Comparisons of dual isogenic human iPSC pairs identify functional alterations directly caused by an epilepsy associated SCN1A mutation.

Over 1250 mutations in SCN1A, the Nav1.1 voltage-gated sodium channel gene, are associated with seizure disorders including GEFS+. To evaluate how a specific mutation, independent of genetic background, causes seizure activity we generated two pairs of isogenic human iPSC lines by CRISPR/Cas9 gene editing. One pair is a control line from an unaffected sibling, and the mutated control carrying the GEFS+ K1270T SCN1A mutation. The second pair is a GEFS+ patient line with the K1270T mutation, and the corrected patient line. By comparing the electrophysiological properties in inhibitory and excitatory iPSC-derived neurons from these pairs, we found the K1270T mutation causes cell type-specific alterations in sodium current density and evoked firing, resulting in hyperactive neural networks. We also identified differences associated with genetic background and interaction between the mutation and genetic background. Comparisons within and between dual pairs of isogenic iPSC-derived neuronal cultures provide a novel platform for evaluating cellular mechanisms underlying a disease phenotype and for developing patient-specific anti-seizure therapies.

Imaging of effector memory T cells during a delayed-type hypersensitivity reaction and suppression by Kv1.3 channel block.

Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.

The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.

Ca(2+)-release-activated Ca(2+) (CRAC) channels underlie sustained Ca(2+) signalling in lymphocytes and numerous other cells after Ca(2+) liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim and Orai, that together form the molecular basis for CRAC channel activity. Stim senses depletion of the ER Ca(2+) store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane, and Orai embodies the pore of the plasma membrane calcium channel. A close interaction between Stim and Orai, identified by co-immunoprecipitation and by Förster resonance energy transfer, is involved in the opening of the Ca(2+) channel formed by Orai subunits. Most ion channels are multimers of pore-forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. Here we show, by biochemical analysis after cross-linking in cell lysates and intact cells and by using non-denaturing gel electrophoresis without cross-linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single-molecule imaging of green fluorescent protein (GFP)-tagged Orai expressed in Xenopus oocytes showed predominantly two-step photobleaching, again consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca(2+) store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca(2+)-selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.

Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states.

We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in mammalian cells. Orai1 was detected predominantly as dimers under resting conditions and as tetramers when coexpressed with C-STIM1 to activate Ca(2+) influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca(2+), redistribution of STIM1 to the plasma membrane, and STIM1/Orai1 puncta formation, and may cause the channel to be in the activated state. Consistent with this possibility, Orai1 was found predominantly as a dimer when coexpressed with STIM1 in living cells under resting conditions. We further show that Orai3, like Orai1, is dimeric under resting conditions and is predominantly tetrameric when activated by C-STIM1. Interestingly, a dimeric Orai3 stoichiometry was found both before and during application of 2-aminoethyldiphenyl borate (2-APB) to activate a nonselective cation conductance in its STIM1-independent mode. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca(2+)-selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.

Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai.

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) and Orai. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells. The overexpression of Stim and Orai together markedly increases CRAC current. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.

Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation.

For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+ concentration near the interface, indicating Ca2+ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+ signaling during subsequent antigen encounter.

Interneuron Dysfunction in a New Mouse Model of SCN1A GEFS.

Advances in genome sequencing have identified over 1300 mutations in the SCN1A sodium channel gene that result in genetic epilepsies. However, it still remains unclear how most individual mutations within SCN1A result in seizures. A previous study has shown that the K1270T (KT) mutation, linked to genetic epilepsy with febrile seizure plus (GEFS+) in humans, causes heat-induced seizure activity associated with a temperature-dependent decrease in GABAergic neuron excitability in a Drosophila knock-in model. To examine the behavioral and cellular effects of this mutation in mammals, we introduced the equivalent KT mutation into the mouse (Mus musculus) Scn1a (Scn1aKT) gene using CRISPR/Cas9 and generated mutant lines in two widely used genetic backgrounds: C57BL/6NJ and 129X1/SvJ. In both backgrounds, mice homozygous for the KT mutation had spontaneous seizures and died by postnatal day (P)23. There was no difference in mortality of heterozygous KT mice compared with wild-type littermates up to six months old. Heterozygous mutants exhibited heat-induced seizures at ∼42°C, a temperature that did not induce seizures in wild-type littermates. In acute hippocampal slices at permissive temperatures, current-clamp recordings revealed a significantly depolarized shift in action potential threshold and reduced action potential amplitude in parvalbumin (PV)-expressing inhibitory CA1 interneurons in Scn1aKT/+ mice. There was no change in the firing properties of excitatory CA1 pyramidal neurons. These results suggest that a constitutive decrease in inhibitory interneuron excitability contributes to the seizure phenotype in the mouse model.

