Characterisation of Na(v) types endogenously expressed in human SH-SY5Y neuroblastoma cells.

The human neuroblastoma cell line SH-SY5Y is a potentially useful model for the identification and characterisation of Na(v) modulators, but little is known about the pharmacology of their endogenously expressed Na(v)s. The aim of this study was to determine the expression of endogenous Na(v) α and ß subunits in SH-SY5Y cells using PCR and immunohistochemical approaches, and pharmacologically characterise the Na(v) isoforms endogenously expressed in this cell line using electrophysiological and fluorescence approaches. SH-SY5Y human neuroblastoma cells were found to endogenously express several Na(v) isoforms including Na(v)1.2 and Na(v)1.7. Activation of endogenously expressed Na(v)s with veratridine or the scorpion toxin OD1 caused membrane depolarisation and subsequent Ca(2+) influx through voltage-gated L- and N-type calcium channels, allowing Na(v) activation to be detected with membrane potential and fluorescent Ca(2) dyes. µ-Conotoxin TIIIA and ProTxII identified Na(v)1.2 and Na(v)1.7 as the major contributors of this response. The Na(v)1.7-selective scorpion toxin OD1 in combination with veratridine produced a Na(v)1.7-selective response, confirming that endogenously expressed human Na(v)1.7 in SH-SY5Y cells is functional and can be synergistically activated, providing a new assay format for ligand screening.

Differential evolution of voltage-gated sodium channels in tetrapods and teleost fishes.

The voltage-gated sodium channel (SCN) alpha subunits are large proteins with central roles in the generation of action potentials. They consist of approximately 2,000 amino acids encoded by 24-27 exons. Previous evolutionary studies have been unable to reconcile the proposed gene duplication schemes with the species distribution and molecular phylogeny of the genes. We have carefully annotated the complete SCN gene sequences, correcting numerous database errors, for a broad range of vertebrate species and analyzed their phylogenetic relationships. We have also compared the chromosomal positions of the SCN genes relative to adjacent gene families. Our studies show that the ancestor of the vertebrates probably had a single sodium channel gene with two characteristic AT-AC introns, the second of which is unique to vertebrate SCN genes. This ancestral gene, located close to a HOX gene cluster, was quadrupled along with HOX in the two rounds of basal vertebrate tetraploidizations to generate the ancestors of the four channels SCN1A, SCN4A, SCN5A, and SCN8A. The third tetraploidization in the teleost fish ancestor doubled this set of genes and all eight are still present in at least three of four investigated teleost fish genomes. In tetrapods, the gene family expanded by local duplications before the radiation of amniotes, generating the cluster SCN5A, SCN10A, and SCN11A on one chromosome and the cluster SCN1A, SCN2A, SCN3A, and SCN9A on a different chromosome. In eutherian mammals, a tenth gene, SCN7A, arose in a local duplication in the SCN1A gene cluster. The SCN7A gene has undergone rapid evolution and has lost the ability to cause action potentials-instead, it functions as a sodium sensor. The three genes in the SCN5A cluster were translocated from the HOX-bearing chromosome in a mammalian ancestor along with several adjacent genes. This evolutionary scenario is supported by the adjacent TGF-ß receptor superfamily (comprised of five distinct families) and the cysteine-serine-rich nuclear protein gene family as well as the HOX clusters. The independent expansions of the SCN repertoires in tetrapods and teleosts suggest that the functional diversification may differ between the two lineages.

An emerging role for voltage-gated Na+ channels in cellular migration: regulation of central nervous system development and potentiation of invasive cancers.

Voltage-gated Na(+) channels (VGSCs) exist as macromolecular complexes containing a pore-forming alpha subunit and one or more beta subunits. The VGSC alpha subunit gene family consists of 10 members, which have distinct tissue-specific and developmental expression profiles. So far, four beta subunits (beta1-beta4) and one splice variant of beta1 (beta1A, also called beta1B) have been identified. VGSC beta subunits are multifunctional, serving as modulators of channel activity, regulators of channel cell surface expression, and as members of the immunoglobulin superfamily, cell adhesion molecules (CAMs). beta subunits are substrates of beta-amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase, yielding intracellular domains (ICDs) that may further modulate cellular activity via transcription. Recent evidence shows that beta1 regulates migration and pathfinding in the developing postnatal CNS in vivo. The alpha and beta subunits, together with other components of the VGSC signaling complex, may have dynamic interactive roles depending on cell/tissue type, developmental stage, and pathophysiology. In addition to excitable cells like nerve and muscle, VGSC alpha and beta subunits are functionally expressed in cells that are traditionally considered nonexcitable, including glia, vascular endothelial cells, and cancer cells. In particular, the alpha subunits are up-regulated in line with metastatic potential and are proposed to enhance cellular migration and invasion. In contrast to the alpha subunits, beta1 is more highly expressed in weakly metastatic cancer cells, and evidence suggests that its expression enhances cellular adhesion. Thus, novel roles are emerging for VGSC alpha and beta subunits in regulating migration during normal postnatal development of the CNS as well as during cancer metastasis.

