A potential cost of evolving epibatidine resistance in poison frogs.

BACKGROUND: Some dendrobatid poison frogs sequester the toxin epibatidine as a defense against predators. We previously identified an amino acid substitution (S108C) at a highly conserved site in a nicotinic acetylcholine receptor ß2 subunit of dendrobatid frogs that decreases sensitivity to epibatidine in the brain-expressing α4ß2 receptor. Introduction of S108C to the orthologous high-sensitivity human receptor similarly decreased sensitivity to epibatidine but also decreased sensitivity to acetylcholine, a potential cost if this were to occur in dendrobatids. This decrease in the acetylcholine sensitivity manifested as a biphasic acetylcholine concentration-response curve consistent with the addition of low-sensitivity receptors. Surprisingly, the addition of the ß2 S108C into the α4ß2 receptor of the dendrobatid Epipedobates anthonyi did not change acetylcholine sensitivity, appearing cost-free. We proposed that toxin-bearing dendrobatids may have additional amino acid substitutions protecting their receptors from alterations in acetylcholine sensitivity. To test this, in the current study, we compared the dendrobatid receptor to its homologs from two non-dendrobatid frogs. RESULTS: The introduction of S108C into the α4ß2 receptors of two non-dendrobatid frogs also does not affect acetylcholine sensitivity suggesting no additional dendrobatid-specific substitutions. However, S108C decreased the magnitude of neurotransmitter-induced currents in Epipedobates and the non-dendrobatid frogs. We confirmed that decreased current resulted from fewer receptors in the plasma membrane in Epipedobates using radiolabeled antibodies against the receptors. To test whether S108C alteration of acetylcholine sensitivity in the human receptor was due to (1) adding low-sensitivity binding sites by changing stoichiometry or (2) converting existing high- to low-sensitivity binding sites with no stoichiometric alteration, we made concatenated α4ß2 receptors in stoichiometry with only high-sensitivity sites. S108C substitutions decreased maximal current and number of immunolabeled receptors but no longer altered acetylcholine sensitivity. CONCLUSIONS: The most parsimonious explanation of our current and previous work is that the S108C substitution renders the ß2 subunit less efficient in assembling/trafficking, thereby decreasing the number of receptors in the plasma membrane. Thus, while ß2 S108C protects dendrobatids against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4ß2 receptor function.

Rapid evolution of a voltage-gated sodium channel gene in a lineage of electric fish leads to a persistent sodium current.

Most weakly electric fish navigate and communicate by sensing electric signals generated by their muscle-derived electric organs. Adults of one lineage (Apteronotidae), which discharge their electric organs in excess of 1 kHz, instead have an electric organ derived from the axons of specialized spinal neurons (electromotorneurons [EMNs]). EMNs fire spontaneously and are the fastest-firing neurons known. This biophysically extreme phenotype depends upon a persistent sodium current, the molecular underpinnings of which remain unknown. We show that a skeletal muscle-specific sodium channel gene duplicated in this lineage and, within approximately 2 million years, began expressing in the spinal cord, a novel site of expression for this isoform. Concurrently, amino acid replacements that cause a persistent sodium current accumulated in the regions of the channel underlying inactivation. Therefore, a novel adaptation allowing extreme neuronal firing arose from the duplication, change in expression, and rapid sequence evolution of a muscle-expressing sodium channel gene.

Convergence of ion channel genome content in early animal evolution.

Multicellularity has evolved multiple times, but animals are the only multicellular lineage with nervous systems. This fact implies that the origin of nervous systems was an unlikely event, yet recent comparisons among extant taxa suggest that animal nervous systems may have evolved multiple times independently. Here, we use ancestral gene content reconstruction to track the timing of gene family expansions for the major families of ion-channel proteins that drive nervous system function. We find that animals with nervous systems have broadly similar complements of ion-channel types but that these complements likely evolved independently. We also find that ion-channel gene family evolution has included large loss events, two of which were immediately followed by rounds of duplication. Ctenophores, cnidarians, and bilaterians underwent independent bouts of gene expansion in channel families involved in synaptic transmission and action potential shaping. We suggest that expansions of these family types may represent a genomic signature of expanding nervous system complexity. Ancestral nodes in which nervous systems are currently hypothesized to have originated did not experience large expansions, making it difficult to distinguish among competing hypotheses of nervous system origins and suggesting that the origin of nerves was not attended by an immediate burst of complexity. Rather, the evolution of nervous system complexity appears to resemble a slow fuse in stem animals followed by many independent bouts of gene gain and loss.