A functional single-nucleotide polymorphism in the TRPC6 gene promoter associated with idiopathic pulmonary arterial hypertension.

BACKGROUND: Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role in the development of idiopathic pulmonary arterial hypertension (IPAH), whereas a rise in cytosolic Ca2+ concentration triggers PASMC contraction and stimulates PASMC proliferation. Recently, we demonstrated that upregulation of the TRPC6 channel contributes to proliferation of PASMCs isolated from IPAH patients. This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC6 gene promoter that are associated with IPAH and have functional significance in regulating TRPC6 activity in PASMCs. METHODS AND RESULTS: Genomic DNA was isolated from blood samples of 237 normal subjects and 268 IPAH patients. Three biallelic SNPs, -361 (A/T), -254(C/G), and -218 (C/T), were identified in the 2000-bp sequence upstream of the transcriptional start site of TRPC6. Although the allele frequencies of the -361 and -218 SNPs were not different between the groups, the allele frequency of the -254(C-->G) SNP in IPAH patients (12%) was significantly higher than in normal subjects (6%; P<0.01). Genotype data showed that the percentage of -254G/G homozygotes in IPAH patients was 2.85 times that of normal subjects. Moreover, the -254(C-->G) SNP creates a binding sequence for nuclear factor-kappaB. Functional analyses revealed that the -254(C-->G) SNP enhanced nuclear factor-kappaB-mediated promoter activity and stimulated TRPC6 expression in PASMCs. Inhibition of nuclear factor-kappaB activity attenuated TRPC6 expression and decreased agonist-activated Ca2+ influx in PASMCs of IPAH patients harboring the -254G allele. CONCLUSIONS: These results suggest that the -254(C-->G) SNP may predispose individuals to an increased risk of IPAH by linking abnormal TRPC6 transcription to nuclear factor-kappaB, an inflammatory transcription factor.

Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes.

The adaptive immune response is initiated in secondary lymphoid organs by contact between antigen-bearing dendritic cells (DCs) and antigen-specific CD4+ T cells. However, there is scant information regarding the single cell dynamics of this process in vivo. Using two-photon microscopy, we imaged the real-time behavior of naive CD4+ T cells and in vivo-labeled DCs in lymph nodes during a robust T cell response. In the first 2 h after entry into lymph nodes, T cells made short-lived contacts with antigen-bearing DCs, each contact lasting an average of 11-12 min and occurring mainly on dendrites. Altered patterns of T cell motility during this early stage of antigen recognition promoted serial engagement with several adjacent DCs. Subsequently, T cell behavior progressed through additional distinct stages, including long-lived clusters, dynamic swarms, and finally autonomous migration punctuated by cell division. These observations suggest that the immunological synapse in native tissues is remarkably fluid, and that stable synapses form only at specific stages of antigen presentation to T cells. Furthermore, the serial nature of these interactions implies that T cells activate by way of multiple antigen recognition events.

STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Cystic fibrosis transmembrane conductance regulator (CFTR) functionality is dependent on coatomer protein I (COPI).

