Upregulated Membrane Expression of a Conserved Voltage-

Gated Sodium Channel, Nav1.4a, and Electrical Organ Discharge in Electric Mouse, P. pikachu Melissa L. Amrein1, Charles Soong1*, and Ningjian Liang2 1 Department of Pathology and Laboratory Medicine, University of Brish Columbia, Vancouver, Brish Columbia, Canada 2 Department of Food, Nutrion, and Health, University of Brish Columbia, Vancouver, Brish Columbia, Canada

Abstract Electrocytes contain membrane proteins that allow the polarizaon of the plasma membrane, thereby allowing the generaon of in . It has been long established how electricity is generated in the electric , but recent studies found similar electrocytes to be acve in electric mice. We aimed to study the

basis behind electric discharge in a land . We found that the voltage-gated sodium channel, Nav1.4a, was expressed in electric organs of the electric mouse, Pokemon pikachu and the electric eel, Electrophorus

electricus. However, Nav1.4a was not expressed in the muscle cells of E. electricus while it was expressed in the muscle cells of P. pikachu and other rodents. We also found that P. pikachu and E. electricus shared similar amino acid substuons at the nonconserved region of this protein. Voltage-clamp technique gave insight on the much greater potenal differences generated by P. pikachu compared to electric eel and finally,

microscopy analysis revealed greater Nav1.4a numbers in P. pikachu, potenally correlang with aforemenoned greater electric potenal generaon, which perhaps lead to its capability to discharge electricity readily through air.

Citaon: Amrein ML, Soong C, and Liang N (2013) Upregulated expression of a conserved voltage-gated sodium channel, Nav1.4a, and electrical organ discharge in electric mouse, P. pikachu. PLoS Biol 11(3): e1001501. doi:10.1152/journal.p.bio.1001501 Academic Editor: Thomas Clausen, University of Toronto, Canada Received July 30, 2012; Accepted January 13, 2013; Published March 2, 2013 Copyright: © 2013 Amrein et al. This is an open-access arcle distributed under the terms of the Creave Commons Aribuon License, which permits unrestricted use, distribuon, and reproducon in any medium, provided the original author and source are credited. Funding: The authors acknowledge the Naonal Instute of Health (NIH) for grant NIH220251, Naonal and the Canadian Instutes of Health Research (CIHR) for grant CIHR0444921. The funders had no role in study design, data collecon and analysis, decision to publish, or preparaon of the manuscript. Compeng Interests: The authors have declared that no compeng interests exist Abbreviaons: EO, ; EOD, electric organ discharge; Nav1.4, voltage-gated sodium channel 4; qRT-PCR, quantave reverse transcriptase PCR; ELISA, enzyme-linked immunoadsorbant assay; BSA, bovine serum albumin. * E-mail: [email protected]

Introducon permeability along the way. From there, Voltage-gated Na+- permeable channels then evolved. Nav channels are The nervous and muscular systems developed shortly the basis for electrical excitability in animals [2]. aer the appearance of the first mulcellular organisms, The polarizaon of membranes is indeed key to approximately 650 million years ago [1] . In animals with a animals that generate electricity. Cells called electrocytes nervous system, the neurons generate acon potenals have evolved key muscle membrane proteins that (AP) and form circuits: furthermore, they possess the polarize the plasma membrane. The electric ssue in capability of releasing neurotransmiers, innervang these animals contain membrane proteins that are muscles and direcng behaviour [2]. The foundaon of homologous to those found in other excitable ssues, and electrical excitability in animals is built upon the voltage- especially in the electric eel where electric ssues are gated channel. The simplest forms, which include invaluable for studying membrane excitability. The potassium leak and voltage-dependent (K+) channels, electric eel, , is capable of appeared 3 billion years ago in bacteria and exist in all generang electrical potenal of up to 600 volts by means organisms today [3]. Voltage-gated ion channels provide a of its three well-defined electric organs. The main organ resng potenal and repolarize membranes that have generates high voltage discharges and runs from the been excited. These simple ion channels have, however, viscera of the eel down to the tail, while the adjacent evolved further from the six transmembrane (6TM) organs emit low electric charges and probably serve in helices K+ channels into 4x6TM channels, obtaining Ca2+ facilitang the eel’s sense of electrolocaon [4].

