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Comp. Biochem. Physiol. Vol. 119A, No. 1, pp. 225–241, 1998 ISSN 1095-6433/98/$19.00Copyright 1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(97)00414-5

REVIEW

Electrophorus electricus as a Model System for theStudy of Membrane Excitability

Anthony L. Gotter,* Marcia A. Kaetzel, and John R. DedmanDepartment of Molecular and Cellular Physiology, University of Cincinnati College of Medicine,

Cincinnati, OH, U.S.A.

ABSTRACT. The stunning sensations produced by electric fish, particularly the electric eel, Electrophorus elec-tricus, have fascinated scientists for centuries. Within the last 50 years, however, electric cells of Electrophorushave provided a unique model system that is both specialized and appropriate for the study of excitable cellmembrane electrophysiology and biochemistry. Electric tissue generates whole animal electrical discharges bymeans of membrane potentials that are remarkably similar to those of mammalian neurons, myocytes and secre-tory cells. Electrocytes express ion channels, ATPases and signal transduction proteins common to these otherexcitable cells. Action potentials of electrocytes represent the specialized end function of electric tissue whereasother excitable cells use membrane potential changes to trigger sophisticated cellular processes, such as myofila-ment cross-bridging for contraction, or exocytosis for secretion. Because electric tissue lacks these functions andthe proteins associated with them, it provides a highly specialized membrane model system. This review examinesthe basic mechanisms involved in the generation of the electrical discharge of the electric eel and the membraneproteins involved. The valuable contributions that electric tissue continues to make toward the understandingof excitable cell physiology and biochemistry are summarized, particularly those studies using electrocytes as amodel system for the study of the regulation of membrane excitability by second messengers and signal transduc-tion pathways. comp biochem physiol 119A;1:225–241, 1998. 1998 Elsevier Science Inc.

KEY WORDS. Electrophorus electricus, excitable membranes, Na1 channels, acetylcholine receptor, acetylcho-linesterase, Na1/K1-ATPase, calmodulin, calmodulin-dependent protein kinase II

INTRODUCTION electric eel helped to contribute to the understanding ofelectricity. In 1775, John Walsh used the eel as an electrical

The electric eel, Electrophorus electricus, is capable of gener-potential source much like a powerful battery. Among nu-

ating an electrical potential of up to 600 volts, making itmerous experiments, one investigation included ten persons

the greatest producer of bioelectricity in the animal king- holding hands in a circle where the first and last ones tou-dom (50). Electric organs of these teleost fish develop from

ched the head and tail regions, respectively, of a single largeskeletal muscle and retain most of the biochemical and mor-

electric eel. All ten people received a severe shock. By hold-phological properties of the muscle sarcolemma (62). Elec-

ing materials such as brass chains, iron bars, glass, wood andtrocytes, however, have evolved excessive amounts of key

silk between two of the subjects, these investigators weremuscle membrane proteins that are polarized to particular

able to determine the relative conductivities of various sub-domains on their plasma membranes. This polarization en-stances (142). Since that time, the use of electric tissue of

ables these cells to produce transcellular potentials thatthe electric eel as a model for excitable cell membranes has

summate to generate a powerful whole-animal electrical dis-been realized.charge. The electric eel uses this voluntary production of

Because electric tissue contains membrane proteins ho-electricity as an effective mechanism to stun prey and ward-

mologous to other excitable tissues, it provides an appro-off predators.

priate model system in which to study excitable membraneIn the time of Benjamin Franklin and the Leyden jar, thebiochemistry and electrophysiology. Electric tissue ex-presses high levels of membrane receptors, channels and

Address reprint requests to: John R. Dedman, Department of Molecular andATPases, and has been implemented as a tissue source forCellular Physiology, University of Cincinnati College of Medicine, P.O.

Box 670576, Cincinnati, OH 45267-0576. Tel. (513) 558-4145; Fax (513) the purification of these proteins for biochemical structure/558-5738; E-mail:[email protected]. function analyses. Electrocytes, containing excessive

*Present address: Laboratory of Developmental Chronobiology, Harvardamounts of these membrane proteins, are also well suitedMedical School, Boston, MA 02114, U.S.A.

Received 13 August 1996; 5 March 1997; accepted 13 March 1997. for the study of membrane voltage and current changes of

226 A. L. Gotter et al.

excitable cells. Electrophorus electric tissue is specialized rival of the stimulus (11); and 3) synaptic transmissionbetween central medullary neurons and those of thesolely for membrane excitability. It does not contract or se-

crete compounds, and contains minimal amounts of pro- electromotor nucleus are mediated both chemically, by neu-rotransmitters, and electrically, through gap-junctions (86).teins involved in these processes. Electrocytes, therefore,

furnish a simplified system when compared to biochemically Presumably, synaptic transmission of neurons innervatingthe proximal electric organ is predominantly neurotrans-complex cells such as myocytes, neurons or secretory cells.

Electric tissue of the electric eel has proved to be an invalu- mitter-mediated and slow, whereas electrical coupling pre-vails in neurons innervating more distant regions.able system used to further the understanding of membrane

function of excitable cells. As the study of membrane elec- Electrophorus possesses three well defined electric organs.The main electric organ generates powerful high voltagetrophysiology now begins to focus on the regulation of

membrane proteins by signal transduction pathways, elec- discharges, whereas the tonically active Hunter’s organ andSach’s organ emit low voltage discharges and are thoughttrocytes of Electrophorus will continue to provide a special-

ized cell type in which to examine the modulation of mem- to be involved in electrolocation (11). The main organ ex-tends from behind the peritoneal cavity of the viscera downbrane potentials and ionic currents by intracellular second

messengers. the tail of the animal where it gives rise to Sach’s organ(Fig. 1A). This latter electric organ, which is more translu-cent and has less densely packed electrocytes, occupies the

