Colloquium 9: Structural and Molecular Dissection of the Node of Ranvier. Sodium channel β subunits: multifunctional molecules at the node of Ranvier

Voltage‐gated sodium channels are unique in that they combine action potential conduction with cell adhesion. Mammalian sodium channels are heterotrimers, composed of a central, pore‐forming α subunit and two auxiliary β subunits. The α subunits are members of a large gene family containing the voltage‐gated sodium, potassium, and calcium channels. Sodium channel α subunits form a gene subfamily with at least 11 members. Mutations in sodium channel α subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome (LQT), and hyperkalemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode the sodium channel β subunits with at least one alternative splice product. Unlike the pore‐forming α subunits, the sodium channel β subunits are not structurally related to β subunits of calcium and potassium channels. Sodium channel β subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. We have shown that β subunits also function as cell adhesion molecules (CAMs) in terms of interaction with extracellular matrix molecules, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. A mutation in SCN1B has been shown to cause GEFS + 1 epilepsy in human families. We propose that the sodium channel signalling complex at nodes of Ranvier involves β subunits as channel modulators as well as CAMs, other CAMs such as neurofascin and contactin, RPTPβ, and extracellular matrix molecules such as tenascin.     

Repolarisation and vulnerability to re-entry in the human heart with short QT syndrome arising from KCNQ1 mutation--a simulation study

Idiopathic short QT syndrome (SQTS) is a recently identified, genetically heterogeneous condition characterised by abbreviated QT intervals and an increased susceptibility to arrhythmia and sudden death. This simulation study identifies mechanisms by which cellular electrophysiological changes in the SQT2 (slow delayed rectifier, I_Ks, -linked) SQTS variant increases arrhythmia risk. The channel kinetics of the V307L mutation of the KCNQ1 subunit of the I_Ks channel were incorporated into human ventricular action potential (AP) models and into 1D and 2D transmural tissue simulations. Incorporating the V307L mutation into simulations reproduced defining features of the SQTS: abbreviation of the QT interval, and increases in T wave amplitude and T_peak–T_end duration. In the single-cell model, the V307L mutation abbreviated ventricular cell AP duration at 90% repolarisation (APD₉₀) and increased the maximal transmural voltage heterogeneity (δV) during APs; this resulted in augmented transmural heterogeneity of APD₉₀ and of the effective refractory period (ERP). In the intact tissue model, the vulnerable window for unidirectional conduction block was also increased. In 2D tissue the V307L mutation facilitated and maintained reentrant excitation. Thus, in SQT2 increases in transmural heterogeneity of APD, δV, ERP and an increased vulnerable window for unidirectional conduction block generate an electrical substrate favourable to reentrant arrhythmia.     

Modified autonomic regulation in mice with a P/Q-type calcium channel mutation

Recent genetic analyses revealed an important association between P/Q-type channels and hereditary neurological disorders. The α1 subunit of P/Q-type channels is coded by a single CaV2.1 gene. Since calcium entry via neuronal calcium channels is essential for neurotransmission, P/Q-type channels may play an important role in cardiac autonomic neurotransmission. To elucidate the physiological importance of P/Q-type channels in autonomic nerve control, we used rolling Nagoya (tg^rol) mice, which have a mutation in the CaV2.1 gene and decreased P/Q-type channel currents with reduced voltage sensitivity.The tg^rol mice demonstrated unmodified expression of other calcium channel subunits. Electrocardiogram and echocardiographic analyses revealed decreased heart rate. Furthermore, ω-agatoxin IVA, a P/Q-type channel inhibitor, decreased heart rate and ejection fraction only in wild-type mice, thus suggesting a significant involvement of P/Q-type channels in chronotropic regulation. Atrium contraction analyses revealed a minor but significant role for P/Q-type channels in sympathetic and parasympathetic nerve regulation.     

