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Comp. Biochem. Physiol. Vol. 119A, No. 1, pp. 225–241, 1998 ISSN 1095-6433/98/$19.

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Copyright  1998 Elsevier Science Inc. All rights reserved. PII S1095-6433(97)00414-5

REVIEW
Electrophorus electricus as a Model System for the
Study of Membrane Excitability
Anthony L. Gotter,* Marcia A. Kaetzel, and John R. Dedman
Department 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 Electrophorus
have provided a unique model system that is both specialized and appropriate for the study of excitable cell
membrane electrophysiology and biochemistry. Electric tissue generates whole animal electrical discharges by
means 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 other
excitable cells. Action potentials of electrocytes represent the specialized end function of electric tissue whereas
other 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 and
the proteins associated with them, it provides a highly specialized membrane model system. This review examines
the basic mechanisms involved in the generation of the electrical discharge of the electric eel and the membrane
proteins involved. The valuable contributions that electric tissue continues to make toward the understanding
of excitable cell physiology and biochemistry are summarized, particularly those studies using electrocytes as a
model 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 of


electricity. 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 it
merous 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 large
skeletal 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 and
trocytes, however, have evolved excessive amounts of key
silk between two of the subjects, these investigators were
muscle 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 that
the 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 membrane
In the time of Benjamin Franklin and the Leyden jar, the
biochemistry and electrophysiology. Electric tissue ex-
presses high levels of membrane receptors, channels and
Address reprint requests to: John R. Dedman, Department of Molecular and
Cellular Physiology, University of Cincinnati College of Medicine, P.O. ATPases, and has been implemented as a tissue source for
Box 670576, Cincinnati, OH 45267-0576. Tel. (513) 558-4145; Fax (513) the purification of these proteins for biochemical structure/
558-5738; E-mail:john.dedman@uc.edu. function analyses. Electrocytes, containing excessive
*Present address: Laboratory of Developmental Chronobiology, Harvard
Medical School, Boston, MA 02114, U.S.A. amounts of these membrane proteins, are also well suited
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 transmission
solely for membrane excitability. It does not contract or se- between central medullary neurons and those of the
crete compounds, and contains minimal amounts of pro- electromotor nucleus are mediated both chemically, by neu-
teins involved in these processes. Electrocytes, therefore, rotransmitters, and electrically, through gap-junctions (86).
furnish a simplified system when compared to biochemically Presumably, synaptic transmission of neurons innervating
complex cells such as myocytes, neurons or secretory cells. the proximal electric organ is predominantly neurotrans-
Electric tissue of the electric eel has proved to be an invalu- mitter-mediated and slow, whereas electrical coupling pre-
able system used to further the understanding of membrane vails in neurons innervating more distant regions.
function of excitable cells. As the study of membrane elec- Electrophorus possesses three well defined electric organs.
trophysiology now begins to focus on the regulation of The main electric organ generates powerful high voltage
membrane proteins by signal transduction pathways, elec- discharges, whereas the tonically active Hunter’s organ and
trocytes of Electrophorus will continue to provide a special- Sach’s organ emit low voltage discharges and are thought
ized cell type in which to examine the modulation of mem- to be involved in electrolocation (11). The main organ ex-
brane potentials and ionic currents by intracellular second tends from behind the peritoneal cavity of the viscera down
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 and
OF THE ELECTRICAL DISCHARGE
dorsal to the long swimming fin on the ventral surface of
The electric eel is well evolved to produce high voltage the animal. In cross section, the electric organs are seen to
electrical discharges. In fact, the electric organs dominate occupy the ventral two-thirds of the animal, while the swim
the mass of the posterior 80% of the animal, while the vis- bladder, spinal cord, blood vessels and skeletal muscles are
cera is crowded into the anterior 20% (58,63). The muscu- located in the dorsal section (Fig. 1B). Hunter’s organ is
lar digestive tract is noteworthy in that it protracts caudally, partially delineated from the main organ by two sets of skel-
but bends back toward the mouth before terminating at the etal muscles, running along the lower edge of the main or-
anus located just behind the head, anterior to the small pec- gan. In immature electric eels, the electric organ is seen to
toral fins. Like the majority of teleost fish, the electric eel develop from this skeletal muscle tissue and is thought to
also possesses a swim bladder that extends along the length arise from embryonic myocyte precursor cells (62).