1 Nav1.4a Expression in P. pikachu

Author Summary higher potenal. We assume a similar set up for electric mice, except that the current circuit is closed by flowing Many species of are able to generate weak or strong electric discharges, either for communication or through the air. The lower conductance of air in electric for stunning predator or prey. The electric organ, made mice led us to study the key difference that allows of electrocytes, is responsible for generating electric Pikachu to do so. discharge. Electrocytes are thought to be derived from Over the past few decades it has been reported that neuronal and muscle cells. The voltage-gated sodium certain mouse strains are also capable of generang channel, Nav1.4a, is found to be absent in the muscle electricity in a similar fashion to the E. electricus [5]. The cells, but is highly expressed in the electric organs of most prominent of which is the species of mouse electric . In our study, we looked at Na 1.4a in a v Pokemon pikachu, which has already been the subject of species of mouse, P. pikachu, that also generates electricity, but through air instead of water. We several other studies. Ash Ketchum et al [6]. have shown compared this electric mouse with electric eel as well as the existence of electric organs in P. pikachu’s cheeks and tails, where stacks of electrocytes have been found. This nonelectric rodents. Here, we found that Nav1.4a is expressed in both the muscles and electric organs of P. has prompted our research group to further invesgate P. pikachu. In terms of the amino acid sequence, the pikachu’s electrocytes to look for a common ancestral line channel protein of P. pikachu was more similar to the from which mouse and separate. More specifically, electric eel rather than the rodents. We then observed we are interested in the Nav1.4 gene that is conserved in that P. pikachu possessed much greater numbers of the electric mouse and thus, we compare sequences to Nav1.4a and generated a much higher potential compared to the electric eel, which may explain its possibly find homologues between mouse and eel Nav1.4 ability to discharge electricity through air. gene and how the difference contribute to the gene’s funcon (especially regarding the fact that electric mouse is able to generate electricity on land). Further, we Columns of cells run the length of the main organ and are invesgate how the Pikachu electrocytes generate the separated by electrically insulang septa. Electrocytes more massive amounts of electricity in comparison to themselves are mulnucleated syncya, which are electrophorus electricus and we look at the voltage stacked one aer another to make up those producon by means of voltage-clamp technique. Finally, aforemenoned columns, thereby allowing the we are interested in comparing the cell morphology, using producon of pronounced fluctuaon in membrane confocal microscopy imaging, to provide an answer potenal. Figure 1 displays an example of how whether cell size plays a role in electric mice generang electrocytes are stacked and cumulavely produce a higher voltages.

Figure 1. Physiological basis of electric organ discharge (EOD). Electrocytes are stacked together in series. The simultaneous smulaon of the electrocytes allow the transcellular potenals to summate. One electrocyte in E. electricus produces a intracellular potenal of 150 mV. By stacking in series, potenals summate over the electrocytes. In this figure, three electrocytes of E. electricus build up to a cumulave transcellular potenal of 450 mV. For electric fish, the current flows in posterior to anterior direcon, and the circuit is closed by flowing out of the water, back to the tail. Derived from Bennet et al, with permission [4].

2 Nav1.4a Expression in P. pikachu

Results Nonconserved amino acid sequences in the

Nav1.4a of P. Pikachu are similar to the E. Nav1.4a is expressed in the muscles and EO of electricus variant P. pikachu Next, we compared the amino acid sequences of the

To assess whether Nav1.4a is also expressed in the EO Nav1.4a of the four species. Overall, the majority of the of P. Pikachu, we first measured its transcript levels of regions contained conserved amino acid sequences for

Nav1.4a in EO and muscles, and compared that with the the four species (data not shown). This is in agreement transcript levels in E. electricus (Fig. 2A). As we predicted, with previous studies, which have shown that a large like E. electricus, Nav1.4a was highly expressed in the EO poron of the protein contain conserved sequences [7]. It of P. Pikachu. Studies using ELISA are in agreement with has been suggested that evoluon acts to maintain this this finding (Fig. 2B). Previous studies have shown that conservaon, as amino acid substuons in many regions