Convergent Substitutions in a Sodium Channel Suggest Multiple Origins of Toxin Resistance in Poison Frogs.

Complex phenotypes typically have a correspondingly multifaceted genetic component. However, the genotype-phenotype association between chemical defense and resistance is often simple: genetic changes in the binding site of a toxin alter how it affects its target. Some toxic organisms, such as poison frogs (Anura: Dendrobatidae), have defensive alkaloids that disrupt the function of ion channels, proteins that are crucial for nerve and muscle activity. Using protein-docking models, we predict that three major classes of poison frog alkaloids (histrionicotoxins, pumiliotoxins, and batrachotoxins) bind to similar sites in the highly conserved inner pore of the muscle voltage-gated sodium channel, Nav1.4. We predict that poison frogs are somewhat resistant to these compounds because they have six types of amino acid replacements in the Nav1.4 inner pore that are absent in all other frogs except for a distantly related alkaloid-defended frog from Madagascar, Mantella aurantiaca. Protein-docking models and comparative phylogenetics support the role of these replacements in alkaloid resistance. Taking into account the four independent origins of chemical defense in Dendrobatidae, phylogenetic patterns of the amino acid replacements suggest that 1) alkaloid resistance in Nav1.4 evolved independently at least seven times in these frogs, 2) variation in resistance-conferring replacements is likely a result of differences in alkaloid exposure across species, and 3) functional constraint shapes the evolution of the Nav1.4 inner pore. Our study is the first to demonstrate the genetic basis of autoresistance in frogs with alkaloid defenses.

Voltage-gated sodium channel gene repertoire of lampreys: gene duplications, tissue-specific expression and discovery of a long-lost gene.

Studies of the voltage-gated sodium (Nav) channels of extant gnathostomes have made it possible to deduce that ancestral gnathostomes possessed four voltage-gated sodium channel genes derived from a single ancestral chordate gene following two rounds of genome duplication early in vertebrates. We investigated the Nav gene family in two species of lampreys (the Japanese lamprey Lethenteron japonicum and sea lamprey Petromyzon marinus) (jawless vertebrates-agnatha) and compared them with those of basal vertebrates to better understand the origin of Nav genes in vertebrates. We noted six Nav genes in both lamprey species, but orthology with gnathostome (jawed vertebrate) channels was inconclusive. Surprisingly, the Nav2 gene, ubiquitously found in invertebrates and believed to have been lost in vertebrates, is present in lampreys, elephant shark (Callorhinchus milii) and coelacanth (Latimeria chalumnae). Despite repeated duplication of the Nav1 family in vertebrates, Nav2 is only in single copy in those vertebrates in which it is retained, and was independently lost in ray-finned fishes and tetrapods. Of the other five Nav channel genes, most were expressed in brain, one in brain and heart, and one exclusively in skeletal muscle. Invertebrates do not express Nav channel genes in muscle. Thus, early in the vertebrate lineage Nav channels began to diversify and different genes began to express in heart and muscle.

South American Weakly Electric Fish (Gymnotiformes) Are Long-Wavelength-Sensitive Cone Monochromats.