of the animal dorsal to the main electric organ and ventral Close examination of the main electric organ reveals
to the spinal cord (58). rectangular columns of cells running the length of the elec-
The central nervous system of the electric eel includes a tric organ (Fig. 1C). Each column of cells is delineated by
small teleostean brain and a spinal cord that extends the electrically insulating septa. Electrocytes are multinucleated
length of the tail (58,102). Control and coordination of the syncytiums, much like the skeletal muscle cells from which
electrical discharge is orchestrated by the central control they are derived. They are large ribbon-like cells having
nucleus located in the ventral medial region of the medulla dimensions of up to 4 cm in length by 1.5 mm in diameter
oblongata (25,133). Central efferent neurons extend cau- and 100 µm in thickness (63). They extend laterally from
dally down the spinal cord and synapse on specialized mo- the midline to the skin and are stacked one after another
torneurons of the electromotor nucleus occupying the dor- along their flat axis to make-up long columns of cells ex-
sal medial region along the length of the spinal cord. Axons tending in the longitudinal direction of the electric organ
from somata of electromotor neurons radiate into the elec- (Fig. 1). Light and electron microscopy has revealed the
tric organs and innervate electrocytes (12). In order to gen- unique morphology of these flattened cells. The posterior
erate a synchronized electrical discharge, individual electro- membrane is innervated and flat relative to the rostral non-
cytes within the entire electric organ must be stimulated innervated membrane which has large undulations (Fig. 2).
simultaneously. This synchrony is achieved by delaying the However, small papillae have been observed on the inner-
activation of proximal portions of the electric organ to such vated membrane where electromotor neurons form synapses
that these electrocytes are stimulated at the same time as similar to neuromuscular junctions (76). Electrocyte mor-
those of more distal regions of the electric organ (25). Evi- phology reflects the specialized physiological function of
dence exists for three possible mechanisms of this delay: 1) electrocytes, which is to produce pronounced fluctuations
Electromotor neurons innervating proximal regions of the in membrane potential. As shown in the hematoxylin and
electric organ are of smaller diameter than those innervat- eosin stained section in the micrograph of Fig. 2, electro-
ing distal regions, and therefore conduct action potentials cytes have a simple regular morphology where they are
more slowly (11); 2) proximal portions of the electric organ stacked one after another along the long axis of the animal.
are innervated by electromotor neurons that wind a more This staining also demonstrates the biochemical simplicity
devious path to these electrocytes, thereby delaying the ar- of electric tissue relative to adjacent skeletal muscle. Intense
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 through
the middle portion of the eel, drawn such that the anterior surface is nearest the reader. (C) Columns of electrocytes extend
the length of the electric organ. In this panel, the flatter, posterior surface of each electrocyte would be innervated by numerous
electromotor 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 of
ment of macromolecules necessary for contraction, while contractile tissue, as well as the muscle-specific intermedi-
most of the staining of electrocytes appears at the plasma ate filament protein, desmin, further demonstrating the my-
membrane, indicative of its specialized electrophysiological ocyte origin of electrocytes (7,26).