ELECTROPHORUS remainder of the caudal portion. The smaller Hunter’s or-ANATOMY AND GENERATION gan is located subjacent to the other electric organs andOF THE ELECTRICAL DISCHARGE dorsal to the long swimming fin on the ventral surface of

the animal. In cross section, the electric organs are seen toThe electric eel is well evolved to produce high voltageelectrical discharges. In fact, the electric organs dominate occupy the ventral two-thirds of the animal, while the swim

bladder, spinal cord, blood vessels and skeletal muscles arethe mass of the posterior 80% of the animal, while the vis-cera is crowded into the anterior 20% (58,63). The muscu- located in the dorsal section (Fig. 1B). Hunter’s organ is

partially delineated from the main organ by two sets of skel-lar digestive tract is noteworthy in that it protracts caudally,but bends back toward the mouth before terminating at the etal muscles, running along the lower edge of the main or-

gan. In immature electric eels, the electric organ is seen toanus located just behind the head, anterior to the small pec-toral fins. Like the majority of teleost fish, the electric eel develop from this skeletal muscle tissue and is thought to

arise from embryonic myocyte precursor cells (62).also possesses a swim bladder that extends along the lengthof the animal dorsal to the main electric organ and ventral Close examination of the main electric organ reveals

rectangular columns of cells running the length of the elec-to the spinal cord (58).The central nervous system of the electric eel includes a tric organ (Fig. 1C). Each column of cells is delineated by

electrically insulating septa. Electrocytes are multinucleatedsmall teleostean brain and a spinal cord that extends thelength of the tail (58,102). Control and coordination of the syncytiums, much like the skeletal muscle cells from which

they are derived. They are large ribbon-like cells havingelectrical discharge is orchestrated by the central controlnucleus located in the ventral medial region of the medulla dimensions of up to 4 cm in length by 1.5 mm in diameter

and 100 µm in thickness (63). They extend laterally fromoblongata (25,133). Central efferent neurons extend cau-dally down the spinal cord and synapse on specialized mo- the midline to the skin and are stacked one after another

along their flat axis to make-up long columns of cells ex-torneurons of the electromotor nucleus occupying the dor-sal medial region along the length of the spinal cord. Axons tending in the longitudinal direction of the electric organ

(Fig. 1). Light and electron microscopy has revealed thefrom somata of electromotor neurons radiate into the elec-tric organs and innervate electrocytes (12). In order to gen- unique morphology of these flattened cells. The posterior

membrane is innervated and flat relative to the rostral non-erate a synchronized electrical discharge, individual electro-cytes within the entire electric organ must be stimulated innervated membrane which has large undulations (Fig. 2).

However, small papillae have been observed on the inner-simultaneously. This synchrony is achieved by delaying theactivation of proximal portions of the electric organ to such vated membrane where electromotor neurons form synapses

similar to neuromuscular junctions (76). Electrocyte mor-that these electrocytes are stimulated at the same time asthose of more distal regions of the electric organ (25). Evi- phology reflects the specialized physiological function of

electrocytes, which is to produce pronounced fluctuationsdence exists for three possible mechanisms of this delay: 1)Electromotor neurons innervating proximal regions of the in membrane potential. As shown in the hematoxylin and

eosin stained section in the micrograph of Fig. 2, electro-electric organ are of smaller diameter than those innervat-ing distal regions, and therefore conduct action potentials cytes have a simple regular morphology where they are

stacked one after another along the long axis of the animal.more slowly (11); 2) proximal portions of the electric organare innervated by electromotor neurons that wind a more This staining also demonstrates the biochemical simplicity

of electric tissue relative to adjacent skeletal muscle. Intensedevious path to these electrocytes, thereby delaying the ar-

Electrophorus—A Model Membrane System 227

FIG. 1. Anatomy of the electric eel. (A) Diagram illustrating the anatomical orientation of electric organs. (B) A section throughthe middle portion of the eel, drawn such that the anterior surface is nearest the reader. (C) Columns of electrocytes extendthe length of the electric organ. In this panel, the flatter, posterior surface of each electrocyte would be innervated by numerouselectromotor neurons (not shown). Heavy dark horizontal lines depict insulating septa delineating columns of electrocytes.Light blue shading represents the interior of electrocytes exposed in the cross-section.

staining is seen in muscle tissue which contains a compli- expresses two isoforms of actin that are characteristic ofcontractile tissue, as well as the muscle-specific intermedi-ment of macromolecules necessary for contraction, while

most of the staining of electrocytes appears at the plasma ate filament protein, desmin, further demonstrating the my-ocyte origin of electrocytes (7,26).membrane, indicative of its specialized electrophysiological

function. To increase surface area, electrocyte membranes Electrocytes generate electrical discharges using ionchannels, receptors, and ATPases, which are polarized tohave invaginations reminiscent of muscle T-tubules or ca-

veole (Fig. 3). The majority of the cytoplasm is devoid of the two major membranes of the cell (Fig. 4A). The nonin-nervated membrane exhibits a high concentration of theorganelles, and contains a loose filamentous network along

with glycogen granules. Organelles involved in protein syn- Na1/K1-ATPase (6,130) and resting current channelswhich together are responsible for the 285 mV resting po-thesis, such as nuclei, the endoplasmic reticulum, the golgi

apparatus, and mitochondria, are localized near both inner- tential. The bulk of resting current that maintains this po-tential is thought to be carried by K1 ions (67,121), but avated and noninnervated membranes, further suggesting

that the bulk of protein in the electrocyte is needed at the chloride current has not been conclusively ruled-out. Chlo-ride currents are thought to be present in electrocytes ofcell surface (77,134). This subcellular morphology is likely

to be maintained by cytoskeletal proteins characteristic of Sternopygus, a gymnotid closely related to the electric eel,where substitution of chloride with chloride channel imper-skeletal muscle cells. In fact, differentiated electric tissue


FIG. 2. Histological comparison of electrocytes and skeletalmuscle. A 4 mm section through Electrophorus main organelectrocytes (E) and adjacent skeletal muscle tissue (SK)stained with hematoxylin and eosin. This micrograph is ori-ented similarly to Fig. 1C, such that the relatively flat inner-vated membranes of electrocytes lay to the left while theundulated, noninnervated surface is to the right. Bar 5 50mm.