Losses of cone opsin genes are noted in animals that are nocturnal or rely on senses other than vision. We investigated the cone opsin repertoire of night-active South American weakly electric fish. We obtained opsin gene sequences from genomic DNA of 3 gymnotiforms (Eigenmannia virescens, Sternopygus macrurus, Apteronotus albifrons) and the assembled genome of the electric eel (Electrophorus electricus). We identified genes for long-wavelength-sensitive (LWS) and medium-wavelength-sensitive cone opsins (RH2) and rod opsins (RH1). Neither of the 2 short-wavelength-sensitive cone opsin genes were found and are presumed lost. The fact that Electrophorus has a complete repertoire of extraretinal opsin genes and conservation of synteny with the zebrafish (Danio rerio) for genes flanking the 2 short-wavelength-sensitive opsin genes supports the supposition of gene loss. With microspectrophotometry and electroretinograms we observed absorption spectra consistent with RH1 and LWS but not RH2 opsins in the retinal photoreceptors of E. virescens. This profile of opsin genes and their retinal expression is identical to the gymnotiform's sister group, the catfish, which are also nocturnally active and bear ampullary electroreceptors, suggesting that this pattern likely occurred in the common ancestor of gymnotiforms and catfish. Finally, we noted an unusual N-terminal motif lacking a conserved glycosylation consensus site in the RH2 opsin of gymnotiforms, a catfish and a characin (Astyanax mexicanus). Mutations at this site influence rhodopsin trafficking in mammalian photoreceptors and cause retinitis pigmentosa. We speculate that this unusual N terminus may be related to the absence of the RH2 opsin in the cones of gymnotiforms and catfish.

Ionic mechanisms of microsecond-scale spike timing in single cells.

Electric fish image their environments and communicate by generating electric organ discharges through the simultaneous action potentials (APs) of electric organ cells (electrocytes) in the periphery. Steatogenys elegans generates a biphasic electrocyte discharge by the precisely regulated timing and waveform of APs generated from two excitable membranes present in each electrocyte. Current-clamp recordings of electrocyte APs reveal that the posterior membrane fires first, followed ∼30 µs later by an AP on the anterior membrane. This delay was maintained even as the onset of the first AP was advanced >5 ms by increasing stimulus intensity and across multiple spikes during bursts of APs elicited by prolonged stimulation. Simultaneous cell-attached loose-patch recordings of Na(+) currents on each membrane revealed that activation voltage for Na(+) channels on the posterior membrane was 10 mV hyperpolarized compared with Na(+) channels on the anterior membrane, with no differences in activation or inactivation kinetics. Computational simulations of electrocyte APs demonstrated that this difference in Na(+) current activation voltage was sufficient to maintain the proper firing order and the interspike delay. A similar difference in activation threshold has been reported for the Na(+) currents of the axon initial segment compared with somatic Na(+) channels of pyramidal neurons, suggesting convergent evolution of spike initiation and timing mechanisms across different systems of excitable cells.

Expression evolution facilitated the convergent neofunctionalization of a sodium channel gene.

Ion channels have played a substantial role in the evolution of novel traits across all of the domains of life. A fascinating example of a novel adaptation is the convergent evolution of electric organs in the Mormyroid and Gymnotiform electric fishes. The regulated currents that flow through ion channels directly generate the electrical signals which have evolved in these fish. Here, we investigated how the expression evolution of two sodium channel paralogs (Scn4aa and Scn4ab) influenced their convergent molecular evolution following the teleost-specific whole-genome duplication. We developed a reliable assay to accurately measure the expression stoichiometry of these genes and used this technique to analyze relative expression of the duplicate genes in a phylogenetic context. We found that before a major shift in expression from skeletal muscle and neofunctionalization in the muscle-derived electric organ, Scn4aa was first downregulated in the ancestors of both electric lineages. This indicates that underlying the convergent evolution of this gene, there was a greater propensity toward neofunctionalization due to its decreased expression relative to its paralog Scn4ab. We investigated another derived muscle tissue, the sonic organ of Porichthys notatus, and show that, as in the electric fishes, Scn4aa again shows a radical shift in expression away from the ancestral muscle cells into the evolutionarily novel muscle-derived tissue. This study presents evidence that expression downregulation facilitates neofunctionalization after gene duplication, a pattern that may often set the stage for novel trait evolution after gene duplication.