function. To increase surface area, electrocyte membranes Electrocytes generate electrical discharges using ion
have invaginations reminiscent of muscle T-tubules or ca- channels, receptors, and ATPases, which are polarized to
veole (Fig. 3). The majority of the cytoplasm is devoid of the two major membranes of the cell (Fig. 4A). The nonin-
organelles, and contains a loose filamentous network along nervated membrane exhibits a high concentration of the
with glycogen granules. Organelles involved in protein syn- Na1 /K1-ATPase (6,130) and resting current channels
thesis, such as nuclei, the endoplasmic reticulum, the golgi which together are responsible for the 285 mV resting po-
apparatus, and mitochondria, are localized near both inner- tential. The bulk of resting current that maintains this po-
vated and noninnervated membranes, further suggesting tential is thought to be carried by K1 ions (67,121), but a
that the bulk of protein in the electrocyte is needed at the chloride current has not been conclusively ruled-out. Chlo-
cell surface (77,134). This subcellular morphology is likely ride currents are thought to be present in electrocytes of
to be maintained by cytoskeletal proteins characteristic of Sternopygus, a gymnotid closely related to the electric eel,
skeletal muscle cells. In fact, differentiated electric tissue where substitution of chloride with chloride channel imper-
228 A. L. Gotter et al.

FIG. 2. Histological comparison of electrocytes and skeletal


muscle. A 4 mm section through Electrophorus main organ
electrocytes (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 the
undulated, noninnervated surface is to the right. Bar 5 50
mm.

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 of
producing 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,
cal stimulation with depolarizing current is sufficient to pro- cytoplasmic filaments. Bars: (A) 1.0 mm (B) 0.5 mm. Con-
duce action potentials on this membrane. For this reason, tributed by Robert R. Scully, University of Texas Health Sci-
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 marine
electric 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 the
innervated membrane. The noninnervated membrane contains no voltage-gated Na1 channels and maintains the 285 mV
resting 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 of
electric tissue. (B) Since each cell is stimulated simultaneously, electrocyte transcellular potentials summate. The potentials
of three electrocytes culminate to produce 450 mV. Currents generated by stimulated electrocytes flow down electrocyte
columns in the posterior to anterior direction. The circuit is closed by current flowing out the head of the eel, through the
water, and back into the tail region.
230 A. L. Gotter et al.

electrical stimulation (18,49). In eel electric tissue, inward durations between 2–3 msec, values similar to those of neu-
rectifying K1 channels, as well as measurable amounts of rons and skeletal muscle cells. Action potentials are pro-
Na1 /K1-ATPase, also exist in the innervated membrane duced on the electrocyte membrane via an increase in Na1
(92,130). The resting potential of the innervated mem- conductance, analogous to what occurs along an unmyelin-
brane is maintained primarily by inward rectifying K1 chan- ated axon.
nels and secondarily by leak conductance channels selective On the other hand, the innervated membranes of electro-
for either K 1 or Cl2 (93). cytes of Torpedo, the electric ray, resemble modified motor
Upon electromotor neuron stimulation, acetylcholine is endplates containing excessive amounts of the AchR, but
released onto postsynaptic regions of the posterior mem- little Na1 channel protein. These fish belong to the elasmo-
brane opening AchRs (Fig. 4A). The resulting endplate po- branch order, compared to Electrophorus, which is a teleost,
tentials activate voltage-gated Na1 channels, producing ac- and the existence of electric tissue in these two animals
tion potentials similar to those of neurons and myocytes. presumably represents convergent evolution (49). Because
Electrophorus electrocytes have a high density of Na1 chan- the electric ray provides a specialized model for skeletal
nels enabling action potentials of large amplitude to be pro- muscle motor-endplates, they have been used along with
duced. As the innervated membrane depolarizes, inward electric tissue of Electrophorus for functional and biochemi-
rectifying K1 channels close, further augmenting the rate of cal studies of cholinergic synaptic transmission. However,
rise and amplitude of the action potential. The membrane Electrophorus electrocytes, which express Na1 channels, of-
potential is repolarized by Na 1 channels closing, due to in- fer a more general model for other excitable cell mem-
activation, and from the conductance of Cl2 and K1 branes. These electrocytes are also more easily manipulated
through leak channels. Meanwhile, the noninnervated for dissection and functional studies.