meant methyl sulfate results in an increase in resting mem-brane resistance (40). The noninnervated membrane con-tains no voltage-gated Na1 channels and is incapable ofproducing action potentials (63). On the other hand, nico-tinic acetylcholine receptors (AchRs), acetylcholinesterase,and Na1 channels are preferentially localized to the poste-

FIG. 3. Transmission electron microscopy of the noninner-rior, innervated membrane and impart chemical and electri- vated (A) and innervated (B) regions of electrocytes show-cal excitability to this face of the cell (10,17,34,42,63). ing numerous membrane invaginations. GC, glycocalyx;Chemical stimulation with AchR agonists or direct electri- NT, nerve terminus; T, tubular membrane invaginations; F,

cytoplasmic filaments. Bars: (A) 1.0 mm (B) 0.5 mm. Con-cal stimulation with depolarizing current is sufficient to pro-tributed by Robert R. Scully, University of Texas Health Sci-duce action potentials on this membrane. For this reason,ence Center at Houston (47).eel electric tissue provides an appropriate model for electri-

cally excitable membranes. This contrasts with electric tis-sue of some other electric fish, such as Torpedo, the marineelectric ray, which express few Na1 channels on their in-nervated membranes, and cannot be activated with direct

FIG. 4. Diagrammatic representation of electrocytes. The left surface of each cell represents the posterior innervated mem-brane. (A) At rest, both the innervated and noninnervated membrane exhibit a potential of 285 mV. When stimulated,activated AchRs generate endplate potentials, triggering Na1 channel-mediated action potentials peaking at 165 mV on theinnervated membrane. The noninnervated membrane contains no voltage-gated Na1 channels and maintains the 285 mVresting potential. The result is a transcellular potential difference of approximately 150 mV. The presence of an L-type Ca21

channel has not yet been supported by experimental evidence, but is included in this diagram given the myogenic origin ofelectric tissue. (B) Since each cell is stimulated simultaneously, electrocyte transcellular potentials summate. The potentialsof three electrocytes culminate to produce 450 mV. Currents generated by stimulated electrocytes flow down electrocytecolumns in the posterior to anterior direction. The circuit is closed by current flowing out the head of the eel, through thewater, and back into the tail region.


electrical stimulation (18,49). In eel electric tissue, inward durations between 2–3 msec, values similar to those of neu-rons and skeletal muscle cells. Action potentials are pro-rectifying K1 channels, as well as measurable amounts of

Na1/K1-ATPase, also exist in the innervated membrane duced on the electrocyte membrane via an increase in Na1

conductance, analogous to what occurs along an unmyelin-(92,130). The resting potential of the innervated mem-brane is maintained primarily by inward rectifying K1 chan- ated axon.

On the other hand, the innervated membranes of electro-nels and secondarily by leak conductance channels selectivefor either K1 or Cl2 (93). cytes of Torpedo, the electric ray, resemble modified motor

endplates containing excessive amounts of the AchR, butUpon electromotor neuron stimulation, acetylcholine isreleased onto postsynaptic regions of the posterior mem- little Na1 channel protein. These fish belong to the elasmo-

branch order, compared to Electrophorus, which is a teleost,brane opening AchRs (Fig. 4A). The resulting endplate po-tentials activate voltage-gated Na1 channels, producing ac- and the existence of electric tissue in these two animals

presumably represents convergent evolution (49). Becausetion potentials similar to those of neurons and myocytes.Electrophorus electrocytes have a high density of Na1 chan- the electric ray provides a specialized model for skeletal

muscle motor-endplates, they have been used along withnels enabling action potentials of large amplitude to be pro-duced. As the innervated membrane depolarizes, inward electric tissue of Electrophorus for functional and biochemi-

cal studies of cholinergic synaptic transmission. However,rectifying K1 channels close, further augmenting the rate ofrise and amplitude of the action potential. The membrane Electrophorus electrocytes, which express Na1 channels, of-

fer a more general model for other excitable cell mem-potential is repolarized by Na1 channels closing, due to in-activation, and from the conductance of Cl2 and K1 branes. These electrocytes are also more easily manipulated

for dissection and functional studies.through leak channels. Meanwhile, the noninnervatedmembrane containing an abundance of resting channels Due to their large size, individual Electrophorus electro-

cytes can be isolated in order to measure the total flux ofmaintains a potential of approximately 285 mV. At thepeak of stimulation, a transcellular potential difference re- ions as well as ionic currents across both the innervated and

noninnervated membranes. Schoffeniels (121) designed in-sults, and the electrocyte effectively acts as a battery of ap-proximately 150 mV (Fig. 4B). Insulating septa on either novative pieces of equipment to take advantage of the flat-

tened dimensions of these cells. Individual electrocytes wereside of the electrocyte prevent this potential from beingshort circuited by current flowing around the outside of cell. mounted and sealed over rectangular windows in lucite

sheets, such that the cell separated two different bath solu-Instead, the resulting current flows along the entire columnof electrocytes, out the head region of the eel, through the tions. With this method, the total flux of radiolabeled Na1

and K1 ions into and out of single electrocytes could besurrounding water and back into the electric organ at thetail of the animal. As mentioned earlier, every electrocyte measured. Acetylcholine agonists and 2,4-dinitrophenol al-

tered the fluxes of these cations demonstrating the activityof the electric organ is activated simultaneously. Since elec-trocytes are stacked one after another down the length of of AchRs and transport via the Na1/K1-ATPase, respec-

tively, in an intact cell (121). This method of mountinga column, their transcellular potentials summate, as do bat-teries in series. For example, three electrocytes each produc- electrocytes between two different solutions was later modi-

fied to record Na1 and K1 currents across the innervateding transcellular potentials of 150 mV will yield a total po-tential of 450 mV (Fig. 4B). In order for an electric eel to membrane. The rising phase of the electrocyte action po-

tential was shown to be due to an inward movement of Na1produce a whole animal discharge of 600 V, at least 4000electrocytes need to be activated at once. Large electric eels, ions, as demonstrated for other electrically excitable cells