BACKGROUND INFORMATION: Cystic fibrosis results from mutations in the ABC transporter CFTR (cystic fibrosis transmembrane conductance regulator), which functions as a cAMP-regulated anion channel. The most prevalent mutation in CFTR, the Phe(508) deletion, results in the generation of a trafficking and functionally deficient channel. The cellular machineries involved in modulating CFTR trafficking and function have not been fully characterized. In the present study, we identified a role for the COPI (coatomer protein I) cellular trafficking machinery in the development of the CFTR polypeptide into a functional chloride channel. To examine the role of COPI in CFTR biosynthesis, we employed the cell line ldlF, which harbours a temperature-sensitive mutation in epsilon-COP, a COPI subunit, to inhibit COPI function and then determined whether the CFTR polypeptide produced from the transfected gene developed into a cAMP-regulated chloride channel. RESULTS: When COPI was inactivated in the ldlF cells by an elevated temperature pulse (39 degrees C), the CFTR polypeptide was detected on the cell surface by immunofluorescence microscopy and cell-surface biotinylation. Therefore, CFTR proceeded upstream in the secretory pathway in the absence of COPI function, a result demonstrated previously by others. In contrast, electrophysiological measurements indicated an absence of cAMP-stimulated chloride efflux, suggesting that channel function was impaired. In comparison, expression of CFTR at the same elevated temperature (39 degrees C) in an epsilon-COP-rescued cell line [ldlF(ldlF)], which has an introduced wild-type epsilon-COP gene in addition to the mutant epsilon-COP gene, showed restoration of cAMP-stimulated channel activity, confirming the requirement of COPI for channel function. CONCLUSIONS: These results therefore suggest that generation of the folded-functional conformation of CFTR requires COPI.

State-dependent block of Orai3 TM1 and TM3 cysteine mutants: insights into 2-APB activation.

After endoplasmic reticulum (ER) Ca(2+) store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca(2+)-selective Ca(2+) release-activated Ca(2+) (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1-Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB-activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd(2+)). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB-activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd(2+)-induced block that is state dependent: Cd(2+) only blocks 2-APB-activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB-activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore.

Orai3 TM3 point mutation G158C alters kinetics of 2-APB-induced gating by disulfide bridge formation with TM2 C101.

After endoplasmic reticulum (ER) Ca(2+) store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident STIM proteins to form the Ca(2+)-selective Ca(2+) release-activated Ca(2+) (CRAC) channel. However, in the absence of Ca(2+) store depletion and STIM interaction, the mammalian homologue Orai3 can be activated by 2-aminoethyl diphenylborinate (2-APB), resulting in a nonselective cation conductance characterized by biphasic inward and outward rectification. Here, we use site-directed mutagenesis and patch-clamp analysis to better understand the mechanism by which 2-APB activates Orai3. We find that point mutation of glycine 158 in the third transmembrane (TM) segment to cysteine, but not alanine, slows the kinetics of 2-APB activation and prevents complete channel closure upon 2-APB washout. The "slow" phenotype exhibited by Orai3 mutant G158C reveals distinct open states, characterized by variable reversal potentials. The slow phenotype can be reversed by application of the reducing reagent bis(2-mercaptoethylsulfone) (BMS), but in a state-dependent manner, only during 2-APB activation. Moreover, the double mutant C101G/G158C, in which an endogenous TM2 cysteine is changed to glycine, does not exhibit altered kinetics of 2-APB activation. We suggest that a disulfide bridge, formed between the introduced cysteine at TM3 position 158 and the endogenous cysteine at TM2 position 101, hinders transitions between Orai3 open and closed states. Our data provide functional confirmation of the proximity of these two residues and suggest a location within the Orai3 protein that is sensitive to the actions of 2-APB.

Ca2+ signals in CD4+ T cells during early contacts with antigen-bearing dendritic cells in lymph node.

T cell activation by APC requires cytosolic Ca(2+) ([Ca(2+)](i)) elevation. Using two-photon microscopy, we visualized Ca(2+) signaling and motility of murine CD4(+) T cells within lymph node (LN) explants under control, inflammatory, and immunizing conditions. Without Ag under basal noninflammatory conditions, T cells showed infrequent Ca(2+) spikes associated with sustained slowing. Inflammation reduced velocities and Ca(2+) spiking in the absence of specific Ag. During early Ag encounter, most T cells engaged Ag-presenting dendritic cells in clusters, and showed increased Ca(2+) spike frequency and elevated basal [Ca(2+)](i). These Ca(2+) signals persisted for hours, irrespective of whether T cells were in contact with visualized dendritic cells. We propose that sustained increases in basal [Ca(2+)](i) and spiking frequency constitute a Ca(2+) signaling modality that, integrated over hours, distinguishes immunogenic from basal state in the native lymphoid environment.

Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity.

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.