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1.  Ancient association between cation leak channels and Mid1 proteins is conserved in fungi and animals 
Neuronal resting potential can tune the excitability of neural networks, affecting downstream behavior. Sodium leak channels (NALCN) play a key role in rhythmic behaviors by helping set, or subtly changing neuronal resting potential. The full complexity of these newly described channels is just beginning to be appreciated, however. NALCN channels can associate with numerous subunits in different tissues and can be activated by several different peptides and second messengers. We recently showed that NALCN channels are closely related to fungal calcium channels, which they functionally resemble. Here, we use this relationship to predict a family of NALCN-associated proteins in animals on the basis of homology with the yeast protein Mid1, the subunit of the yeast calcium channel. These proteins all share a cysteine-rich region that is necessary for Mid1 function in yeast. We validate this predicted association by showing that the Mid1 homolog in Drosophila, encoded by the CG33988 gene, is coordinately expressed with NALCN, and that knockdown of either protein creates identical phenotypes in several behaviors associated with NALCN function. The relationship between Mid1 and leak channels has therefore persisted over a billion years of evolution, despite drastic changes to both proteins and the organisms in which they exist.
doi:10.3389/fnmol.2014.00015
PMCID: PMC3945613  PMID: 24639627
Drosophila; Cch1; RNAi; Social Space Index; Circadian Rhythms; phenocopy
2.  Phylogeny Unites Animal Sodium Leak Channels with Fungal Calcium Channels in an Ancient, Voltage-Insensitive Clade 
Molecular Biology and Evolution  2012;29(12):3613-3616.
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 (Cav), and sodium (Nav) 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 Nav/Cav 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.
doi:10.1093/molbev/mss182
PMCID: PMC3494271  PMID: 22821012
NALCN; Cch1; maximum likelihood; pore motif
3.  Expansion of Voltage-dependent Na+ Channel Gene Family in Early Tetrapods Coincided with the Emergence of Terrestriality and Increased Brain Complexity 
Molecular Biology and Evolution  2010;28(4):1415-1424.
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.
doi:10.1093/molbev/msq325
PMCID: PMC3058772  PMID: 21148285
sodium channel; tetrapods; amniotes; terrestriality; gene duplication; brain
4.  Circadian and Social Cues Regulate Ion Channel Trafficking 
PLoS Biology  2009;7(9):e1000203.
Electric fish strengthen their communication signals nightly and during social encounters by rapidly trafficking ion channels into cell membranes, demonstrating a direct relationship between environmental stimuli, channel trafficking, and behavior.
Electric fish generate and sense electric fields for navigation and communication. These signals can be energetically costly to produce and can attract electroreceptive predators. To minimize costs, some nocturnally active electric fish rapidly boost the power of their signals only at times of high social activity, either as night approaches or in response to social encounters. Here we show that the gymnotiform electric fish Sternopygus macrurus rapidly boosts signal amplitude by 40% at night and during social encounters. S. macrurus increases signal magnitude through the rapid and selective trafficking of voltage-gated sodium channels into the excitable membranes of its electrogenic cells, a process under the control of pituitary peptide hormones and intracellular second-messenger pathways. S. macrurus thus maintains a circadian rhythm in signal amplitude and adapts within minutes to environmental events by increasing signal amplitude through the rapid trafficking of ion channels, a process that directly modifies an ongoing behavior in real time.
Author Summary
Excitable cells, such as neurons and muscle cells, control behavior by generating action potentials, electrical signals that propagate along the cell membrane. Action potentials are generated when the cell allows charged molecules (ions) such as sodium and potassium to move across the membrane through specialized proteins called ion channels. By changing the number of ion channels in the plasma membrane, excitable cells can rapidly remodel their functional characteristics, potentially causing changes in behavior. To gain an understanding of how environmental events cause the remodeling of excitable cell membranes and the resulting behavioral adaptations, we studied the electric communication/navigation signals of an electric fish, Sternopygus macrurus. High amplitude signals facilitate communication and electrolocation, but are energetically costly and more detectable by those predators that can detect electrical signals. We found that Sternopygus increase signal amplitude at night, when they are active, and increase signal amplitude rapidly during social encounters. Electrocytes, the cells that produce the signal, rapidly boost the signal amplitude when they allow more sodium to cross the cell membrane, thereby generating larger action potentials. To increase sodium currents during the action potential, electrocytes rapidly insert additional sodium channels into the cell membrane in response to hormones released into circulation by the pituitary. By adding new ion channels to the electrocyte membrane only during periods of activity or social encounters and removing these channels during inactive periods, these animals can save energy and reduce predation risks associated with communication.