Expression of AmphiNaC, a new member of the amiloride-sensitive sodium channel related to degenerins and epithelial sodium channels in amphioxus.

Degenerins and amiloride-sensitive Na+ channels form a new family of cationic ion channels (DEG/NaC). DEG/NaC family emerged as common denominator within a metazoan mechanosensory apparatus. In this study, we characterized a new member of such family in amphioxus, Branchiostoma floridae. The AmphiNaC cDNA sequence encodes a protein showing amino acid residues characteristic of DEG/NaC family, such as two hydrophobic domains surrounding a large extracellular loop that includes cystein-rich domains; nevertheless its predicted sequence is quite divergent from other family members. AmphiNaC is expressed at early larval stage in some putative sensory epidermal cells in the middle of the body and in neurons of the posterior cerebral vesicle, as well as in some ventrolateral and mediolateral neurons of the neural tube. In late larvae, AmphiNaC expression is maintained in some neurons of the neural tube, and it is expressed in putative sensory epidermal cells of rostrum and mouth. The analysis of AmphiNaC gene expression pattern suggests that it might be involved in neurotransmission and sensory modulation.

High-affinity potassium and sodium transport systems in plants.

All living cells have an absolute requirement for K+, which must be taken up from the external medium. In contrast to marine organisms, which live in a medium with an inexhaustible supply of K+, terrestrial life evolved in oligotrophic environments where the low supply of K+ limited the growth of colonizing plants. In these limiting conditions Na+ could substitute for K+ in some cellular functions, but in others it is toxic. In the vacuole, Na+ is not toxic and can undertake osmotic functions, reducing the total K+ requirements and improving growth when the lack of K+ is a limiting factor. Because of these physiological requirements, the terrestrial life of plants depends on high-affinity K+ uptake systems and benefits from high-affinity Na+ uptake systems. In plants, both systems have received extensive attention during recent years and a clear insight of their functions is emerging. Some plant HAK transporters mediate high-affinity K+ uptake in yeast, mimicking K+ uptake in roots, while other members of the same family may be K+ transporters in the tonoplast. In parallel with the HAK transporters, some HKT transporters mediate high-affinity Na+ uptake without cotransporting K+. HKT transporters have two functions: (i) to take up Na+ from the soil solution to reduce K+ requirements when K+ is a limiting factor, and (ii) to reduce Na+ accumulation in leaves by both removing Na+ from the xylem sap and loading Na+ into the phloem sap.

Tetrodotoxin-sensitive Na+ channels and muscarinic and purinergic receptors identified in human erythroid progenitor cells and red blood cell ghosts.

This article concerns the identification of different types of voltage-gated Na(+) channels and of muscarinic and purinergic receptors that are expressed in human erythroid precursor cells and red cell ghosts. We analyzed, by RT-PCR, RNA that was extracted from purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes in 7 to 14 days. These extracts were free of white cell and platelet contamination. Two types of voltage-gated, tetrodotoxin-sensitive Na(+) channels were found. These were Na(v)1.4 and Na(v)1.7, the former known to be present in skeletal muscle and the latter in peripheral nerve. By using a pan Na(+) channel antibody and Western blotting, an immunoreactive channel was detected in ghosts of human red blood cells, consistent with the expression of these two channels. The transcripts for four of the five known subtypes of muscarinic receptors were also identified, including subtypes M2, M3, M4, and M5, whereas subtype M1 was not found. Expression was also detected for the purinergic type receptors P2X(1), P2X(4), P2X(7), and P2Y(1) whereas types P2Y(2), P2Y(4), and P2Y(6) were not found. We also searched for but did not find transcripts for hBNP-1, a type 1b human brain sodium phosphate cotransporter, and cystic fibrosis transmembrane conductance regulator (CFTR). Implications regarding the presence of these different types of channels and receptors in human red blood cells and their functional significance are discussed.

Modulation of sodium currents in rat sensory neurons by nucleotides.