Nav1.4a expression is lost in the muscles of many strains crical for fundamental properes of Na channels lead to of electric fish, presumably in exchange for its expression oen fatal neuromuscular diseases in human [8-9]. We in the their dedicated EO [7]. This is consistent with the zoomed in our analysis on four intracellular loops located low levels of Nav1.4 we observed at the transcript and in Domain II, Domain III, and between Domain III and IV protein levels of muscle ssues in E. electricus. (Fig. 3A). These loops have been shown to play roles in

Interesngly, while highly expressed in the EO, Nav1.4a of sodium channel inacvaon, a vital step in the cell P. Pikachu was expressed at a level comparable to that of electrophysiology and also a determinant step of rate of two other rodents, Raus novigicus and Mus musculus inacvaon [10].

(Fig. 2A). This suggests that phenotypically, Nav1.4a of P. The Glycine to Cysteine substuon in the S4-S5 loop pikachu resembles both electric fishes and rodents. in Domain II creates an addional disulfide interacon, which may affect the conformaonal changes of the sodium channel (Fig. 3B). For the S4-S5 loop in Domain III, A 15 an Asparagine residue is highly conserved for mammals, which forms part of the receptor for inacvaon. In both E. electricus and P. pikachu, this residue is replaced with 10 posively charged Arginine and Lysine. There is no evidence showing phenotypic consequences for 5 differences between an Arginine or a Lysine residue Substuon for the highly conserved Proline with Cysteine in the interdomain linker has been shown to 0 perturb channel inacvaon in rats [8]. Overall, the Relave mRNA expression Ee Mm Rn Pp amino acid sequence at the intracellular loops for P. Muscle EO pikachu resembled more of E. electricus than the rodents, which are the perceived closer relaves to pikachu. B 10 Individual electrocytes from P. pikachu 8 generates greater transcellular potenal

6 compared to E. electricus

4 Since Nav1.4a proteins between E. electricus and P. pikachu are similar in terms of amino acid sequence, we 2

Absorbance (450 nm) next sought to study the physiological property of individual electrocyte isolated from E. electricus (Fig. 4A) 0 and P. pikachu (Fig. 4B). Potenal Difference in individual Ee Mm Rn Pp electrocytes were analyzed. Two pieces of informaon Muscle EO were generated from Fig. 4. The first informaon was that Potenal Difference generated from P. pikachu Figure 2. Comparison of Nav1.4a expression in muscle and EO electrocyte was on average 865 mV which was 4.7 mes across four species. A. Relave Nav1.4a mRNA abundance measured by qRT-PCR. Each sample was normalized to beta- higher than what E. electricus electrocytes generated (150 mV). The second informaon is that there was a 10-fold acon. B. Relave abundance of Nav1.4a protein measured by ELISA. N=5. Ee, Electrophorus electricus; Mm, Mus musculus; Rn, difference in electric discharge duraon between P. Raus norvegicus; Pp, Pokemon pikachu. pikachu (0.01 s) and E. electricus (0.1 s). This data indicates that pikachu’s electrocytes generate higher elec-

3 Nav1.4a Expression in P. pikachu tric potenal difference in a higher frequency than E. electrocytes from E. electricus and Fig. 5B for P. pikachu electricus. aer immunostaining. The stained area were normalized with the nuclei counts for quanficaon. Electrocytes from P. pikachu had significantly greater amounts of Nav1.4a is highly expressed on the membranes of the electrocytes of P. pikachu compared to stained area per nuclei, indicang that at the single cell level, the electrocytes of P. pikachu contained more E. electricus membrane-bound Nav1.4a than E. electricus. We next sought to determine the cause for the greater transcellular potenal and frequency of EOD of P. pikachu Discussion compared to E. electricus. EO from both species were isolated and immunostained for Nav1.4a. Using confocal While the existence of mice, with EO, possessing the microscopy, the image secons containing ion channel- capabilies of generang electric discharges had been rich side of the membrane were analyzed. Fig. 5A shows idenfied decades earlier, the mechanism behind electric the representave confocal microscopy image of discharge through air was poorly understood. We began A A