(93).containing more electrocytes in series, produce greaterwhole animal potentials. This is analogous to a flashlight This technique of isolating electrocytes to measure mem-

brane currents has also been used to further understand thein which a larger number of batteries arranged in series gen-erates a brighter source of light. electrophysiological properties of nicotinic acetylcholine

receptors. Electrocytes were stimulated with bath applica-tion of AchR agonists or by activation of innervating elec-

THE ELECTRIC ORGAN tromotor neurons in order to determine the kinetics of re-AS A MODEL SYSTEM FOR THE ceptor gating and how processes associated with this gatingSTUDY OF EXCITABLE CELL MEMBRANES depend on various agonists and membrane potentialElectrophysiological Contributions (69,126). Other studies used the isolated electrocyte prepa-

ration to examine the binding site properties of AchRs byElectric tissue of Electrophorus provides a specialized modelsystem for the study of membrane excitability and bioelec- using photoisomizable agonists and antagonists, reagents

whose action was not limited by diffusion (66,68). Thesetricity. Keynes and Martins-Ferreira (63) performed the firstcomprehensive study of membrane potentials produced by experiments contributed to the understanding of the mech-

anisms of elementary receptor channel gating events, evenelectrocytes. They found resting potentials of about 285mV, and action potentials peaking at about 165 mV with before the advent of patch-clamp technology.


The activity of individual frog muscle channel proteins channel is associated with additional low molecular weightβ subunits (8,53), further demonstrating the simplicity ofin a small area of membrane was first observed by using the

patch-clamp technique (95). To date, this is the only eel electric tissue model system. For functional studies, puri-fied electric eel Na1 channel could be reconstituted intomethod capable of measuring the activity of individual pro-

tein molecules. This technology was also applied to isolated lipid vesicles where the uptake of radiolabeled Na1 couldbe blocked by tetrodotoxin, and activated by batrachotoxinelectrocytes to measure single channel AchR and Na1 cur-

rents. Electrocyte single-channel AchR currents are identi- and veratridine (30,114). Lipid vesicles containing purifiedelectric eel Na1 channels were also fused to planar bilayerscal to those of other excitable cells in their voltage and

temperature dependence. However, currents from electric in order to measure single channel conductances even inthe absence of activating agents. Single channel conduc-eel electrocytes are more homogeneous compared to other

cells that display complex distributions of channels with tances and voltage-dependent activation of incorporatedelectric eel Na1 channels were similar to Na1 channels ofvarious open times and conductances (106). This property

indicated that Electrophorus expresses AchRs made up of a patch-clamped skeletal muscle myocytes and neurons (124).The electric organ also provided a valuable source of Na1single combination of subunits, demonstrating the utility of

electrocytes as a simplified model system. Na1 channel ion channels for the determination of the protein’s primary, sec-ondary, and tertiary structure. The eel Na1 channel was theselectivity and gating currents were investigated with simi-

lar preparations. The innervated membrane of eel electro- first voltage-gated ion channel to be cloned and sequenced.After purification, trypsin-generated Na1 channel peptidecytes display a high density of these channel proteins, and

macroscopic Na1 currents can be recorded through mem- fragments were sequenced in order to synthesize oligonucle-otide probes. These probes were then used to screen an Elec-brane patches of small diameter. Because of the large popu-

lation of channels within a single patch, charge movements trophorus cDNA library. This approach was used to identify,clone and sequence Na1 channel mRNA’s containing anassociated with structural shifts of the protein during open-

ing of the channel gate could be measured. Each channel open reading frame corresponding to 1,820 amino acids(97). The sequence exhibited four homologous repeats,was calculated to shift 1.3 charges upon opening of the

channel, a value similar to Na1 channels of nerve and mus- each of which contained a cluster of positively charged resi-dues, a motif emulated by other voltage-gated cation chan-cle (123). Using the same technique, the relative selectivity

of electric eel Na1 channels for Na1 versus K1 ions was nels to be sequenced later (Fig. 5). Oligonucleotide probesfashioned from the electric eel Na1 channel sequence werefound to vary substantially from different sample membrane

patches taken from the same cell (125). Because electro- later used to determine the primary structure of three iso-forms of the mammalian brain protein (96). The sequencescytes are thought to express a single Na1 channel isoform

with no associated auxiliary subunits, these investigations of these channels were then used to clone and sequence therat skeletal muscle Na1 channel. Oocytes injected with thissuggested that channel function could be modified by post-

translational modifications such as glycosylation, acylation, mRNA exhibited Na1 currents with gating and pharmaco-logical properties similar to rat muscle fibers (137). Theand phosphorylation (1), a theme that is being extensively

investigated in other excitable cells (71). electric eel channel is most closely related to the skeletalmuscle channel, while Na1 channels from brain tissue pos-ses an additional 202 amino acids between the first and sec-

Contributions to Excitable Cell Membrane Biochemistry ond homologous repeat domains. Models for the orientationof the Na1 channel within the membrane proposed thatNa1 CHANNEL. Electrophorus electric tissue expresses

excessive amounts of membrane ion channels, receptors, each homologous domain contained 6 to 8 transmembranehelices, and assigned cytoplasmic orientations to the N- andand ATPases to achieve its specialized electrophysiological

function. For this reason, it has been used extensively as C-termini. These models also proposed that the fourth posi-tively charged amphipathic transmembrane helix (the S4a tissue source for the purification of membrane proteins

involved in excitable cell function. The voltage-gated Na1 segment) of each homologous repeat was responsible forvoltage-dependent gating of the channel (Fig. 5). This S4channel from Electrophorus was the first to be purified, and

was accomplished by its ability to specifically bind toxins segment has been postulated to rotate while shifting to amore extracellular position upon membrane depolarization,such as tetrodotoxin and saxitoxin and lectins that bind

negatively charged sialic acid carbohydrate residues thereby opening the channel (8,136). To test these struc-tural models, antibodies were raised against selected Elec-(1,59,88). The purified protein was found to consist of a

single functional subunit that migrates on SDS PAGE at trophorus Na1 channel sequences to determine the topo-graphical orientation of different regions of the protein withapproximately 260 kDa. Roughly 30% of the mass of the

protein was found to be due to carbohydrate modification, respect to the membrane. The C-terminus and the regionbetween domains II and III were identified as cytoplasmic,as the channel’s molecular weight was shifted upon deglyco-

sylation (70). The existence of a single α subunit is in con- since antibodies directed against these regions labeled right-side out Na1 channel-containing vesicles only after permea-trast to the purified protein form other tissues where the


structure of the Electrophorus Na1 channel continueto prove invaluable to the study of the biochemistry of allvoltage-gated cation channels, since these other proteinshave been found to have similar structural motifs.