The effects of various nucleotides on the fast tetrodotoxin-sensitive (f-TTX-S) and the slow tetrodotoxin-resistant (s-TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were investigated using the patch-clamp technique. Nucleoside triphosphates (NTPs; ATP, GTP, UTP and CTP) and nucleoside diphosphates (NDPs; ADP, GDP, UDP and CDP) decreased f-TTX-S current, whereas they increased s-TTX-R current, when currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV. NTPs and NDPs shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction in both types of sodium currents. Most of them also increased the maximum conductance of s-TTX-R current. ITP, a derivative of ribonucleotide, and dTTP, a deoxyribonucleotide, modulated both types of sodium currents similarly to NTPs and NDPs. However, nucleoside monophosphates (NMPs; AMP, GMP, UMP and CMP) and adenosine had little or no effect on either type of sodium current. Therefore, it seems that nucleotides, regardless of the kind of base, should have two or more phosphates to be able to modulate sodium currents in DRG neurons. Extracellular nucleotides with di- or tri-phosphates would influence the perception by modulating sodium currents in sensory neurons. Particularly, the increase of the maximum conductance and the hyperpolarizing shift of the conductance-voltage relationship of s-TTX-R sodium current would result in an intensified nociception.

A delta-conotoxin from Conus ermineus venom inhibits inactivation in vertebrate neuronal Na+ channels but not in skeletal and cardiac muscles.

We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA inhibits Na+ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na+ channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na+ channels without acting on Na+ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na+ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na+ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation.

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.

The impact of phorbol ester on the regulation of amiloride-sensitive epithelial sodium channel in alveolar type ii epithelial cells.

Amiloride-sensitive sodium channel (ENaC) plays an important role in recovery from pulmonary edema. Recently, it has been shown that an activation of protein kinase C (PKC) could affect the mRNA expression of ENaC in rat parotid gland cells and A6 distal nephron epithelial cells. To determine whether an activation of PKC would regulate the mRNA expression or the function of ENaC, we stimulated rat alveolar type II epithelial cells with phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, at a concentration of 100 nM. The mRNA expression of alpha-, beta-, and gamma-ENaC subunits and amiloride-sensitive current were measured. PMA inhibited the mRNA expression of all 3 ENaC subunits (alpha-ENaC: 56.0% +/- 12.1%; beta-ENaC: 62.6% +/- 15.9%; gamma-ENaC: 68.5% +/- 10.6%, respectively) and amiloride-sensitive current (control = 7.0 +/- 1.5 microA/cm(2); PMA = 1.7 +/- 0.9 microA/cm(2)) significantly at 24 hours. On the other hand, 4alpha-phorbol didecanoate 4alpha-PDD, inactive form of PMA, had no inhibitory effect on alpha- and gamma-ENaC expression or amiloride-sensitive current. However, no significant difference was seen in beta-ENaC expression between PMA and 4alpha-PDD. GF 109203X, a wide-range PKC inhibitor, blocked the inhibitory effect of PMA on all ENaC subunits mRNA expression. These results suggest that an activation of PKC may play an important role in the regulation of ENaC mRNA expression and function.

Modular assembly of voltage-gated channel proteins: a sequence analysis and phylogenetic study.

Voltage-sensitive cation-selective ion channels of the voltage-gated ion channel (VGC) superfamily were examined by a combination of sequence alignment and phylogenetic tree construction procedures. Segments of the alpha-subunits of K+-selective channels homologous to the structurally elucidated KcsA channel of Streptomyces lividans were multiply aligned, and this alignment provided the database for computer-assisted structural analyses and phylogenetic tree construction. Similar analyses were conducted with the four homologous repeats of the alpha-subunits from representative Ca2+- and Na+-selective channels, as well as with the ensemble of K+, Ca2+ and Na+ channels. In both the single subunit of the K+ channels and the individual repeats of the Ca2+ and Na+ channels, the analyses suggest the occurrence of at least two tandemly arranged modules corresponding to the predicted voltage-sensor domain and the pore domain. The phylogenetic analyses reveal strict clustering of segments according to cation-selectivity and repeat unit. We surmise that the pore module of the prokaryotic K+ channel was the primordial polypeptide upon which other modules were superimposed during evolution in order to generate phenotypic diversity. These observations may prove applicable to all members of the VGC family yet to be discovered throughout the prokaryotic and eukaryotic kingdoms.

Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons.