doi:10.1371/journal.pbio.1000203
PMCID: PMC2741594  PMID: 19787026
5.  Toxin-Resistant Sodium Channels: Parallel Adaptive Evolution across a Complete Gene Family 
Molecular Biology and Evolution  2008;25(6):1016-1024.
Approximately 75% of vertebrate proteins belong to protein families encoded by multiple evolutionarily related genes, a pattern that emerged as a result of gene and genome duplications over the course of vertebrate evolution. In families of genes with similar or related functions, adaptation to a strong selective agent should involve multiple adaptive changes across the entire gene family. However, we know of no evolutionary studies that have explicitly addressed this point. Here, we show how 4 taxonomically diverse species of pufferfishes (Tetraodontidae) each evolved resistance to the guanidinium toxins tetrodotoxin (TTX) and saxitoxin (STX) via parallel amino acid replacements across all 8 sodium channels present in teleost fish genomes. This resulted in diverse suites of coexisting sodium channel types that all confer varying degrees of toxin resistance, yet show remarkable convergence among genes and phylogenetically diverse species. Using site-directed mutagenesis and expression of a vertebrate sodium channel, we also demonstrate that resistance to TTX/STX is enhanced up to 15-fold by single, frequently observed replacements at 2 sites that have not previously been implicated in toxin binding but show similar or identical replacements in pufferfishes and in distantly related vertebrate and nonvertebrate animals. This study presents an example of natural selection acting upon a complete gene family, repeatedly arriving at a diverse but limited number of adaptive changes within the same genome. To be maximally informative, we suggest that future studies of molecular adaptation should consider all functionally similar paralogs of the affected gene family.
doi:10.1093/molbev/msn025
PMCID: PMC2877999  PMID: 18258611
adaptation; parallel evolution; gene families; sodium channels; tetrodotoxin; saxitoxin
6.  A novel Na+ channel splice form contributes to the regulation of an androgen-dependent social signal 
Na+ channels are often spliced but little is known about the functional consequences of splicing. We have been studying the regulation of Na+ current inactivation in an electric fish model in which systematic variation in the rate of inactivation of the electric organ Na+ current shapes the electric organ discharge (EOD), a sexually-dimorphic, androgen-sensitive communication signal. Here we examine the relationship between a Na+ channel (Nav1.4b), which has two splice forms, and the waveform of the EOD. One splice form (Nav1.4bL) possesses a novel first exon that encodes a 51 amino acid N terminal extension. This is the first report of a Na+ channel with alternative splicing in the N terminal. This N terminal is present in zebrafish suggesting its general importance in regulating Na+ currents in teleosts. The extended N terminal significantly speeds fast inactivation, shifts steady state inactivation, and dramatically enhances recovery from inactivation, essentially fulfilling the functions of a β subunit. Both splice forms are equally expressed in muscle in electric fish and zebrafish but Nav1.4bL is the dominant form in the electric organ implying electric organ-specific transcriptional regulation. Transcript abundance of Nav1.4bL in the electric organ is positively correlated with EOD frequency and lowered by androgens. Thus, shaping of the EOD waveform involves the androgenic regulation of a rapidly inactivating splice form of a Na+ channel. Our results emphasize the role of splicing in the regulation of a vertebrate Na+ channel and its contribution to a known behavior.
