Ionic gradients across cell membranes (bioelectricity) are utilized by all organisms. Some fish have developed extreme adaptations of bioelectricity with the evolution of electric organ systems. It is thought that electric organs have evolved independently six or seven times in fish and can be classified as either weak or strong, which is reflective of the size and function of the organs within the fish. For example, Gymnotids are weakly electrogenic and only possess accessory electric organs used for electroreception and electrolocation . In contrast, Torpedinid and Electrophorous are strongly electrogenic and possess organs that account for approximately one-third of the organism's mass and are used for generation of electric shocks for predation or protection .
Developmental studies have shown most electric organs are derived from muscle anlage tissue; the exception is the neurogenic development of the Sternarcus electric organ. Several basic differences exist amongst myogenic-derived electric organs. The location of the myogenic-derived electric organs varies from gill (Torpedo), tail (Raja, Gnathonemus, Gymnarchus, Gymnotus), and ocular muscle (Astroscopus). Strong electrogenic organs lose the characteristic myofibrils and sarcomeres during transdifferentiation of the organ. In contrast, weakly electrogenic Gymnarchids and Mormyrids maintain the myofibrillar structures into adulthood . Organs differ in the ability to initiate and propagate an action potential. Generally, marine fish possess organs with electrically inexcitable membranes (lacking voltage-sensitive sodium channels), whereas fresh water fish have organs that are electrically excitable (have voltage-sensitive sodium channels). Succinctly put, the degree of muscle likeness of precursor cells differs among electrogenic fish families. These anatomical differences may represent an evolutionary divergence required for the performance of strong and weak electric organs.
The research presented here focuses on Torpedo californica (Pacific electric ray), a cartilaginous fish within the Chondrichthyes class and Torpedinidae family. This species evolved an electric organ capable of generating approximately 45-50 V (electron motive force 110 mV), released in 414 monophasic discharges that last 3-5 ms each, with a total power output up to 1 kW [4–6]. An electrocyte from the electric organ of Torpedo nobiliana (Atlantic Torpedo with similar length but twice the weight of T. californica) measures 5-7 mm in diameter by 10-30 μm thick and 500-1,000 electrocytes are stacked into columns, all with ventrally innervated and dorsally non-innervated membranes aligned . Approximately 50 A of current has been measured from the parallel stacks composing the electric organ of T. nobiliana, and about 1 A measured from the series-aligned electrocytes of Electrophorous. The postsynaptic membrane of the electric organ in Torpedo is rich in nicotinic acetylcholine receptors (AChR) and is multi-innervated with dendrites from four large, heavily myelinated neurons descending from the electric lobe of the brain. The non-innervated membrane is extensively invaginated into structures called caniculi that may be reminiscent of skeletal muscle T tubules . The electrocytes are multinucleated and filled with a gelatinous cytoplasm with an extensive filamentous network. The electrocyte itself has low internal resistance with low resistance across the non-innervated membrane . Insulating septa, extracellular matrix components, blood vessels, nerves, and amoeboid cells have also been described in intercellular regions .
Proteins that were originally identified in the Torpedo electric organ and subsequently studied in higher vertebrates include agrin, dynein, chloride channel, and rapsyn [9–12]. Also identified in the electric organ are α, β, δ, and γ AChR subunits, α and β dystroglycan, dystrophin, syntrophin, dystrobrevin, receptor tyrosine kinase, tyrosine protein kinase fyn, protein tyrosine kinase fyk, and desmin [13–23]. The electric organ has been used to define the structure and function of creatine kinase and AChR pore [24, 25]. These proteins also are characterized at the mammalian neuromuscular junction (NMJ) or are components of skeletal muscle, which is consistent with the Torpedo electric organ representing an extreme adaptation of muscle tissue and the NMJ. Thus, the electric organ has served as a model to study the NMJ. However, the number of NMJ proteins described in current mouse, cell culture, and Drosophila studies demands a closer look at how the innervated membrane of Torpedo electrocytes relates to the NMJ.