B NILIKIICNSVGAL GMKVVVRALLGAIPS GGEDLFMTEEQ LGSKKAAKCIPRPSNVVQ Ee NILIKIICNSVGAL GMKVVVKALLGAIPS GGEDLFMTEEQ LGSKKAAKCIPRPSNVVL Pp NMLIKIIGNSVGAL GMRVVVNALLGAIPS GGKDIFMTEEQ LGSKKPQKPIPRPQNKIQ Mm NMLIKIIGNSVGAL GMRVVVNALLGAIPS GGKDIFMTEEQ LGSKKPQKPIPRPQNKIQ Rn

Figure 3. Comparison of interspecies amino acid sequence of Nav1.4a. A. Schemac drawing of the domains of Nav1.4a. B. Four variable regions of the pepde (S4-S5 linker of Domain II, S4-S5 linker of Domain III, two interdomain linker of Domains III-IV) were compared in greater detail. Ee, Electrophorus electricus; Mm, Mus musculus; Rn, Raus norvegicus; Pp, Pokemon pikachu.

A B

Figure 4. Time-dependent electric potenal difference in individual electrocytes. A. Measurement of potenal difference across electrocytes in E. electricus. B. Measurement of potenal difference across electrocytes in P. pikachu. Representave measurements from one electrocyte from each species are shown. Other data not shown. Measurements were repeated for 20 electrocytes per species.

4 Nav1.4a Expression in P. pikachu

rodent with gained ability to generate electricity. A The analysis of amino acid substuons confirmed that

the Nav1.4a channels in the EO of P. pikachu are more similar to other electric fish, and less similar to the rodent families. The amino acid substuons shared by P. pikachu and E. electricus are mostly at regions associated with channel inacvaon. This perhaps shapes the difference between the ion channels funconing as a mode for relaying informaon in the body versus a mode Ee Pp for generang electricity. The sequence similarity between the Nav1.4a in P. pikachu and E. electricus, however, would not explain how one is able to induce electric discharge over air while the B other is limited to water. Therefore, funconal studies on 10 individual electrocytes were performed to measure the differences in the potenal generated by EO in P. pikachu 7.5 and E. electricus. We have observed that the electrocytes for P. pikachu generated much greater trancellular potenal at a higher frequency. Immunostaining was 5 then performed to confirm that P. pikachu electrocytes have far greater abundance of Nav1.4a. It is possible that by having more sodium channels, a greater amount of 2.5 sodium enter the electrocytes once signaled, leading

Rao of stained area to a faster ion influx, resulng in higher frequency, as well

0 as greater ion influx, and resulng in higher potenal. The possible consequence of having electrocytes each Ee Pp generang a transcellular potenal of nearly 1 V is that, summed together, the enre EO of P. pikachu may Figure 5. Immunostaining of Nav1.4a in E. electricus and P. pikachu. A. Representave confocal microscope images of the generate up to thousands of voltages of electricity. It may two species at 20X magnificaon. Blue: nucleus; Red: Nav1.4a. be this high magnitude of voltage that allows this strain of B. Image-based quanficaon. Following color segmentaon, electric mouse to discharge electricity through air. the area of red was normalized to the number of nuclei present. Future work would revolve around confirming some of For the box plot, the lower whisker, lower box, upper box, and the hypotheses stated above. We will first need to st th uppwer whisker each represents the 1 to 4 quarle of the determine whether there is a direct correlaon between obtained data set. N=20. the number of Nav1.4a and the transcellular potenal. One way to test this is to treat electrocytes with small molecule inhibitors of sodium channels, at various our study focusing on a voltage-gated sodium channel dosages, and measure whether there are changes in protein, Nav1.4a, which was expressed in the EO of all potenal associated with the treatment. Secondly we will known marine electric organisms. We found that Nav1.4a need to study the physiology of the EO of P. pikachu as a was also expressed in the EO of P. pikachu, located at its whole. It is possible that the surrounding matrix, or cheeks and tail. Interesngly, while electric fishes have supporve ssues in P. pikachu are allowing for the lost the Nav1.4a expression in the muscle cells, P. pikachu generaon of greater potenal. Nevertheless, our work maintains its expression. Therefore from the standpoint has created a potenal link between the molecular of ssue-specific expression, P. pikachu shares both the biology of marine electric fish and an electric mouse. This characteriscs of an electric fish as well as its closely- may open new fields of research on electric mouse using related rodents. In the case of electric fish, by losing ion methods previously developed for electric fish. channel expression in muscle they have sacrificed some funcons of their muscle in return for the greater benefit of electrocommunicaon and aacking predators or preys through electric discharge. P. pikachu resides in areas where there is high compeon for physical fitness, required for constantly baling other species. Therefore, it is not illogical that P. pikachu has been selected to possess both phenotypes – maintaining the molity of a