doi:10.1523/JNEUROSCI.2783-08.2008
PMCID: PMC2615813  PMID: 18784298
androgen; electric organ; Neuroethology; oocyte; sodium channel; splice variant
7.  Regulation and modulation of electric waveforms in gymnotiform electric fish 
Weakly electric gymnotiform fish specialize in the regulation and modulation of the action potentials that make up their multi-purpose electric signals. To produce communication signals, gymnotiform fish modulate the waveforms of their electric organ discharges (EODs) over timescales spanning ten orders of magnitude within the animal's life cycle: developmental, reproductive, circadian, and behavioral. Rapid changes lasting milliseconds to seconds are the result of direct neural control of action potential firing in the electric organ. Intermediate-term changes taking minutes to hours result from the action of melanocortin peptides, the pituitary hormones that induce skin darkening and cortisol release in many vertebrates. Long-term changes in the EOD waveform taking days to weeks result from the action of sex steroids on the electrocytes in the electric organ as well as changes in the neural control structures in the brain. These long-term changes in the electric organ seem to be associated with changes in the expression of voltage-gated ion channels in two gene families. Electric organs express multiple voltage-gated sodium channel genes, at least one of which seems to be regulated by androgens. Electric organs also express multiple subunits of the shaker (Kv1) family of voltage-gated potassium channels. Expression of the Kv1 subtype has been found to vary with the duration of the waveform in the electric signal. Our increasing understanding of the mechanisms underlying precise control of electric communication signals may yield significant insights into the diversity of natural mechanisms available for modifying the performance of ion channels in excitable membranes. These mechanisms may lead to better understanding of normal function in a wide range of physiological systems and future application in treatment of disease states involving pathology of excitable membranes.
doi:10.1007/s00359-006-0101-1
PMCID: PMC2430267  PMID: 16437223
Androgens; Electrogenesis; Phenotypic plasticity
8.  Isolation and Characterization of CvIV4: A Pain Inducing α- Scorpion Toxin 
PLoS ONE  2011;6(8):e23520.
Background
Among scorpion species, the Buthidae produce the most deadly and painful venoms. However, little is known regarding the venom components that cause pain and their mechanism of action. Using a paw-licking assay (Mus musculus), this study compared the pain-inducing capabilities of venoms from two species of New World scorpion (Centruroides vittatus, C. exilicauda) belonging to the neurotoxin-producing family Buthidae with one species of non-neurotoxin producing scorpion (Vaejovis spinigerus) in the family Vaejovidae. A pain-inducing α-toxin (CvIV4) was isolated from the venom of C. vittatus and tested on five Na+ channel isoforms.
Principal Findings
C. vittatus and C. exilicauda venoms produced significantly more paw licking in Mus than V. spinigerus venom. CvIV4 produced paw licking in Mus equivalent to the effects of whole venom. CvIV4 slowed the fast inactivation of Nav1.7, a Na+ channel expressed in peripheral pain-pathway neurons (nociceptors), but did not affect the Nav1.8-based sodium currents of these neurons. CvIV4 also slowed the fast inactivation of Nav1.2, Nav1.3 and Nav1.4. The effects of CvIV4 are similar to Old World α-toxins that target Nav1.7 (AahII, BmK MI, LqhIII, OD1), however the primary structure of CvIV4 is not similar to these toxins. Mutant Nav1.7 channels (D1586A and E1589Q, DIV S3–S4 linker) reduced but did not abolish the effects of CvIV4.
Conclusions
This study: 1) agrees with anecdotal evidence suggesting that buthid venom is significantly more painful than non-neurotoxic venom; 2) demonstrates that New World buthids inflict painful stings via toxins that modulate Na+ channels expressed in nociceptors; 3) reveals that Old and New World buthids employ similar mechanisms to produce pain. Old and New World α-toxins that target Nav1.7 have diverged in sequence, but the activity of these toxins is similar. Pain-inducing toxins may have evolved in a common ancestor. Alternatively, these toxins may be the product of convergent evolution.
doi:10.1371/journal.pone.0023520
PMCID: PMC3160894  PMID: 21887265

Results 1-8 (8)