membrane containing an abundance of resting channels Due to their large size, individual Electrophorus electro-
maintains a potential of approximately 285 mV. At the cytes can be isolated in order to measure the total flux of
peak of stimulation, a transcellular potential difference re- ions as well as ionic currents across both the innervated and
sults, and the electrocyte effectively acts as a battery of ap- noninnervated membranes. Schoffeniels (121) designed in-
proximately 150 mV (Fig. 4B). Insulating septa on either novative pieces of equipment to take advantage of the flat-
side of the electrocyte prevent this potential from being tened dimensions of these cells. Individual electrocytes were
short circuited by current flowing around the outside of cell. mounted and sealed over rectangular windows in lucite
Instead, the resulting current flows along the entire column sheets, such that the cell separated two different bath solu-
of electrocytes, out the head region of the eel, through the tions. With this method, the total flux of radiolabeled Na1
surrounding water and back into the electric organ at the and K1 ions into and out of single electrocytes could be
tail of the animal. As mentioned earlier, every electrocyte measured. Acetylcholine agonists and 2,4-dinitrophenol al-
of the electric organ is activated simultaneously. Since elec- tered the fluxes of these cations demonstrating the activity
trocytes are stacked one after another down the length of of AchRs and transport via the Na1 /K1-ATPase, respec-
a column, their transcellular potentials summate, as do bat- tively, in an intact cell (121). This method of mounting
teries in series. For example, three electrocytes each produc- electrocytes between two different solutions was later modi-
ing transcellular potentials of 150 mV will yield a total po- fied to record Na1 and K1 currents across the innervated
tential of 450 mV (Fig. 4B). In order for an electric eel to membrane. The rising phase of the electrocyte action po-
produce a whole animal discharge of 600 V, at least 4000 tential was shown to be due to an inward movement of Na1
electrocytes need to be activated at once. Large electric eels, ions, as demonstrated for other electrically excitable cells
containing more electrocytes in series, produce greater (93).
whole animal potentials. This is analogous to a flashlight This technique of isolating electrocytes to measure mem-
in which a larger number of batteries arranged in series gen- brane currents has also been used to further understand the
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 gating
STUDY OF EXCITABLE CELL MEMBRANES depend on various agonists and membrane potential
Electrophysiological Contributions (69,126). Other studies used the isolated electrocyte prepa-
Electric tissue of Electrophorus provides a specialized model ration to examine the binding site properties of AchRs by
system for the study of membrane excitability and bioelec- using photoisomizable agonists and antagonists, reagents
tricity. Keynes and Martins-Ferreira (63) performed the first whose action was not limited by diffusion (66,68). These
comprehensive study of membrane potentials produced by experiments contributed to the understanding of the mech-
electrocytes. They found resting potentials of about 285 anisms of elementary receptor channel gating events, even
mV, and action potentials peaking at about 165 mV with before the advent of patch-clamp technology.
Electrophorus—A Model Membrane System 231

The activity of individual frog muscle channel proteins channel is associated with additional low molecular weight
in a small area of membrane was first observed by using the β subunits (8,53), further demonstrating the simplicity of
patch-clamp technique (95). To date, this is the only eel electric tissue model system. For functional studies, puri-
method capable of measuring the activity of individual pro- fied electric eel Na1 channel could be reconstituted into
tein molecules. This technology was also applied to isolated lipid vesicles where the uptake of radiolabeled Na 1 could
electrocytes to measure single channel AchR and Na1 cur- be blocked by tetrodotoxin, and activated by batrachotoxin
rents. Electrocyte single-channel AchR currents are identi- and veratridine (30,114). Lipid vesicles containing purified
cal to those of other excitable cells in their voltage and electric eel Na1 channels were also fused to planar bilayers
temperature dependence. However, currents from electric in order to measure single channel conductances even in
eel electrocytes are more homogeneous compared to other the absence of activating agents. Single channel conduc-
cells that display complex distributions of channels with tances and voltage-dependent activation of incorporated
various open times and conductances (106). This property electric eel Na1 channels were similar to Na1 channels of
indicated that Electrophorus expresses AchRs made up of a patch-clamped skeletal muscle myocytes and neurons (124).
