Evolution and divergence of sodium channel genes in vertebrates

Invertebrate species possess one or two Na+ channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na+ channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na+ channel gene followed by tandem duplications of Na+ channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na+ channel genes in a teleost fish. Using an approach designed to clone all of the Na+ channel genes in a genome, we found six Na+ channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na+ channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na+ channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na+ channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na+ channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than {D[A(BC)]}.

Antillatoxin is a marine cyanobacterial toxin that potently activates voltage-gated sodium channels

Antillatoxin (ATX) is a lipopeptide derived from the pantropical marine cyanobacterium Lyngbya majuscula. ATX is neurotoxic in primary cultures of rat cerebellar granule cells, and this neuronal death is prevented by either N-methyl-d-aspartate (NMDA) receptor antagonists or tetrodotoxin. To further explore the potential interaction of ATX with voltage-gated sodium channels, we assessed the influence of tetrodotoxin on ATX-induced Ca2+ influx in cerebellar granule cells. The rapid increase in intracellular Ca2+ produced by ATX (100 nM) was antagonized in a concentration-dependent manner by tetrodotoxin. Additional, more direct, evidence for an interaction with voltage-gated sodium channels was derived from the ATX-induced allosteric enhancement of [3H]batrachotoxin binding to neurotoxin site 2 of the α subunit of the sodium channel. ATX, moreover, produced a strong synergistic stimulation of [3H]batrachotoxin binding in combination with brevetoxin, which is a ligand for neurotoxin site 5 on the voltage-gated sodium channel. Positive allosteric interactions were not observed between ATX and either α-scorpion toxin or the pyrethroid deltamethrin. That ATX interaction with voltage-gated sodium channels produces a gain of function was demonstrated by the concentration-dependent and tetrodotoxin-sensitive stimulation of 22Na+ influx in cerebellar granule cells exposed to ATX. Together these results demonstrate that the lipopeptide ATX is an activator of voltage-gated sodium channels. The neurotoxic actions of ATX therefore resemble those of brevetoxins that produce neural insult through depolarization-evoked Na+ load, glutamate release, relief of Mg2+ block of NMDA receptors, and Ca2 + influx.

Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons.

Recent evidence indicates that long-chain polyunsaturated fatty acids (PUFAs) can prevent cardiac arrhythmias by a reduction of cardiomyocyte excitability. This was shown to be due to a modulation of the voltage-dependent inactivation of both sodium (INa) and calcium (ICa) currents. To establish whether PUFAs also regulate neuronal excitability, the effects of PUFAs on INa and ICa were assessed in CA1 neurons freshly isolated from the rat hippocampus. Extracellular application of PUFAs produced a concentration-dependent shift of the voltage dependence of inactivation of both INa and ICa to more hyperpolarized potentials. Consequently, they accelerated the inactivation and retarded the recovery from inactivation. The EC50 for the shift of the INa steady-state inactivation curve was 2.1 +/- 0.4 microM for docosahexaenoic acid (DHA) and 4 +/- 0.4 microM for eicosapentaenoic acid (EPA). The EC50 for the shift on the ICa inactivation curve was 2.1 +/- 0.4 for DHA and > 15 microM for EPA. Additionally, DHA and EPA suppressed both INa and ICa amplitude at concentrations > 10 microM. PUFAs did not affect the voltage dependence of activation. The monounsaturated oleic acid and the saturated palmitic acid were virtually ineffective. The combined effects of the PUFAs on INa and ICa may reduce neuronal excitability and may exert anticonvulsive effects in vivo.

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.

The heart communicates with the kidney exclusively through the guanylyl cyclase-A receptor: acute handling of sodium and water in response to volume expansion.

Disruption of guanylyl cyclase-A (GC-A) results in mice displaying an elevated blood pressure, which is not altered by high or low dietary salt. However, atrial natriuretic peptide (ANP), a proposed ligand for GC-A, has been suggested as critical for the maintenance of normal blood pressure during high salt intake. In this report, we show that infusion of ANP results in substantial natriuresis and diuresis in wild-type mice but fails to cause significant changes in sodium excretion or urine output in GC-A-deficient mice. ANP, therefore, appears to signal through GC-A in the kidney. Other natriuretic/diuretic factors could be released from the heart. Therefore, acute volume expansion was used as a means to cause release of granules from the atrium of the heart. That granule release occurred was confirmed by measurements of plasma ANP concentrations, which were markedly elevated in both wild-type and GC-A-null mice. After volume expansion, urine output as well as urinary sodium and cyclic GMP excretion increased rapidly and markedly in wild-type mice, but the rapid increases were abolished in GC-A-deficient animals. These results strongly suggest that natriuretic/diuretic factors released from the heart function exclusively through GC-A.

Expression of a renal type I sodium/phosphate transporter (NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions.

Two distinct molecular types (I and II) of renal proximal tubular brush border Na+/Pi cotransporters have been identified by expression cloning on the basis of their capacity to induce Na+-dependent Pi influx in tracer experiments. Whereas the type II transporters (e.g., NaPi-2 and NaPi-3) resemble well known characteristics of brush border Na+/Pi cotransport, little is known about the properties of the type I transporter (NaPi-1). In contrast to type II, type I transporters produced electrogenic transport only at high extracellular Pi concentrations (> or =3 mM). On the other hand, expression of NaPi-1 induced a Cl- conductance in Xenopus laevis oocytes, which was inhibited by Cl- channel blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > niflumic acid >> 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid]. Further, the Cl- conductance was inhibited by the organic anions phenol red, benzylpenicillin (penicillin G), and probenecid. These organic anions induced outwardly directed currents in the absence of Cl-. In tracer studies, we observed uptake of benzylpenicillin with a Km of 0.22 mM; benzylpenicillin uptake was inhibited by NPPB and niflumic acid. These findings suggest that the type I Na+/Pi cotransporter functions also as a novel type of anion channel permeable not only for Cl- but also for organic anions. Such an apical anion channel could serve an important role in the transport of Cl- and the excretion of anionic xenobiotics.