Electric tissue continues to serve as an excellent modelsystem in which to study the regulation of Na1 channels bypost-translational modifications such as phosphorylation. Inelectrocytes of Sternopygus, a weakly electric fish closely re-lated to Electrophorus, the frequency of the repetitive elec-tric organ discharge is inversely proportional to the durationof the action potential. Treatment of these fish with theandrogen, dihydrotestosterone, not only decreases the fre-quency of the electric organ discharge, but also lengthensthe duration of the action potential due to Na1 currentsthat inactivate more slowly (39). This suggests that thesechannels may be modulated by androgen-dependent post-translational modifications, since only one isoform of thechannel protein is known to be expressed in electric tissue,without auxiliary subunits (1,97). Numerous membrane re-ceptors and ion channels have been found to be phosphory-

FIG. 5. Topographical model of the eel Na1 channel withinlated by protein kinases resulting in modulation of channelthe plasma membrane. The orientation of the protein hasgating and conductance (71). The electric eel Na1 channelbeen proposed from sequence determination, hydropathy

plot data (97), and epitope mapping (45,46,85). Transmem- has been found to be phosphorylated on at least three sitesbrane segments containing positive charges deliniate the after the protein is purified in the presence of phosphatasevoltage sensors of the channel. The relative locations of inhibitors, demonstrating that these post-translationalmonoclonal antibody epitopes and PKA phosphorylation

modifications exist in vivo [Fig. 5, (35)]. Phosphorylation ofsites are indicated. Reference numbers and phosphorylatedthe Na1 channel of excised eel electrocyte membraneserine residues are indicated in parentheses (S6, S444,

S1680) (35). Newer models of the channel suggest that the patches by purified cAMP-dependent protein kinase (PKA)loops connecting transmembrane segments 5 and 6 of each results in an 80% reduction of Na1 conductance and a shifthomologous repeat bend into the plane of the membrane in the voltage of activation to more positive potentials (36).bilayer (not shown).

In a more physiological study using intact electrocytes ofSternopygus, McAnelly and Zakon (81) showed that appli-cation of 8-Br-cAMP activated endogenous PKA to doublebilization, and the cytoplasmic side of electrocyte mem-

branes as observed by electron microscopy (45,46). Anti- the peak magnitude of Na1 currents with no appreciableshift in the voltage of activation. In mammalian brain tis-bodies against part of the S4 segment of the electric eel Na1

channel modified the gating characteristics of mammalian sue, phosphorylation of Na1 channels by PKA results in adecrease in conductance (72). Given the degree of homol-neuronal Na1 currents when applied extracellularly. The

voltage-dependence of the rate of activation and inactiva- ogy between the electric eel Na1 channel and the skeletalmuscle channel, modulation of the myocyte channel by thistion was accelerated, suggesting that this region of the chan-

nel is, in fact, involved in voltage gating (85). The binding kinase seems likely, and remains to be determined.of this antibody to the extracellular portion of rat brain Na1

channels was enhanced with depolarization, further sup- NICOTINIC ACETYLCHOLINE RECEPTOR (AchR). The util-ity of Electrophorus electric tissue as a source for the purifi-porting models of the channel that predict an extracellular

shift of the voltage sensor upon channel activation (119). cation and characterization of the AchR was recognized asearly as 1960, when Ehrenpreis produced an enrichedThe model for the Na21 channel presented in Fig. 5 is

continually changing in light of accumulating evidence. As d-tubocurine receptor preparation. Even without chroma-tography and gel electrophoresis methods, an acetylcholine-with other voltage-gated cation channels, the loops of each

homologous repeat connecting transmembrane segments 5 binding fraction could be isolated with ammonium sulfateprecipitation and equilibrium dialysis techniques (33). Puri-and 6 have been postulated to bend into the ion channel

pore instead of the extracellular configuration presented in fications of the AchR were done later by various protocolsutilizing differential centrifugation and detergent extractionFig. 5. Synthetic peptides corresponding to portions of these

loop regions can enter the hydrophobic membrane environ- followed by ion exchange, gel filtration and affinity chroma-tography. The solubilized, non-denatured receptor was ini-ment and associate with one another, consistent with the

hypothesis that these segments of the channel bend into tially isolated from other acetylcholine-binding proteins,such as acetylcholinesterase, by gel filtration and found tothe pore of the protein (109). These studies elucidating the


be a 260 kDa macromolecule (16,43,104). Affinity resins (EAMG), has been used as a useful model for the humandisease, since it is also marked by altered motor function,of acetylcholine analogs, or cobra venom toxin coupled to

Sepharose were used to purify the receptor further and en- and on the ultrastructural level, by separated nerve terminiand degenerated postsynaptic regions at neuromuscularabled the identification of the acetylcholine-binding α-sub-

unit as a 41–44 kDa protein (16,64,104). Copurifying poly- junctions. Myocyte electrophysiology is also altered inEAMG where endplate potentials and action potential am-peptides having molecular weights of 50 to 65 kDa were

later identified as the β, γ, and δ-subunits that comprise the plitudes are reduced, if present at all (37,107). When puri-fied Electrophorus or Torpedo AchR is injected into rabbitspentameric holoenzyme (22,61,73). Monoclonal antibodies

generated against the acetylcholine receptor showed some to produce EAMG, antibodies are produced predominatelyagainst a specific extracellular region of the α-subunit calledcross-reactivity between each of these subunits, suggesting

that these polypeptides have at least limited sequence ho- the main immunogenic region (MIR) (138,139). Antibod-ies in sera from patients with myesthenia gravis cross-reactmology (138). At about the same time, electric tissue of

various species of Torpedo was also used to purify the recep- with the MIR of Electrophorus and Torpedo AchRs, sug-gesting that the autoimmune response is directed againsttor and yielded similar results (120,129,139).