single combination of subunits, demonstrating the utility of The electric organ also provided a valuable source of Na1
electrocytes as a simplified model system. Na1 channel ion channels for the determination of the protein’s primary, sec-
selectivity and gating currents were investigated with simi- ondary, and tertiary structure. The eel Na1 channel was the
lar preparations. The innervated membrane of eel electro- first voltage-gated ion channel to be cloned and sequenced.
cytes display a high density of these channel proteins, and After purification, trypsin-generated Na1 channel peptide
macroscopic Na 1 currents can be recorded through mem- fragments were sequenced in order to synthesize oligonucle-
brane patches of small diameter. Because of the large popu- otide probes. These probes were then used to screen an Elec-
lation of channels within a single patch, charge movements trophorus cDNA library. This approach was used to identify,
associated with structural shifts of the protein during open- clone and sequence Na1 channel mRNA’s containing an
ing of the channel gate could be measured. Each channel open reading frame corresponding to 1,820 amino acids
was calculated to shift 1.3 charges upon opening of the (97). The sequence exhibited four homologous repeats,
channel, a value similar to Na 1 channels of nerve and mus- each of which contained a cluster of positively charged resi-
cle (123). Using the same technique, the relative selectivity dues, a motif emulated by other voltage-gated cation chan-
of electric eel Na 1 channels for Na 1 versus K1 ions was nels to be sequenced later (Fig. 5). Oligonucleotide probes
found to vary substantially from different sample membrane fashioned from the electric eel Na1 channel sequence were
patches taken from the same cell (125). Because electro- later used to determine the primary structure of three iso-
cytes are thought to express a single Na1 channel isoform forms of the mammalian brain protein (96). The sequences
with no associated auxiliary subunits, these investigations of these channels were then used to clone and sequence the
suggested that channel function could be modified by post- rat skeletal muscle Na 1 channel. Oocytes injected with this
translational modifications such as glycosylation, acylation, mRNA exhibited Na 1 currents with gating and pharmaco-
and phosphorylation (1), a theme that is being extensively logical properties similar to rat muscle fibers (137). The
investigated in other excitable cells (71). electric eel channel is most closely related to the skeletal
muscle 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 orientation
1
Na CHANNEL. Electrophorus electric tissue expresses of the Na 1 channel within the membrane proposed that
excessive amounts of membrane ion channels, receptors, each homologous domain contained 6 to 8 transmembrane
and ATPases to achieve its specialized electrophysiological helices, and assigned cytoplasmic orientations to the N- and
function. For this reason, it has been used extensively as C-termini. These models also proposed that the fourth posi-
a tissue source for the purification of membrane proteins tively charged amphipathic transmembrane helix (the S4
involved in excitable cell function. The voltage-gated Na 1 segment) of each homologous repeat was responsible for
channel from Electrophorus was the first to be purified, and voltage-dependent gating of the channel (Fig. 5). This S4
was accomplished by its ability to specifically bind toxins segment has been postulated to rotate while shifting to a
such as tetrodotoxin and saxitoxin and lectins that bind more extracellular position upon membrane depolarization,
negatively charged sialic acid carbohydrate residues thereby opening the channel (8,136). To test these struc-
(1,59,88). The purified protein was found to consist of a tural models, antibodies were raised against selected Elec-
single functional subunit that migrates on SDS PAGE at trophorus Na 1 channel sequences to determine the topo-
approximately 260 kDa. Roughly 30% of the mass of the graphical orientation of different regions of the protein with
protein was found to be due to carbohydrate modification, respect to the membrane. The C-terminus and the region
as the channel’s molecular weight was shifted upon deglyco- between domains II and III were identified as cytoplasmic,
sylation (70). The existence of a single α subunit is in con- since antibodies directed against these regions labeled right-
trast to the purified protein form other tissues where the side out Na1 channel-containing vesicles only after permea-
232 A. L. Gotter et al.

structure of the Electrophorus Na1 channel continue


to prove invaluable to the study of the biochemistry of all
voltage-gated cation channels, since these other proteins
have been found to have similar structural motifs.