Differential expression of T-type calcium channels in P/Q-type calcium channel mutant mice with ataxia and absence epilepsy

Mutations in P/Q-type calcium channels generate common phenotypes in mice and humans, which are characterized by ataxia, paroxysmal dyskinesia, and absence seizures. Subsequent functional changes of T-type calcium channels in thalamus are observed in P/Q-type calcium channel mutant mice and these changes play important roles in generation of absence seizures. However, the changes in T-type calcium channel function and/or expression in the cerebellum, which may be related to movement disorders, are still unknown. The leaner mouse exhibits severe ataxia, paroxysmal dyskinesia, and absence epilepsy due to a P/Q-type calcium channel mutation. We investigated changes in T-type calcium channel expression in the leaner mouse thalamus and cerebellum using quantitative real-time polymerase chain reaction (qRT-PCR) and quantitative in situ hybridization histochemistry (ISHH). qRT-PCR analysis showed no change in T-type calcium channel alpha 1G subunit (Cav3.1) expression in the leaner thalamus, but a significant decrease in alpha 1G expression in the whole leaner mouse cerebellum. Interestingly, quantitative ISHH revealed differential changes in alpha 1G expression in the leaner cerebellum, where the granule cell layer showed decreased alpha 1G expression while Purkinje cells showed increased alpha 1G expression. To confirm these observations, the granule cell layer and the Purkinje cell layer were laser capture microdissected separately, then analyzed with qRT-PCR. Similar to the observation obtained by ISHH, the leaner granule cell layer showed decreased alpha 1G expression and the leaner Purkinje cell layer showed increased alpha 1G expression. These results suggest that differential expression of T-type calcium channels in the leaner cerebellum may be involved in the observed movement disorders.     

Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs

Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is deltaF508-CFTR, which inefficiently traffics to the surfaces of most cells. The deltaF508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and deltaF508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment deltaF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and deltaF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of deltaF508-CFTR channels in excised membrane patches by 10-15-fold under conditions in which wild-type channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the deltaF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface.     

The zona pellucida-initiated acrosome reaction: defect due to mutations in the sperm glycine receptor/Cl(-) channel

The mammalian sperm acrosome reaction (AR) is essential to fertilization, and the egg zona pellucida (ZP) is generally believed to be an in vivo initiator of the fertilizing sperm AR. Previously a neuronal glycine receptor/Cl(-) channel (GlyR) was detected on the plasma membrane of mammalian sperm and earlier pharmacological studies suggested that this receptor/channel is important to the ZP-initiated AR. Here, sperm from mice with mutations in the neuronal GlyR alpha or beta subunits (spasmodic and spastic) were shown to be deficient in their ability to undergo the AR initiated in vitro by glycine or by solubilized ZP from mouse eggs. However, both spontaneous and calcium ionophore (A23187)-initiated AR were unaffected. The ZP-initiated AR in wild-type sperm was maximal after 2 h of capacitation, but capacitation of sperm from spasmodic mice for up to 3 h did not result in significant ZP-initiated AR. Similar results were observed when sperm from wild-type and spastic mice were compared. Testis from mice with the beta subunit mutation contained truncated beta subunit mRNAs. Moreover, a monoclonal antibody against GlyR completely blocked ZP initiation of AR in normal mouse sperm. Our results are consistent with an essential role for the sperm GlyR in the ZP-initiated AR.     

Decreased calretinin expression in cerebellar granule cells in the leaner mouse

We investigated calretinin expression in cerebellar granule cells of 30-day-old leaner mice to understand possible changes in calcium homeostasis due to the calcium channel mutation that these mice carry. Quantitative in situ hybridization histochemistry showed decreased calretinin mRNA expression in the leaner cerebellum. Immunohistochemical staining also revealed decreased calretinin immunoreactivity in the leaner cerebellum. To exclude the effect of granule cell loss that occurs in the leaner mouse when comparing cerebellar calretinin expression, the number of granule cells per unit area in the cerebellum was compared to the wild-type cerebellum. Granule cell counts per unit area of cerebellum revealed similar numbers of granule cells present in wild-type and leaner mice. Laser capture microdissection (LCM) was employed to obtain an equal number of granule cells from wild-type and leaner mice. Western blot analysis with LCM-procured cerebellar granule cells showed decreased calretinin expression in leaner granule cells. These results indicate that there is an absolute decrease in calretinin expression in leaner granule cells even when granule cell loss is taken into account. Decreased calretinin expression in leaner granule cells may contribute to altered calcium buffering capacity. This alteration could be an adaptive change due to the calcium channel dysfunction, and may result in abnormal neuronal excitability and gene expression.     