Studies utilizing electric tissue of Electrophorus and Tor- this region of the receptor (140). Monoclonal antibodies tothe MIR cross-react with the receptor from numerous spe-pedo have together provided a wealth of knowledge about

the structure, pharmacology, and clinical importance of this cies indicating that this region is evolutionarily well con-served, and is important for the function of the receptor asligand-gated ion channel (44,82,101). Complete primary

structures for each of the four subunits of the AchR were well as the induction of myesthenia gravis (138,139).determined by screening Torpedo electric organ cDNA li-braries with degenerate oligonucleotide probes correspond- ACETYLCHOLINESTERASE (AchE). AchE terminates the

acetylcholine signal within synapses between electromotoring to partial amino acid sequences of purified receptor sub-units. The four different fully processed mRNA sequences neurons and electrocytes, as well as in the mammalian neu-

romuscular junction, by hydrolyzing the neurotransmitterexhibited considerable homology, and encoded polypep-tides of 461, 493, 489, and 522 amino acids in length for into choline and acetate. Inhibitors of AchE prolong the

lifetime of acetylcholine leading to the overstimulation andthe α, β, γ, and δ subunits, respectively (24,98–100). Tor-pedo and Electrophorus mRNA’s encode the functional eventual desensitization of nicotinic AchRs. Exposure to

anti-AchE compounds found in pesticides and chemicalreceptor as confirmed by oocyte studies that detectedan increase in acetylcholine-induced conductance changes warfare agents induce acute and chronic alterations in neu-

romuscular and central nervous system function. Anti-following microinjection of mRNA (4,65,89). From hy-drophobicity plots and examination of glycosylation and AchE agents at lower concentrations have been used thera-

peutically to treat glaucoma, Alzheimer’s dementia, and my-affinity-labeling sites, a general model for the topology ofeach subunit within the membrane was proposed. It pre- esthenia gravis—diseases marked by compromised acetyl-

choline release or attenuated postsynaptic AchR densitydicted that each subunit was anchored in the membrane byfour hydrophobic segments that make-up the cation chan- (37,87,92).

Because of its pronounced capacity for chlolinergic syn-nel of the full pentamer. The model also proposed that 52%of the protein, including the N and C-termini, protrude into aptic transmission, Electrophorus electric tissue was used as

a source for the purification and characterization of AchE.the extracellular space, and provide sites for glycosylation,as well as acetylcholine binding by the α-subunit (24,100). Initial purifications, taking as much as nine days to perform,

utilized numerous ammonium sulfate and magnesium sulfateThis structural model has been continually modified in lightof investigations using monoclonal antibodies directed precipitations, ion exchange chromatography and lengthy

toluene washes to isolate the soluble form of the proteinagainst various sequences of the receptor for epitope map-ping (27,108,110). (52,116). It was later recognized that the native enzyme was

in fact anchored in the postsynaptic membrane by a gly-In the process of producing antibodies to the Electropho-rus AchR for structural studies, investigators noticed that colsyl-phosphatidylinositol linkage, since AchE activity

could be released into a soluble form in response to phos-rabbits injected with the antigen developed symptoms simi-lar to the human neuromuscular disease, myesthenia gravis pholipase C treatment (31,110,112,113). Investigators then

began using detergents and 1 M NaCl to obtain the intact(107). This autoimmune disease is characterized by a defectin neuromuscular transmission that is responsible for protein in a soluble form (24). Affinity chromatography,

utilizing agents that structurally resembled acetylcholine ormarked muscle weakness and fatigue (143). When purifiedElectrophorus AchR receptor is injected into rabbits, the an- anti-AchE agents coupled to Sepharose, were successful in

purifying the protein more rapidly (19,32,78,141). The puri-tibodies generated by the animal’s immune system alsocross-react with similar regions of the rabbit’s own AchR fied protein migrated at 80 kDa on SDS PAGE, correspond-

ing to a single functional subunit that associates into tetra-protein, triggering an inflammatory response. This phenom-enon, termed experimental autoimmune myesthenia gravis mers, octomers, and dodecamers (105). These affinity


chromatography purifications also contributed to the under- in native membranes (29,146). The existance of two iso-forms of the Na1/K1-ATPase have now been postulated tostanding of the active site structure and properties that were

later shown to be important for interactions of therapeutic exist in electrocyte membranes and appear to be differen-tially distributed between the innervated and noninner-and toxic anti-AchE agents.

X-ray diffraction analysis has been performed on crystals vated membranes (54).With functional Na1/K1-ATPase purified from electricof AchE purified from electric tissue of Electrophorus and

Torpedo californica (122,131,132). The atomic structure of eel electric tissue and other tissues with a high capacity foractive transport as models, a reaction scheme was compiledthe Torpedo enzyme has been solved to 2.8 A resolution,

and reveals an active site that lies within the center of the for the ATP-driven transport of Na1 and K1 across themembrane (Fig. 6) (74). ATP binding drives the replace-enzyme at the bottom of an ‘‘aromatic gorge.’’ The structure

of the enzyme complexed with AchE inhibitors identified ment of K1 by Na1 on the cytoplasmic side of the membrane(E1 form of the enzyme). Formation of the phosphoenzymeamino acids responsible for specific substrate binding and

in stabilizing the transition state during catalysis (128,131). intermediate then induces a protein conformation changethat favors the release of Na1 into the interstitium (E2-PThe identity of these active site residues was further sup-

ported by studies using transition state analogs and radio- form). Upon extracellular K1 binding, the enzyme depho-sphorylates returning the ATPase to its original conforma-active active-site affinity labels (94,117). The nature of

the active site was further defined by using inhibitors of tion. Figure 6 shows the influence of the cardiac glycoside,ouabain, on this cycle, and also indicates studies done withthe Electrophorus AchE such as nitrosamine and pyridium

salt derivatives, as well as monoclonal antibodies the electric eel Na1/K1-ATPase that contributed to theelucidation of this cycle (3,38,41,56,57,147,148). By(111,127,144,145). These reagents were valuable in de-

termining the kinetics, structural regions, and ionic interac- understanding this reaction mechanism, drugs have beendeveloped to target different partial reactions of the mech-tions involved in the reaction mechanism of the enzyme.

