Iulia Blesneac

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.

Crystallization of the membrane protein hVDAC1 produced in cell-free system

Structural studies of membrane proteins are in constant evolution with the development of new improvements for their expression, purification, stabilization and crystallization. However, none of these methods still provides a universal approach to solve the structure of membrane proteins. Here we describe the crystallization of the human voltage-dependent anion channel-1 produced by a bacterial cell-free expression system. While VDAC structures have been recently solved, we propose an alternative strategy for producing the recombinant protein, which can be applied to other membrane proteins reluctant to expression, purification and crystallization by classical approaches. Despite a lot of efforts to crystallize a cell-free expressed membrane protein, this study is to our knowledge one of the first reports of a successful crystallization. Focusing on expression in a soluble and functional state, in a detergent environment, is the key to get crystals. Although the diffraction of VDAC crystals is limited, the simplicity and the rapidity to set-up and optimize this technology are drastic advantages in comparison to other methods.

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.

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.

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.

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

Significance Ion channels are membrane proteins essential for signal generation and transmission in the nervous system. They are finely regulated, and even small changes in their activity can trigger important physiological or pathological consequences on brain function. A regulatory mechanism of particular importance is phosphorylation. In this study, we assess the impact of phosphorylation on the activity of a specific ion channel, the T-type calcium channel Cav3.2. We show for the first time, to our knowledge, that Cav3.2 is highly phosphorylated in vivo and in a mammalian brain as well as in a human cell line, and we identify many phosphorylation sites critical for the way that the channel opens in response to changes in membrane potential and neuronal excitability. 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.

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.

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.

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

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

ABSTRACT 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.