How to dissect the Node of Ranvier

Voltage-gated sodium channels are unique in that they combine action potential conduction with cell adhesion. Mammalian sodium channels are heterotrimers, composed of a central, pore-forming α subunit and two auxiliary β subunits. The α subunits are members of a large gene family containing the voltage-gated sodium, potassium, and calcium channels. Sodium channel α subunits form a gene subfamily with at least 11 members. Mutations in sodium channel α subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome (LQT), and hyperkalemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode the sodium channel β subunits with at least one alternative splice product. Unlike the pore-forming α subunits, the sodium channel β subunits are not structurally related to β subunits of calcium and potassium channels. Sodium channel β subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. We have shown that β subunits also function as cell adhesion molecules (CAMs) in terms of interaction with extracellular matrix molecules, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. A mutation in SCN1B has been shown to cause GEFS + 1 epilepsy in human families. We propose that the sodium channel signalling complex at nodes of Ranvier involves β subunits as channel modulators as well as CAMs, other CAMs such as neurofascin and contactin, RPTPβ, and extracellular matrix molecules such as tenascin.     

Kinetics of internalization and degradation of N-type voltage-gated calcium channels: role of the alpha2/delta subunit

The contribution of voltage-gated calcium channels to excitable cell function depends, critically, upon the mechanisms that control their expression at the cell surface. While co-assembly of the pore forming alpha(1) and auxiliary beta subunits enhances channel surface expression, the levels are still only 30-40% of those seen with the core alpha(1B)/beta(1b)/alpha(2)delta calcium channel complex. To rationalize this observation, it has been suggested that the alpha(2)/delta subunit might stabilize calcium channel expression at the cell surface. To test this notion, we have resolved the effect of the alpha(2)/delta subunit on the rates of binding, internalization and degradation of defined N-type calcium channel surface complexes expressed in HEK293 cells, through pulse-labeling with the selective, cell impermeable, radioligand [(125)I]-omega-CgTx. Through detailed kinetic and sensitivity analysis we show that alpha(1B)/beta(1b)/alpha(2)delta complexes are internalized slowly (k(int) 0.4/h), whereupon, most become degraded (k(deg) 0.02/h). In contrast, alpha(1B)/beta(1b) complexes are internalized more rapidly (k(int) 0.8/h), following which they are either quickly degraded (k(deg) 0.1/h) or are sequestered slowly (k(tra) 0.1/h) to a pool that is metabolically stable within the time-frame of our experiments (24h). In neither case did we find evidence for recycling via the cell surface. Thus, our data argue for a novel mechanism where complexes lacking an alpha(2)/delta subunit are cleared from the cell surface and are rapidly degraded or stored, possibly for further attempts at complexation as new alpha(2)/delta subunits become available. The slower rate of internalization of complexes containing the alpha(2)/delta subunit rationalizes the stabilizing effect this subunit has upon calcium channel surface expression and suggests a mechanism by which alpha(2)delta mutations may cause severe neurological deficits.     

Differential expression of T‐type calcium channels in P/Q‐type calcium channel mutant mice with ataxia and absence epilepsy

Mutations in P/Q‐type calcium channels generate common phenotypes in mice and humans, which are characterized by ataxia, paroxysmal dyskinesia, and absence seizures. Subsequent functional changes of T‐type calcium channels in thalamus are observed in P/Q‐type calcium channel mutant mice and these changes play important roles in generation of absence seizures. However, the changes in T‐type calcium channel function and/or expression in the cerebellum, which may be related to movement disorders, are still unknown. The leaner mouse exhibits severe ataxia, paroxysmal dyskinesia, and absence epilepsy due to a P/Q‐type calcium channel mutation. We investigated changes in T‐type calcium channel expression in the leaner mouse thalamus and cerebellum using quantitative real‐time polymerase chain reaction (qRT‐PCR) and quantitative in situ hybridization histochemistry (ISHH). qRT‐PCR analysis showed no change in T‐type calcium channel α1G subunit (Cav3.1) expression in the leaner thalamus, but a significant decrease in α1G expression in the whole leaner mouse cerebellum. Interestingly, quantitative ISHH revealed differential changes in α1G expression in the leaner cerebellum, where the granule cell layer showed decreased α1G expression while Purkinje cells showed increased α1G expression. To confirm these observations, the granule cell layer and the Purkinje cell layer were laser capture microdissected separately, then analyzed with qRT‐PCR. Similar to the observation obtained by ISHH, the leaner granule cell layer showed decreased α1G expression and the leaner Purkinje cell layer showed increased α1G expression. These results suggest that differential expression of T‐type calcium channels in the leaner cerebellum may be involved in the observed movement disorders. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