5 Nav1.4a Expression in P. pikachu

Materials and Methods mM CaCl2, 2 mM MgCl2, 1.2 mM K2HPO4, 0.3 mM KH2PO4, 10 mM glucose, pH=7.1). The electrocytes is Nucleic acid extracon placed on the holder horizontally, innervated side down, and part of the innervated membrane, exposed through a Electric organs from the electric eel (Electrophorus window (3 X 0.7.5 mm), is in contact with a stream of electricus) and Pikachu (Pokemon pikachu) were fresh soluon. The rate of flow was such that the soluon extracted. Neighboring muscle ssues from brown rat bathing the innervated side could be changed in a few (Raus norvegicus), house mouse (Mus musculus), and seconds. Potenal differences across either membrane Pikachu were extracted. Total RNA were isolated from were measured between an intracellular glass muscles and electric organs in all species using the microelectrode filled with 3 mM KCl and agar bridges in miRNeasy mini kit (Qiagen). the outside soluons. Electric potenal was calculated through equaons established previously [11]. cDNA synthesis and qRT-PCR Random hexamers were used to reverse transcribe the extracted total RNA into cDNA using the SuperScript III Immunostaining and Confocal Microscopy Electrocytes from electricus and pikachu were mounted first-strand cDNA synthesis kit (Life Technologies). Real- onto glass sides and fixed with 0.4% paraformaldehyde. me PCR was performed with Na 1.4a-specific primer/ v 10% BSA-PBS was used to block the specimen for 20 min. probe set (Universal Probe Library #14, Roche), using the Taqman chemistry (Life Technologies). Beta-acn-specific Goat an-Nav1.4a anbody (Novarus) was applied at 1:100 at room temperature for 1 hour. Aer 3 x 10 min primer / probe set (Universal Probe Library #64, Roche) washes with PBS, Texas Red-conjugated rabbit an-goat was used as endogenous control. anbody was applied at 1:1000 at room temperature for 1

hour. DRAQ5 nuclear stain was applied at 1:10,000 for 10 ELISA Assays min prior to mounng the coverslip. Stained slides were Muscle ssues and electric organs were collected from imaged using a confocal microscope (Nikon). Z-stacks electric eel, mouse, rat, and Pikachu. Levels of Nav1.4a corresponding to different vercal secons of the protein in these ssues and organs were determined specimen were imaged. The Otsu’s image-based using ELISA assay kits from SABioscience according to the segmentaon method was used to quanfy stained nuclei manufacturer’s instrucons. and Nav1.4a area.

RNA-Seq and in amino acid sequence Acknowledgements The cDNA obtained from the previous step were also used for RNA-seq. Barcoded fragmented cDNA libraries were We thank Francisco Bargala, John Avise, and Georg prepared using the TruSeq RNA Sample Prep Kit (Illumina). Strieter for providing previous ssue materials from The libraries were then pooled and sequenced on a MiSeq Pikachu. Thank you also to Francisco Bargala for sequencer with MiSeq Reagent Kit v2 - 300 cycles Kit facilitang the dinner conversaon with excellent wines (Illumina). The obtained sequences are then from his vineyards. demulplexed and joined to recreate the full cDNA sequences of Nav1.4a. In silico translaon was performed Author Contribuons from the cDNA sequences, using ExPASy translate tool, to obtain the amino acid sequences for each species. The author(s) have made the following declaraons about their contribuons: Conceived and designed the Measurement of transcellular potenals experiments: MA CS NL. Performed the experiments MA NL. Analyzed the data: CS NL. Wrote the paper: MA CS Electrocytes were isolated and were mounted in a Lucite NL. holder in which the cell separates two pools of a modified

Ringer's soluon (containing 165 mM NaCl, 2.3 mM KCl, 2

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