Membrane excitability in different tissues is due, in large part, to the selective expression of distinct genes encoding the voltage-dependent sodium channel. Although the predominant sodium channels in brain, skeletal muscle, and cardiac muscle have been identified, the major sodium channel types responsible for excitability within the peripheral nervous system have remained elusive. We now describe the deduced primary structure of a sodium channel, peripheral nerve type 1 (PN1), which is expressed at high levels throughout the peripheral nervous system and is targeted to nerve terminals of cultured dorsal root ganglion neurons. Studies using cultured PC12 cells indicate that both expression and targeting of PN1 is induced by treatment of the cells with nerve growth factor. The preferential localization suggests that the PN1 sodium channel plays a specific role in nerve excitability.

Nonhydrolyzable analogues of GTP activate a new Na+ current in a rat mast cell line.

Whole-cell patch clamp experiments were performed to examine the effects of the nonhydrolyzable GTP analogue, guanosine 5'-3-O-(thio)triphosphate, on membrane currents in rat basophilic leukemia cells. Guanosine 5'-3-O-(thio)triphosphate activated an inward sodium current. This current had a new permeability sequence to monovalent cations and a different pharmacological profile to that of other characterized Na+ channels. Long hyperpolarizing steps revealed that the current declined during the pulse, and the decline was voltage-dependent. Activation of the current required Mg2+ and ATP. The nonhydrolyzable ATP analogues, adenosine 5'-O-(thio)triphosphate and adenosine 5'-(beta,gamma-imino)triphosphate, could not substitute for ATP. Soluble second messengers like cAMP, cGMP, inositol polyphosphates, and Ca2+ did not activate the Na+ current. These results suggest that nonhydrolyzable GTP analogues activate a Na+ current in rat basophilic leukemia cells that is new in terms of its selectivity, pharmacology, and activation mechanism. It may be the prototype for a new family of Na+ channels expressed in certain nonexcitable cells.

Phylogenetic characterization of the epithelial Na+ channel (ENaC) family.

Twenty-one sequenced protein members of the epithelial Na+ channel (ENaC) family have been identified and characterized in terms of their sizes, hydropathy profiles, sequence similarities and phylogenies. These proteins derive from mammals, the frog Xenopus laevis and the worm Caenorhabditis elegans. The eleven sequenced vertebrate proteins fall into four subfamilies designated alpha, beta, gamma and delta. The 10 C. elegans proteins do not cluster with the vertebrate proteins, and they all proved to be distantly related to each other. Nonetheless, the 21 ENaC proteins exhibit the same apparent topology, each with two transmembrane spanning segments separated by a large extracellular loop. All but two ENaC proteins possess highly conserved extracellular domains containing numerous conserved cysteine residues as well as adjacent C-terminal amphipathic transmembrane spanning segments, postulated to contribute to the formation of the hydrophillic pores of these oligomeric channel protein complexes. It is proposed that the well-conserved extracellular domains serve as receptors to control the activities of the channels. A topological model for the ENaC family proteins is presented.

Differential regulation of neuronal sodium channel expression by endogenous and exogenous tyrosine kinase receptors expressed in rat pheochromocytoma cells.

The biological activity of growth factors that act through receptor tyrosine kinases (RTKs) can differ dramatically, depending on both the properties of the RTKs and the cellular environment in which the RTKs are expressed. To determine the ability of different RTKs to elicit ras-independent responses central to neuronal differentiation, we analyzed voltage-dependent sodium (Na) channel expression in rat pheochromocytoma (PC12) cells after activation of a variety of endogenously and exogenously expressed RTKs. In PC12 cells expressing trkB (Ip et al., 1993), the increase in Na current density caused by brain-derived neurotrophic factor (BDNF) was similar to that observed upon activation of endogenous trkA by NGF. BDNF also increased type II Na channel mRNA expression, as did neurotrophin-3 in PC12 cells expressing trkC (Tsoulfas et al., 1993). In contrast, insulin did not increase type II Na channel mRNA expression or Na current density in PC12 cells, while epidermal growth factor (EGF) elicited small, yet reproducible increases in type II Na channel mRNA and Na current density when compared to NGF, even upon coexpression of an EGF receptor/p75 receptor chimera (Yan et al., 1991). Finally, in PC12 cells expressing beta-platelet-derived growth factor (PDGF) receptors (Heasley and Johnson, 1992), PDGF increased type II Na channel mRNA and Na current density to the same extent as NGF. The results show the capabilities of these RTKs in eliciting Na channel expression and the specificity arising due to differences in their intrinsic properties.