Evolution of voltage-gated ion channels at the emergence of Metazoa.

Voltage-gated ion channels are large transmembrane proteins that enable the passage of ions through their pore across the cell membrane. These channels belong to one superfamily and carry pivotal roles such as the propagation of neuronal and muscular action potentials and the promotion of neurotransmitter secretion in synapses. In this review, we describe in detail the current state of knowledge regarding the evolution of these channels with a special emphasis on the metazoan lineage. We highlight the contribution of the genomic revolution to the understanding of ion channel evolution and for revealing that these channels appeared long before the appearance of the first animal. We also explain how the elucidation of channel selectivity properties and function in non-bilaterian animals such as cnidarians (sea anemones, corals, jellyfish and hydroids) can contribute to the study of channel evolution. Finally, we point to open questions and future directions in this field of research.

Adaptive evolution of voltage-gated sodium channels: the first 800 million years.

Voltage-gated Na(+)-permeable (Nav) channels form the basis for electrical excitability in animals. Nav channels evolved from Ca(2+) channels and were present in the common ancestor of choanoflagellates and animals, although this channel was likely permeable to both Na(+) and Ca(2+). Thus, like many other neuronal channels and receptors, Nav channels predated neurons. Invertebrates possess two Nav channels (Nav1 and Nav2), whereas vertebrate Nav channels are of the Nav1 family. Approximately 500 Mya in early chordates Nav channels evolved a motif that allowed them to cluster at axon initial segments, 50 million years later with the evolution of myelin, Nav channels "capitalized" on this property and clustered at nodes of Ranvier. The enhancement of conduction velocity along with the evolution of jaws likely made early gnathostomes fierce predators and the dominant vertebrates in the ocean. Later in vertebrate evolution, the Nav channel gene family expanded in parallel in tetrapods and teleosts (∼9 to 10 genes in amniotes, 8 in teleosts). This expansion occurred during or after the late Devonian extinction, when teleosts and tetrapods each diversified in their respective habitats, and coincided with an increase in the number of telencephalic nuclei in both groups. The expansion of Nav channels may have allowed for more sophisticated neural computation and tailoring of Nav channel kinetics with potassium channel kinetics to enhance energy savings. Nav channels show adaptive sequence evolution for increasing diversity in communication signals (electric fish), in protection against lethal Nav channel toxins (snakes, newts, pufferfish, insects), and in specialized habitats (naked mole rats).

Evolution of sodium channels predates the origin of nervous systems in animals.

Voltage-dependent sodium channels are believed to have evolved from calcium channels at the origin of the nervous system. A search of the genome of a single-celled choanoflagellate (the sister group of animals) identified a gene that is homologous to animal sodium channels and has a putative ion selectivity filter intermediate between calcium and sodium channels. Searches of a wide variety of animal genomes, including representatives of each basal lineage, revealed that similar homologs were retained in most lineages. One of these, the Placozoa, does not possess a nervous system. We cloned and sequenced the full choanoflagellate channel and parts of two placozoan channels from mRNA, showing that they are expressed. Phylogenetic analysis clusters the genes for these channels with other known sodium channels. From this phylogeny we infer ancestral states of the ion selectivity filter and show that this state has been retained in the choanoflagellate and placozoan channels. We also identify key gene duplications and losses and show convergent amino acid replacements at important points along the animal lineage.

A sodium-activated potassium channel supports high-frequency firing and reduces energetic costs during rapid modulations of action potential amplitude.