Electric tissue continues to serve as an excellent model
system in which to study the regulation of Na1 channels by
post-translational modifications such as phosphorylation. In
electrocytes 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 duration
of the action potential. Treatment of these fish with the
androgen, dihydrotestosterone, not only decreases the fre-
quency of the electric organ discharge, but also lengthens
the duration of the action potential due to Na 1 currents
that inactivate more slowly (39). This suggests that these
channels may be modulated by androgen-dependent post-
translational modifications, since only one isoform of the
channel 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 within
the plasma membrane. The orientation of the protein has lated by protein kinases resulting in modulation of channel
been proposed from sequence determination, hydropathy gating and conductance (71). The electric eel Na 1 channel
plot data (97), and epitope mapping (45,46,85). Transmem- has been found to be phosphorylated on at least three sites
brane segments containing positive charges deliniate the after the protein is purified in the presence of phosphatase
voltage sensors of the channel. The relative locations of
inhibitors, demonstrating that these post-translational
monoclonal antibody epitopes and PKA phosphorylation
sites are indicated. Reference numbers and phosphorylated modifications exist in vivo [Fig. 5, (35)]. Phosphorylation of
serine residues are indicated in parentheses (S6, S444, the Na1 channel of excised eel electrocyte membrane
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 shift
homologous 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 of
Sternopygus, McAnelly and Zakon (81) showed that appli-
bilization, and the cytoplasmic side of electrocyte mem- cation of 8-Br-cAMP activated endogenous PKA to double
branes as observed by electron microscopy (45,46). Anti- the peak magnitude of Na1 currents with no appreciable
bodies against part of the S4 segment of the electric eel Na1 shift in the voltage of activation. In mammalian brain tis-
channel modified the gating characteristics of mammalian sue, phosphorylation of Na1 channels by PKA results in a
neuronal Na1 currents when applied extracellularly. The decrease in conductance (72). Given the degree of homol-
voltage-dependence of the rate of activation and inactiva- ogy between the electric eel Na1 channel and the skeletal
tion was accelerated, suggesting that this region of the chan- muscle channel, modulation of the myocyte channel by this
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 Na 1
channels was enhanced with depolarization, further sup- NICOTINIC ACETYLCHOLINE RECEPTOR (AchR). The util-
porting models of the channel that predict an extracellular ity of Electrophorus electric tissue as a source for the purifi-
shift of the voltage sensor upon channel activation (119). cation and characterization of the AchR was recognized as
The model for the Na21 channel presented in Fig. 5 is early as 1960, when Ehrenpreis produced an enriched
continually changing in light of accumulating evidence. As d-tubocurine receptor preparation. Even without chroma-
with other voltage-gated cation channels, the loops of each tography and gel electrophoresis methods, an acetylcholine-
homologous repeat connecting transmembrane segments 5 binding fraction could be isolated with ammonium sulfate
and 6 have been postulated to bend into the ion channel precipitation and equilibrium dialysis techniques (33). Puri-
pore instead of the extracellular configuration presented in fications of the AchR were done later by various protocols
Fig. 5. Synthetic peptides corresponding to portions of these utilizing differential centrifugation and detergent extraction
loop regions can enter the hydrophobic membrane environ- followed by ion exchange, gel filtration and affinity chroma-
ment and associate with one another, consistent with the tography. The solubilized, non-denatured receptor was ini-
hypothesis that these segments of the channel bend into tially isolated from other acetylcholine-binding proteins,