ORAI1 deficiency impairs activated T cell death and enhances T cell survival

ORAI1 is a pore subunit of Ca(2+) release-activated Ca(2+) channels that mediate TCR stimulation-induced Ca(2+) entry. A point mutation in ORAI1 (ORAI1(R91W)) causes SCID in human patients that is recapitulated in Orai1(-/-) mice, emphasizing its important role in the immune cells. In this study, we have characterized a novel function of ORAI1 in T cell death. CD4(+) T cells from Orai1(-/-) mice showed robust proliferation with repetitive stimulations and strong resistance to stimulation-induced cell death due to reduced mitochondrial Ca(2+) uptake and altered gene expression of proapoptotic and antiapoptotic molecules (e.g., Fas ligand, Noxa, and Mcl-1). Nuclear accumulation of NFAT was severely reduced in ORAI1-deficient T cells, and expression of ORAI1 and a constitutively active mutant of NFAT recovered cell death. These results indicate NFAT-mediated cell death pathway as one of the major downstream targets of ORAI1-induced Ca(2+) entry. By expressing various mutants of ORAI1 in wild-type and Orai1(-/-) T cells to generate different levels of intracellular Ca(2+), we have shown that activation-induced cell death is directly proportional to the intracellular Ca(2+) concentration levels. Consistent with the in vitro results, Orai1(-/-) mice showed strong resistance to T cell depletion induced by injection of anti-CD3 Ab. Furthermore, ORAI1-deficient T cells showed enhanced survival after adoptive transfer into immunocompromised hosts. Thus, our results demonstrate a crucial role of the ORAI1-NFAT pathway in T cell death and highlight the important role of ORAI1 as a major route of Ca(2+) entry during activated T cell death.     

An mRNA Encoding a Putative GABA-Gated Chloride Channel Is Expressed in the Human Cardiac Conduction System

Abstract: GABA-gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABA_A receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full-length cDNA. The predicted protein has many of the features shared by other members of the ligand-gated ion channel family. This channel subunit has significant amino acid identity (25–40%) with members of GABA_A and GABA_C receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA _χ. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA _χ subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.     

Effects of Extracellular ATP on Fe2+-Induced Cytotoxicity in PC-12 Cells

Abstract: Extracellular ATP is known to cause a variety of changes, including the alteration of ion fluxes, cell growth, and other physiological activities. Recently, it has been suggested that ATP acts as an excitatory synaptic transmitter, which may produce a Ca²⁺ influx via the activation of a P_2y purinoceptor. Rat pheochromocytoma (PC-12) cells are known to resemble rat sensory neurons and to possess a P_2y purinoceptor. In this study, we demonstrated that extracellular ATP dose-dependently increased PC-12 cell death in the presence of ferrous ions. Voltage-sensitive calcium channel blockers and calpain and xanthine oxidase inhibitors were found to be effective at protecting PC-12 cells from Fe²⁺/ATP-induced lipid peroxidation and cell death. These results suggest that xanthine oxidase activation induced by calpains and subsequent free radical formation may be responsible for Fe²⁺/ATP-induced neuronal cell death.     

Neurotoxic unc-8 mutants encode constitutively active DEG/ENaC channels that are blocked by divalent cations

Ion channels of the DEG/ENaC family can induce neurodegeneration under conditions in which they become hyperactivated. The Caenorhabditis elegans DEG/ENaC channel MEC-4(d) encodes a mutant channel with a substitution in the pore domain that causes swelling and death of the six touch neurons in which it is expressed. Dominant mutations in the C. elegans DEG/ENaC channel subunit UNC-8 result in uncoordinated movement. Here we show that this unc-8 movement defect is correlated with the selective death of cholinergic motor neurons in the ventral nerve cord. Experiments in Xenopus laevis ooctyes confirm that these mutant proteins, UNC-8(G387E) and UNC-8(A586T), encode hyperactivated channels that are strongly inhibited by extracellular calcium and magnesium. Reduction of extracellular divalent cations exacerbates UNC-8(G387E) toxicity in oocytes. We suggest that inhibition by extracellular divalent cations limits UNC-8 toxicity and may contribute to the selective death of neurons that express UNC-8 in vivo.     

