems are often explored in parallel to fulfil this task, or alternately prokaryotic analogues are considered. Although, mitochondrial carriers play key roles in several metabolic pathways, only the structure of the ADP/ATP carrier purified from bovine heart mitochondria was determined so far. More generally, characterisations at the molecular level are restricted to ADP/ATP carrier or the uncoupling protein UCP1, another member of the mitochondrial carrier family, which is abundant in brown adipose tissues. Indeed, mitochondrial carriers have no prokaryotic homologues and very few efficient expression systems were described so far for these proteins. We succeeded in producing UCP1 using a cell free expression system based on E. coli extracts, in quantities that are compatible with structural approaches. The protein was synthesised in the presence of a fluorinated surfactant, which maintains the protein in a soluble form. Further biochemical and biophysical analysis such as size exclusion chromatography, circular dichroism and thermal stability, of the purified protein showed that the protein is non-aggregated, monodisperse and well-folded.

Structural approaches of the mitochondrial carrier family.

The transport of solutes across the inner mitochondrial membrane is highly selective and necessitates membrane proteins mainly from the mitochondrial carrier family (MCF). These carriers are required for the transport of a variety of metabolites implicated in all the important processes occurring within the mitochondrial matrix. Due to its high abundance, the ADP/ATP carrier (AAC) is the member of the family that was studied most. It is the first mitochondrial carrier for which a high-resolution X-ray structure is known. The carrier was crystallized in the presence of a strong inhibitor, the carboxyatractyloside (CATR). The structure gives an insight not only into the overall fold of mitochondrial carriers in general but also into atomic details of the AAC in a conformation that is open toward the intermembrane space (IMS). Molecular dynamics simulations indicate the first events occurring to the carrier after the binding of ADP. A careful analysis of the primary sequences of all the carriers in light with the structure highlights properties of the protein that are related to the substrate.

A simple method for the reconstitution of membrane proteins into giant unilamellar vesicles.

A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.

Single-step production of functional OEP24 proteoliposomes.

The pea chloroplastic outer envelope protein OEP24 is a voltage-dependent channel that can function as a general solute channel in plants. OEP24 is a close functional homologue of VDAC which, in mammalian cells, modulates the permeability of the outer mitochondrial membrane. Here, we describe the production in a one-step reaction of active OEP24 in proteoliposomes or in soluble form using a cell-free expression system. We combine evidence from electrophysiological experiments, biophysical characterization, and biochemical analysis demonstrating that OEP24 is present as a functional channel in liposomes. Thus, production of OEP-containing proteoliposomes may provide a helpful tool for deciphering the role of the OEP family members.