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.

Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity.

Previously, we developed a rat model of persistent mitochondrial dysfunction based upon the chronic partial inhibition of the mitochondrial enzyme cytochrome oxidase (EC 1.9.3.1). Continuous systemic infusion of sodium azide at approximately 1 mg/kg per hr inhibited cytochrome oxidase activity and produced a spatial learning deficit. In other laboratories, glucocorticoids have been reported to exacerbate neuronal damage from various acute metabolic insults. Therefore, we tested the hypothesis that corticosterone, the primary glucocorticoid in the rat, would potentiate the sodium azide-induced learning deficit. To this end, we first identified nonimpairing doses of sodium azide (approximately 0.75 mg/kg per hr) and corticosterone (100-mg pellet, 3-week sustained-release). We now report that chronic co-administration of these individually nonimpairing treatments produced a severe learning deficit. Moreover, the low dose of corticosterone, which did not elevate serum corticosterone, acted synergistically with sodium azide to inhibit cytochrome oxidase activity. The latter result represents a previously unidentified effect of glucocorticoids that provides a candidate mechanism for glucocorticoid potentiation of neurotoxicity induced by metabolic insult. These results may have the clinical implication of expanding the definition of hypercortisolism in patient populations with compromised oxidative metabolism. Furthermore, they suggest that glucocorticoid treatment may contribute to pathology in disease or trauma conditions that involve metabolic insult.

Sodium: a male moth's gift to its offspring.

Males of the moth Gluphisia septentrionis acquire sodium by drinking from mud puddles. Analyses of male and female bodies indicate that such "puddling" behavior enables the male to provide his mate with a nuptial gift of sodium, presumably via the spermatophore. This gift (about 10 microg), amounting to more than half of a puddler male's total body sodium, is in large measure apportioned by the female to her eggs. Puddler-sired eggs contain 2 to 4 times more sodium than those control-sired; this difference is already apparent in eggs laid the night after mating. Paternal endowment of offspring with sodium had not previously been demonstrated for an insect to our knowledge. The potential adaptive significance of such chemical bestowal is evident, given that the foliar diet of G. septentrionis larvae is extremely low in sodium content.

Structure of the sodium channel pore revealed by serial cysteine mutagenesis.

The pores of voltage-gated cation channels are formed by four intramembrane segments that impart selectivity and conductance. Remarkably little is known about the higher order structure of these critical pore-lining or P segments. Serial cysteine mutagenesis reveals a pattern of side-chain accessibility that contradicts currently favored structural models based on alpha-helices or beta-strands. Like the active sites of many enzymes of known structure, the sodium channel pore consists of irregular loop regions.

A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the beta or gamma subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of beta and gamma subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the beta subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.

Cloning of a sodium channel alpha subunit from rabbit Schwann cells.

Overlapping cDNA clones spanning the entire coding region of a Na-channel alpha subunit were isolated from cultured Schwann cells from rabbits. The coding region predicts a polypeptide (Nas) of 1984 amino acids exhibiting several features characteristic of Na-channel alpha subunits isolated from other tissues. Sequence comparisons showed that the Nas alpha subunit resembles most the family of Na channels isolated from brain (approximately 80% amino acid identity) and is least similar (approximately 55% amino acid identity) to the atypical Na channel expressed in human heart and the partial rat cDNA, NaG. As for the brain II and III isoforms, two variants of Nas exist that appear to arise by alternative splicing. The results of reverse transcriptase-polymerase chain reaction experiments suggest that expression of Nas transcripts is restricted to cells in the peripheral and central nervous systems. Expression was detected in cultured Schwann cells, sciatic nerve, brain, and spinal cord but not in skeletal or cardiac muscle, liver, kidney, or lung.

Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats.

We investigated the cellular and molecular events associated with the increase in sodium transport across the alveolar epithelium of rats exposed to hyperoxia (85% O2 for 7 days followed by 100% O2 for 4 days). Alveolar type II (ATII) cell RNA was isolated and probed with a cDNA for one of the rat colonic epithelial sodium channel subunits (alpha rENaC). The alpha rENaC mRNA (3.7-kb transcript) increased 3-fold in ATII cell RNA isolated from rats exposed to 85% O2 for 7 days and 6-fold after 4 days of subsequent exposure to 100% O2. In situ hybridization revealed increased expression of alpha rENaC mRNA transcripts in both airway and alveolar epithelial cells of hyperoxic rats. When immunostained with a polyclonal antibody to kidney sodium channel protein, ATII cells from hyperoxic rats exhibited a significant increase in the amount of immunogenic protein present in both the plasma membrane and the cytoplasm. When patched in the whole-cell mode, ATII cells from hyperoxic rats exhibited amiloride and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (EIPA)-sensitive currents that were 100% higher compared with those obtained from air-breathing rats. Single-channel sodium currents (mean conductance of 25 pS) were seen in ATII cells patched in both the inside-out and cell-attached modes. The number and open probability of these channels increased significantly during exposure to hyperoxia. Exposure to sublethal hyperoxia up-regulated both alpha rENaC mRNA and the functional expression of sodium channels in ATII cells.