Characterization of a novel,dominant negative KCNJ2 mutation associated with Andersen-Tawil syndrome

Andersen-Tawil syndrome is characterized by periodic paralysis, ventricular ectopy, and dysmorphic features. Approximately 60% of patients exhibit loss-of-function mutations in KCNJ2, which encodes the inwardly rectifying K+ channel pore forming subunit Kir2.1. Here, we report the identification of a novel KCNJ2 mutation (G211T), resulting in the amino acid substitution D71Y, in a patient presenting with signs and symptoms of Andersen-Tawil syndrome. The functional properties of the mutant subunit were characterized using voltage-clamp experiments on transiently transfected HEK-293 cells and neonatal mouse ventricular myocytes. Whole-cell current recordings of transfected HEK-293 cells demonstrated that the mutant protein Kir2.1-D71Y fails to form functional ion channels when expressed alone, but co-assembles with wild-type Kir2.1 subunits and suppresses wild-type subunit function. Further analysis revealed that current suppression requires at least two mutant subunits per channel. The D71Y mutation does not measurably affect the membrane trafficking of either the mutant or the wild-type subunit or alter the kinetic properties of the currents. Additional experiments revealed that expression of the mutant subunit suppresses native IK1 in neonatal mouse ventricular myocytes. Simulations predict that the D71Y mutation in human ventricular myocytes will result in a mild prolongation of the action potential and potentially increase cell excitability. These experiments indicate that the Kir2.1-D71Y mutant protein functions as a dominant negative subunit resulting in reduced inwardly rectifying K+ current amplitudes and altered cellular excitability in patients with Andersen-Tawil syndrome.     

Deletion of nine carboxy-terminal residues of the Rubisco small subunit decreases thermal stability but does not eliminate function

A recent X-ray crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the green alga Chlamydomonas reinhardtii lacks 13 carboxy-terminal residues of the small subunit. To determine the importance of this divergent region, a non-sense mutation was created that removes nine residues. This engineered gene was transformed into a Chlamydomonas strain that lacks the small-subunit gene family. The resulting holoenzyme has a normal CO₂/O₂ specificity but decreased carboxylation V_max. Whereas wild-type enzyme retained most of its carboxylase activity after a 10-min incubation at 55°C, the mutant enzyme was inactivated. Thus, although disordered or divergent, the carboxy terminus is required for maximal activity and stability.     

Models of beta-amyloid ion channels in the membrane suggest that channel formation in the bilayer is a dynamic process

Here we model the Alzheimer beta-peptide ion channel with the goal of obtaining insight into the mechanism of amyloid toxicity. The models are built based on NMR data of the oligomers, with the universal U-shaped (strand-turn-strand) motif. After 30-ns simulations in the bilayer, the channel dimensions, shapes and subunit organization are in good agreement with atomic force microscopy (AFM). The models use the Abeta(17-42) pentamer NMR-based coordinates. Extension and bending of the straight oligomers lead to two channel topologies, depending on the direction of the curvature: 1), the polar/charged N-terminal beta-strand of Abeta(17-42) faces the water-filled pore, and the hydrophobic C-terminal beta-strand faces the bilayer (CNpNC; p for pore); and 2), the C-terminal beta-strand faces the solvated pore (NCpCN). In the atomistic simulations in a fully solvated DOPC lipid bilayer, the first (CNpNC) channel preserves the pore and conducts solvent; by contrast, hydrophobic collapse blocks the NCpCN channel. AFM demonstrated open pores and collapsed complexes. The final averaged CNpNC pore dimensions (outer diameter 8 nm; inner diameter approximately 2.5 nm) are in the AFM range (8-12 nm; approximately 2 nm, respectively). Further, in agreement with high-resolution AFM images, during the simulations, the channels spontaneously break into ordered subunits in the bilayer; however, we also observe that the subunits are loosely connected by partially disordered inner beta-sheet, suggesting subunit mobility in the bilayer. The cationic channel has strong selective affinity for Ca(2+), supporting experimental calcium-selective beta-amyloid channels. Membrane permeability and consequent disruption of calcium homeostasis were implicated in cellular degeneration. Consequently, the CNpNC channel topology can sign cell death, offering insight into amyloid toxicity via an ion "trap-release" transport mechanism. The observed loosely connected subunit organization suggests that amyloid channel formation in the bilayer is a dynamic, fluid process involving subunit association, dissociation, and channel rearrangements.