Understanding the mechanism and structure of AchE from anism of ATP-dependent cation translocation. Cardiacglycosides such as ouabain have been known to inhibitelectric tissue will lead to the development of new pharma-

ceuticals aimed at the treatment of cholinergic diseases as Na1/K1-ATPase function and physically interact primarilywith the α-subunit of the electric eel enzyme just as it doeswell as therapies for individuals exposed to toxic anti-AchE

agents. in mammalian isoforms (51,75,115). The mechanism ofouabain inhibition of the ATPase reaction cycle is alsodepicted in Fig. 6. Aluminum and mercury have also beenNa1/K1-ATPase. Electrochemical gradients that dictate

the resting membrane potential of excitable cells are di- shown to inhibit Na1/K1-ATPase function, providing apossible explanation for the toxicity of these cations (5).rectly and indirectly maintained by the Na1/K1-ATPase.

This enzyme utilizes the free energy from ATP hydrolysisto translocate of Na1 out of the cell and K1 into the cell. THIAMINE TRIPHOSPHATASE. Thiamine in the form of its

diphosphate derivative (thiamine diphosphate, TDP) is anClinically, the Na1 /K1-ATPase is the target for cardiac gly-cosides, such as ouabain, to increase myocardial contractil- essential cofactor for enzymes involved in energy metabo-

lism, such as α-ketoglutarate dehydrogenase, transketolaseity. These agents increase intracellular Na1 concentrationswithin cardiac myocytes indirectly elevating Ca21 concen- and the pyruvate dehydrogenase complex. Thiamine is

phosphorylated by various kinases yielding mono-(TMP),trations. This, in turn, augments the Ca21-dependent forceof contraction. di-(TDP) and triphosphate (TTP) forms. As its name im-

plies, thiamine triphosphatase hydrolyzes TTP to TDP, theElectrophorus electrocytes contain relatively massivequantities of Na1 channels and AchRs needed for their spe- cofactor necessary for energy metabolism. Clinically, re-

duced levels of thiamine and its phosphate ester derivativescialized and exaggerated function, and, therefore, requireproportionally large amounts of Na1/K1-ATPase to main- have been observed in patients with Alzheimer’s disease

(79). At the cellular level, TTP has been postulated to regu-tain ionic gradients. For this reason, electric tissue has beenused as a tissue source for the purification and subsequent late chloride conductance, since incubation of rat brain ex-

tracts with thiamine and TDP induces a rise in TTP con-structural study of this enzyme. By measuring Na1-depen-dent ATPase activity and 32P radiolabeling of the protein centrations as well as augmenting chloride permeability

(15). Whether this phenomenon represents a direct effectitself as assays, membrane fractions derived from the nonin-nervated surface of electrocytes were isolated by differential of thiamine derivatives on chloride conductance rather

than an indirect effect through the modulation of energyand sucrose density gradient centrifugation techniques(2,9). Further purification was achieved by solubilizing metabolism remains unclear. Nevertheless, because thia-

mine triphosphatase catalyzes the breakdown of TTP, thismembranes containing the protein with various detergents(20). The purified glycosylated 47 kDa β-subunit and the 94 enzyme may be important in regulating membrane excit-

ability.kDa α-subunit could be reconstituted into proteolipisomesdisplaying the same functional characteristics as the enzyme The main organ of Electrophorus is exceptionally rich in


FIG. 6. Reaction mechanism for the Na1/K1-ATPase. Studies using the eel enzyme that contributed to the understanding ofvarious partial reactions are indicated by their corresponding reference number. Adapted from Lingrel and Kuntzweiler (74).

TTP, and electric tissue has provided a valuable source for including electric tissue Na1 channels as seen in a previoussection. Studies with this cyclic nucleotide in electric tissuethe characterization of thiamine triphosphatase. In purified

membrane preparations, the electric eel enzyme binds TTP are ongoing, and will provide interesting insight into thefunction of this molecule in electrocytes as well as otherat high and low affinity sites with Km’s of about 0.5 µM

and 240 µM, respectively—values close to the physiological excitable cells.Ca21 is another key second-messenger mediating theconcentration of the substrate in this tissue. Interestingly,

the enzyme is activated by monovalent anions (NO32, I2, function of excitable cells. Membrane depolarization in the

form of action potentials is accompanied with a concomi-and SCN2), and inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of voltage-depen- tant influx of Ca21 from the interstitium or intracellular

stores, which in turn triggers various cellular processes suchdent chloride channels and transporters (13). These investi-gations with the electric eel thiamine triphosphatase, there- as secretion and contraction. Ca21 also regulates membrane

excitability by modulating the activity of membrane recep-fore, may provide a link to the increased Cl2 permeabilityassociated with elevated TTP concentrations. Like its mam- tors, ion channels and ATPases by direct interaction with

these proteins and through the activation of Ca21-mediatormalian skeletal muscle counterpart, Electrophorus thiaminetriphosphatase appears to be associated with the membrane proteins. Electric tissue of Electrophorus embryonically de-

velops from skeletal muscle and its membrane properties are(14,80), further suggesting a role for the enzyme in mem-brane electrophysiological function. The exact physiologi- biochemically and functionally similar to that of other ex-

citable cells (62). For this reason, the binding of Ca21 tocal role of thiamine triphosphatase in electrocyte function,however, remains enigmatic. electric tissue membranes was investigated. Ca21-dependent