the pore of the protein (109). These studies elucidating the such as acetylcholinesterase, by gel filtration and found to
Electrophorus—A Model Membrane System 233

be a 260 kDa macromolecule (16,43,104). Affinity resins (EAMG), has been used as a useful model for the human
of acetylcholine analogs, or cobra venom toxin coupled to disease, since it is also marked by altered motor function,
Sepharose were used to purify the receptor further and en- and on the ultrastructural level, by separated nerve termini
abled the identification of the acetylcholine-binding α-sub- and degenerated postsynaptic regions at neuromuscular
unit as a 41–44 kDa protein (16,64,104). Copurifying poly- junctions. Myocyte electrophysiology is also altered in
peptides having molecular weights of 50 to 65 kDa were EAMG where endplate potentials and action potential am-
later identified as the β, γ, and δ-subunits that comprise the plitudes are reduced, if present at all (37,107). When puri-
pentameric holoenzyme (22,61,73). Monoclonal antibodies fied Electrophorus or Torpedo AchR is injected into rabbits
generated against the acetylcholine receptor showed some to produce EAMG, antibodies are produced predominately
cross-reactivity between each of these subunits, suggesting against a specific extracellular region of the α-subunit called
that these polypeptides have at least limited sequence ho- the main immunogenic region (MIR) (138,139). Antibod-
mology (138). At about the same time, electric tissue of ies in sera from patients with myesthenia gravis cross-react
various species of Torpedo was also used to purify the recep- with the MIR of Electrophorus and Torpedo AchRs, sug-
tor and yielded similar results (120,129,139). gesting that the autoimmune response is directed against
Studies utilizing electric tissue of Electrophorus and Tor- this region of the receptor (140). Monoclonal antibodies to
pedo have together provided a wealth of knowledge about the MIR cross-react with the receptor from numerous spe-
the structure, pharmacology, and clinical importance of this cies indicating that this region is evolutionarily well con-
ligand-gated ion channel (44,82,101). Complete primary served, and is important for the function of the receptor as
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
ing to partial amino acid sequences of purified receptor sub- acetylcholine signal within synapses between electromotor
units. The four different fully processed mRNA sequences neurons and electrocytes, as well as in the mammalian neu-
exhibited considerable homology, and encoded polypep- romuscular junction, by hydrolyzing the neurotransmitter
tides of 461, 493, 489, and 522 amino acids in length for into choline and acetate. Inhibitors of AchE prolong the
the α, β, γ, and δ subunits, respectively (24,98–100). Tor- lifetime of acetylcholine leading to the overstimulation and
pedo and Electrophorus mRNA’s encode the functional eventual desensitization of nicotinic AchRs. Exposure to
receptor as confirmed by oocyte studies that detected anti-AchE compounds found in pesticides and chemical
an increase in acetylcholine-induced conductance changes warfare agents induce acute and chronic alterations in neu-
following microinjection of mRNA (4,65,89). From hy- romuscular and central nervous system function. Anti-
drophobicity plots and examination of glycosylation and AchE agents at lower concentrations have been used thera-
affinity-labeling sites, a general model for the topology of peutically to treat glaucoma, Alzheimer’s dementia, and my-
each subunit within the membrane was proposed. It pre- esthenia gravis—diseases marked by compromised acetyl-
dicted that each subunit was anchored in the membrane by choline release or attenuated postsynaptic AchR density
four hydrophobic segments that make-up the cation chan- (37,87,92).
nel of the full pentamer. The model also proposed that 52% Because of its pronounced capacity for chlolinergic syn-
of the protein, including the N and C-termini, protrude into aptic transmission, Electrophorus electric tissue was used as
the extracellular space, and provide sites for glycosylation, a source for the purification and characterization of AchE.