From a developmental perspective, Torpedo electroblasts are derived from the mesodermal layer that gives rise to branchial arches from which the electric organ and gill musculature form. The primordial electric organ first generates 'muscle-like' cells that are multinucleated and have a single striated myofibril, reminiscent of myotubes in skeletal muscle. At this stage, meromyosin is expressed at high levels and the single striated myofibril has a similar diameter to actin-myosin myofibrilar structures composing sarcomeres . As the electroblast transforms into an electrocyte at the onset of electromotor neuron synaptogenesis, Z-disc-like structures disassemble and degenerate completely . It is thought that the electromotor neuron sends signals that induce the degeneration of the myofibril structures, allowing the elongated cells to flatten into thin electrocytes . Desmin, or a light intermediate filament, replaces the myofibril following disassembly, but keratin, a protein typically associated with epithelium, dominates the intracellular architecture [26, 29]. Upon denervation, myofibril-like structures reappear near the synapse but are highly disorganized and short lived . In addition, transcript evidence was shown for myoblast determination protein and myogenic factor 5 expression in adult Torpedo electric organ without evidence of protein expression, suggesting strong post-transcriptional regulation of messenger RNA translation and maintenance of a muscle-like programming . No synapse is observed until late phase of electric organ development when the ventral face of electroblasts develop subneural arches that have increased levels of acetylcholinesterase (AChE) and AChRs that reach 300 times the level in skeletal muscle [27, 28, 30].
From an anatomical perspective, post-transdifferentiation, the electroneuroelectrocyte synapse (electroplate) appears to maintain characteristic synaptic folds and a high density of membrane particles as revealed by electron microscopy and freeze-etch replicas of electric organ tissue [5, 31, 32]. However, the extensive nerve terminal network, formed by four or five electromotor neurons covers nearly the entire postsynaptic membrane, differs from the minute motor neuron connection with a single mammalian myofiber [5, 18].
Despite the electric organ being used as a model for the mammalian NMJ, current literature describes a number of NMJ-associated proteins that have not been characterized in the electric organ. One such protein is low-density lipoprotein receptor-related protein 4 (Lrp4), which forms a complex with muscle-specific tyrosine-protein kinase receptor (MuSK) to facilitate neuronal agrin binding and subsequent initiation of downstream signaling for transcriptional activation of synaptic genes or AChR clustering [33, 34]. It is likely that agrin plays a similar role in the electric organ as in the NMJ, transferring communication between the nerve and postsynaptic tissue, but its downstream target, MuSK, is loosely defined. The published sequence for a tyrosine kinase receptor transcript extracted from the Torpedo electric organ not only encodes extracellular Ig and frizzled domains and intracellular C-terminal tyrosine kinase domains like human MuSK but also encodes a kringle-like domain that is encoded in proteases and Ror receptor tyrosine kinases [20, 35, 36]. The orthology of the Torpedo tyrosine kinase receptor with mammalian MuSK was demonstrated by inducing AChR clustering in the presence and absence of agrin . Furthermore, the cytoplasmic domain of MuSK binds directly to the tetratricopeptide repeat domain of rapsyn, supporting the presence of MuSK and possibly its downstream effectors in the electric organ .
Aside from this knowledge, electrocyte components are undefined mainly because studying the proteome of T. californica is limited. A map of its genome does not currently exist to computationally derive a hypothetical protein profile and public databases contain sparse sequence data for this species. While the Torpedo genome has not yet been reported, the genome sequence likely would be a relatively blunt instrument to understand the highly specialized structure and function of the Torpedo electric organ. For this reason, we sought to understand the molecular components of the electric organ using a combined mRNA (expressed sequence tag (EST)) and proteomics approach.
We have previously reported a preliminary proteome based on two-dimensional matrix-assisted laser desorption/ionization - time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) of soluble proteins and shotgun proteomics of insoluble electric organ fractions in which mass spectral mapping was based on a preliminary library composed of 607 cDNA sequences . More recently, we reported sequencing the transcriptome of T. californica to assemble a Torpedo cDNA library composed of 10,326 sequences assembled into 4,243 non-overlapping contigs . Here, we present a comprehensive electric organ proteome as defined by one-dimensional SDS-PAGE followed by nanospray electrospray ionization quadrupole linear ion-trap tandem mass spectrometry (ESI-LTQ MS/MS) and two-dimensional isoelectric focusing (IEF) SDS-PAGE followed by MALDI-TOF/TOF MS-based approaches of electric organ fractions in which mass spectral mapping was performed using sequences from 10,326 Torpedo cDNA sequences and The Universal Protein Resource (UniProtKB/Swiss-Prot). Our results demonstrate concordance between skeletal muscle, NMJ, and electric organ proteomes. In addition, the electric organ expresses several uncharacterized proteins that may function at a synapse.