We investigated the ionic mechanisms that allow dynamic regulation of action potential (AP) amplitude as a means of regulating energetic costs of AP signaling. Weakly electric fish generate an electric organ discharge (EOD) by summing the APs of their electric organ cells (electrocytes). Some electric fish increase AP amplitude during active periods or social interactions and decrease AP amplitude when inactive, regulated by melanocortin peptide hormones. This modulates signal amplitude and conserves energy. The gymnotiform Eigenmannia virescens generates EODs at frequencies that can exceed 500 Hz, which is energetically challenging. We examined how E. virescens meets that challenge. E. virescens electrocytes exhibit a voltage-gated Na(+) current (I(Na)) with extremely rapid recovery from inactivation (τ(recov) = 0.3 ms) allowing complete recovery of Na(+) current between APs even in fish with the highest EOD frequencies. Electrocytes also possess an inwardly rectifying K(+) current and a Na(+)-activated K(+) current (I(KNa)), the latter not yet identified in any gymnotiform species. In vitro application of melanocortins increases electrocyte AP amplitude and the magnitudes of all three currents, but increased I(KNa) is a function of enhanced Na(+) influx. Numerical simulations suggest that changing I(Na) magnitude produces corresponding changes in AP amplitude and that K(Na) channels increase AP energy efficiency (10-30% less Na(+) influx/AP) over model cells with only voltage-gated K(+) channels. These findings suggest the possibility that E. virescens reduces the energetic demands of high-frequency APs through rapidly recovering Na(+) channels and the novel use of KNa channels to maximize AP amplitude at a given Na(+) conductance.

Phylogeny unites animal sodium leak channels with fungal calcium channels in an ancient, voltage-insensitive clade.

Proteins in the superfamily of voltage-gated ion channels mediate behavior across the tree of life. These proteins regulate the movement of ions across cell membranes by opening and closing a central pore that controls ion flow. The best-known members of this superfamily are the voltage-gated potassium, calcium (Ca(v)), and sodium (Na(v)) channels, which underlie impulse conduction in nerve and muscle. Not all members of this family are opened by changes in voltage, however. NALCN (NA(+) leak channel nonselective) channels, which encode a voltage-insensitive "sodium leak" channel, have garnered a growing interest. This study examines the phylogenetic relationship among Na(v)/Ca(v) voltage-gated and voltage-insensitive channels in the eukaryotic group Opisthokonta, which includes animals, fungi, and their unicellular relatives. We show that NALCN channels diverged from voltage-gated channels before the divergence of fungi and animals and that the closest relatives of NALCN channels are fungal calcium channels, which they functionally resemble.

A naturally occurring amino acid substitution in the voltage-dependent sodium channel selectivity filter affects channel gating.

The pore of sodium channels contains a selectivity filter made of 4 amino acids, D/E/K/A. In voltage sensitive sodium channel (Nav) channels from jellyfish to human the fourth amino acid is Ala. This Ala, when mutated to Asp, promotes slow inactivation. In some Nav channels of pufferfishes, the Ala is replaced with Gly. We studied the biophysical properties of an Ala-to-Gly substitution (A1529G) in rat Nav1.4 channel expressed in Xenopus oocytes alone or with a ß1 subunit. The Ala-to-Gly substitution does not affect monovalent cation selectivity and positively shifts the voltage-dependent inactivation curve, although co-expression with a ß1 subunit eliminates the difference between A1529G and WT. There is almost no difference in channel fast inactivation, but the ß1 subunit accelerates WT current inactivation significantly more than it does the A1529G channels. The Ala-to-Gly substitution mainly influences the rate of recovery from slow inactivation. Again, the ß1 subunit is less effective on speeding recovery of A1529G than the WT. We searched Nav channels in numerous databases and noted at least four other independent Ala-to-Gly substitutions in Nav channels in teleost fishes. Thus, the Ala-to-Gly substitution occurs more frequently than previously realized, possibly under selection for alterations of channel gating.

Old gene duplication facilitates origin and diversification of an innovative communication system--twice.