ATPase activity is present at the cell surface, particularlyin the innervated membrane (135). However, most of theSECOND MESSENGERS AND ASSOCIATED PROTEINS. Elec-

trocytes are particularly well suited for the purpose of study- Ca21 binding to the plasma membrane fraction did not re-quire ATP and consisted of two saturable sites (54,130).ing the modulation of membrane ionic currents by second-

messengers, since they are specialized for membrane excit- Ca21 binding sites were localized by electron microscopy,and found at discrete sites on invaginations of the in-ability and the biochemical processes involved in mem-

brane protein synthesis, targeting, and the regulation of nervated and noninnervated membranes, on cytoplasmicmicrofilaments, and as expected, within mitochondriamembrane proteins. Cyclic AMP activation of PKA can

modulate numerous ion channels and membrane receptors, (28,103). In 1975, a highly abundant Ca21 binding protein


directed against the N-terminal sequence of the rat brainisoform of the kinase, and also exhibits Ca21-independentactivity following autophosphorylation similar to its mam-malian counterpart (48). CaM kinase II phosphorylationpredominates over other modes of second messenger-depen-dent kinase activity, suggesting a role in the specializedfunction of Electrophorus electric tissue (47). Further studiesprobing the contribution of calmodulin and CaM kinase IIin electrocyte function will lend to the understanding ofhow these proteins operate in excitable membrane electro-physiology.

CONCLUDING REMARKS

Electrocytes of Electrophorus provide an excellent model sys-FIG. 7. Immunofluorescent localization of calmodulin

tem for excitable membranes. First, they supply an appro-within main organ electrocytes. Paraffin-embedded 4 mmpriate system that embryonically develops from skeletalsections were probed with anti-calmodulin sheep antibodies.

The location of primary antibodies was visualized with fluo- muscle and retains many of the morphological, biochemical,rescein-labeled rabbit anti-sheep secondary antibodies, and and functional properties of progenitor myocyte mem-photographed using epifluorescence microscopy. Bar 5 50 branes. Electrophysiologically, electrocytes function quitemm.

similar to mammalian neurons and myocytes. As exempli-fied by numerous studies utilizing electric tissue as a sourceof excitable cell proteins for structure, function and pharma-was isolated from Electrophorus electric organ (23). This cal-

cium-dependent regulator protein, later termed calmodulin, cological analysis cited in this review, the biochemistry ofthe electric organ shows considerable conservation withhas been found in nearly every eukaryotic cell including

plants, and has been found to mediate the effects of Ca21 mammalian species. Second, electrocytes are specialized formembrane excitability. These cells are deficient in proteinsin numerous cell functions including cell division, myocyte

contraction, exocytosis, and the regulation of membrane re- involved in processes such as secretion or contraction. Sub-cellular organelles that do exist are localized near the mem-ceptors, ATP-ases and ionic channels (55,83,84,118). Elec-

troporus calmodulin shows sequence and immunological ho- brane, presumably dedicated to the production of proteinsand ATP needed for membrane function. Finally, electro-mology to the mammalian protein, as expected since only

limited amino acid substitutions are observed between the cytes provide an exaggerated system in which membranereceptors, channels and ATPases are abundant. Macro-protein from plants, invertebrates and mammals (21,90).

Roughly 2% of electric organ protein is calmodulin—the molecules important for the function of excitable cellmembrane processes may be scarce in their original tis-highest concentration of any tissue reported (91). Immuno-

fluorescent localization of calmodulin within electric tissue sues such as muscle and nerve, but are found in vastquantities in Electrophorus electric tissue. This is indeed thedemonstrates its abundance within electrocytes (Fig. 7).

Fluorescence is observed throughout the cytoplasm. How- case for proteins such as the Na1 channel, the AchR,AchE, Na1/K1-ATPase, and calmodulin.ever, calmodulin appears concentrated subjacent to both in-

nervated and noninnervated membranes, suggesting a role The electrophysiological function of Electrophorus elec-trocytes is well characterized, as are the membrane proteinsin membrane function.

In an effort to determine the role of calmodulin in elec- responsible for these potential changes. The focus of mem-brane electrophysiology continues to shift toward the regu-trocyte function, and therefore excitable membranes in gen-

eral, calmodulin target proteins have been isolated and lation of ion channels, membrane receptors and ATPases.Electrocytes of the eel now provide a specialized system incharacterized. Among numerous calmodulin-binding pro-

teins, Kaetzel and Dedman (60) isolated an abundant and which to examine these signal transduction pathways. Forexample, the Electrophorus Na1 channel has been shown tonovel 55 kDa protein that bound to calmodulin-Sepharose

with high affinity. This protein was also found in skeletal be regulated by PKA. The electrocyte is also an ideal modelto probe the role of Ca21, calmodulin, and CaM kinase IImuscle, albeit in lesser amounts, suggesting a role for this

protein in skeletal muscle electrophysiology. Another cal- in electrocyte function due to their high activities. The reg-ulatory mechanisms discovered in Electrophorus electrocytesmodulin-target protein isolated in these studies was the 50

kDa calmodulin-dependent protein kinase II (CaM kinase can be evaluated in reconstituted systems such as Xenopusoocytes, similar to what has been done with the electricII). This kinase, implicated in neuron and myocyte func-

tion, was purified from electric tissue and found to have eel Na1 channel and AchR. Such experiments will lead toinvestigations that determine the role of second messenger-properties similar to that of CaM kinase II from mammalian

brain. The electric eel protein cross reacts with antibodies dependent proteins, such as PKA, CaM kinase II or novel


organ of Electrophorus electricus: Substrate-enzyme interac-proteins yet to be discovered, in the electrophysiology oftions. J. Neurochem. 53:738–746;1989.isolated myocytes and neurons. The contribution of these

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