The genetic basis of parallel innovation remains poorly understood due to the rarity of independent origins of the same complex trait among model organisms. We focus on two groups of teleost fishes that independently gained myogenic electric organs underlying electrical communication. Earlier work suggested that a voltage-gated sodium channel gene (Scn4aa), which arose by whole-genome duplication, was neofunctionalized for expression in electric organ and subsequently experienced strong positive selection. However, it was not possible to determine if these changes were temporally linked to the independent origins of myogenic electric organs in both lineages. Here, we test predictions of such a relationship. We show that Scn4aa co-option and rapid sequence evolution were tightly coupled to the two origins of electric organ, providing strong evidence that Scn4aa contributed to parallel innovations underlying the evolutionary diversification of each electric fish group. Independent evolution of electric organs and Scn4aa co-option occurred more than 100 million years following the origin of Scn4aa by duplication. During subsequent diversification of the electrical communication channels, amino acid substitutions in both groups occurred in the same regions of the sodium channel that likely contribute to electric signal variation. Thus, the phenotypic similarities between independent electric fish groups are also associated with striking parallelism at genetic and molecular levels. Our results show that gene duplication can contribute to remarkably similar innovations in repeatable ways even after long waiting periods between gene duplication and the origins of novelty.

A potential cost of evolving epibatidine resistance in poison frogs.

Background: Some poison arrow frogs sequester the toxin epibatidine as a defense against predators. We previously identified a single amino acid substitution (S108C) at a highly conserved site in a neuronal nicotinic acetylcholine receptor (nAChR) ß2 subunit that prevents epibatidine from binding to this receptor. When placed in a homologous mammalian nAChR this substitution minimized epibatidine binding but also perturbed acetylcholine binding, a clear cost. However, in the nAChRs of poison arrow frogs, this substitution appeared to have no detrimental effect on acetylcholine binding and, thus, appeared cost-free. Results: The introduction of S108C into the α4ß2 nAChRs of non-dendrobatid frogs also does not affect ACh sensitivity, when these receptors are expressed in Xenopus laevis oocytes. However, α4ß2 nAChRs with C108 had a decreased magnitude of neurotransmitter-induced currents in all species tested ( Epipedobates anthonyi , non-dendrobatid frogs, as well as human), compared with α4ß2 nAChRs with the conserved S108. Immunolabeling of frog or human α4ß2 nAChRs in the plasma membrane using radiolabeled antibody against the ß2 nAChR subunit shows that C108 significantly decreased the number of cell-surface α4ß2 nAChRs, compared with S108. Conclusions: While S108C protects these species against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4ß2 nAChR function. These results may explain the high conservation of a serine at this site in vertebrates, as well as provide an example of a tradeoff between beneficial and deleterious effects of an evolutionary change. They also provide important clues for future work on assembly and trafficking of this important neurotransmitter receptor.

Expansion of voltage-dependent Na+ channel gene family in early tetrapods coincided with the emergence of terrestriality and increased brain complexity.

Mammals have ten voltage-dependent sodium (Nav) channel genes. Nav channels are expressed in different cell types with different subcellular distributions and are critical for many aspects of neuronal processing. The last common ancestor of teleosts and tetrapods had four Nav channel genes, presumably on four different chromosomes. In the lineage leading to mammals, a series of tandem duplications on two of these chromosomes more than doubled the number of Nav channel genes. It is unknown when these duplications occurred and whether they occurred against a backdrop of duplication of flanking genes on their chromosomes or as an expansion of ion channel genes in general. We estimated key dates of the Nav channel gene family expansion by phylogenetic analysis using teleost, elasmobranch, lungfish, amphibian, avian, lizard, and mammalian Nav channel sequences, as well as chromosomal synteny for tetrapod genes. We tested, and exclude, the null hypothesis that Nav channel genes reside in regions of chromosomes prone to duplication by demonstrating the lack of duplication or duplicate retention of surrounding genes. We also find no comparable expansion in other voltage-dependent ion channel gene families of tetrapods following the teleost-tetrapod divergence. We posit a specific expansion of the Nav channel gene family in the Devonian and Carboniferous periods when tetrapods evolved, diversified, and invaded the terrestrial habitat. During this time, the amniote forebrain evolved greater anatomical complexity and novel tactile sensory receptors appeared. The duplication of Nav channel genes allowed for greater regional specialization in Nav channel expression, variation in subcellular localization, and enhanced processing of somatosensory input.