as well as acetylcholine binding by the α-subunit (24,100). Initial purifications, taking as much as nine days to perform,
This structural model has been continually modified in light utilized numerous ammonium sulfate and magnesium sulfate
of investigations using monoclonal antibodies directed precipitations, ion exchange chromatography and lengthy
against various sequences of the receptor for epitope map- toluene washes to isolate the soluble form of the protein
ping (27,108,110). (52,116). It was later recognized that the native enzyme was
In the process of producing antibodies to the Electropho- in fact anchored in the postsynaptic membrane by a gly-
rus AchR for structural studies, investigators noticed that colsyl-phosphatidylinositol linkage, since AchE activity
rabbits injected with the antigen developed symptoms simi- could be released into a soluble form in response to phos-
lar to the human neuromuscular disease, myesthenia gravis pholipase C treatment (31,110,112,113). Investigators then
(107). This autoimmune disease is characterized by a defect began using detergents and 1 M NaCl to obtain the intact
in neuromuscular transmission that is responsible for protein in a soluble form (24). Affinity chromatography,
marked muscle weakness and fatigue (143). When purified utilizing agents that structurally resembled acetylcholine or
Electrophorus AchR receptor is injected into rabbits, the an- anti-AchE agents coupled to Sepharose, were successful in
tibodies generated by the animal’s immune system also purifying the protein more rapidly (19,32,78,141). The puri-
cross-react with similar regions of the rabbit’s own AchR fied protein migrated at 80 kDa on SDS PAGE, correspond-
protein, triggering an inflammatory response. This phenom- ing to a single functional subunit that associates into tetra-
enon, termed experimental autoimmune myesthenia gravis mers, octomers, and dodecamers (105). These affinity
234 A. L. Gotter et al.

chromatography purifications also contributed to the under- in native membranes (29,146). The existance of two iso-
standing of the active site structure and properties that were forms of the Na 1 /K 1-ATPase have now been postulated to
later shown to be important for interactions of therapeutic exist in electrocyte membranes and appear to be differen-
and toxic anti-AchE agents. tially distributed between the innervated and noninner-
X-ray diffraction analysis has been performed on crystals vated membranes (54).
of AchE purified from electric tissue of Electrophorus and With functional Na 1 /K1-ATPase purified from electric
Torpedo californica (122,131,132). The atomic structure of eel electric tissue and other tissues with a high capacity for
the Torpedo enzyme has been solved to 2.8 Å resolution, active transport as models, a reaction scheme was compiled
and reveals an active site that lies within the center of the for the ATP-driven transport of Na1 and K 1 across the
enzyme at the bottom of an ‘‘aromatic gorge.’’ The structure membrane (Fig. 6) (74). ATP binding drives the replace-
of the enzyme complexed with AchE inhibitors identified ment of K 1 by Na1 on the cytoplasmic side of the membrane
amino acids responsible for specific substrate binding and (E1 form of the enzyme). Formation of the phosphoenzyme
in stabilizing the transition state during catalysis (128,131). intermediate then induces a protein conformation change
The identity of these active site residues was further sup- that favors the release of Na1 into the interstitium (E2-P
ported by studies using transition state analogs and radio- form). Upon extracellular K1 binding, the enzyme depho-
active active-site affinity labels (94,117). The nature of sphorylates returning the ATPase to its original conforma-
the active site was further defined by using inhibitors of tion. Figure 6 shows the influence of the cardiac glycoside,
the Electrophorus AchE such as nitrosamine and pyridium ouabain, on this cycle, and also indicates studies done with
salt derivatives, as well as monoclonal antibodies the electric eel Na 1 /K1-ATPase that contributed to the
(111,127,144,145). These reagents were valuable in de- elucidation of this cycle (3,38,41,56,57,147,148). By
termining the kinetics, structural regions, and ionic interac- understanding this reaction mechanism, drugs have been
tions involved in the reaction mechanism of the enzyme. developed to target different partial reactions of the mech-
Understanding the mechanism and structure of AchE from anism of ATP-dependent cation translocation. Cardiac
electric tissue will lead to the development of new pharma- glycosides such as ouabain have been known to inhibit
ceuticals aimed at the treatment of cholinergic diseases as Na1 /K1-ATPase function and physically interact primarily
well as therapies for individuals exposed to toxic anti-AchE with the α-subunit of the electric eel enzyme just as it does
agents. in mammalian isoforms (51,75,115). The mechanism of
ouabain inhibition of the ATPase reaction cycle is also
1 1
Na /K -ATPase. Electrochemical gradients that dictate depicted in Fig. 6. Aluminum and mercury have also been
the resting membrane potential of excitable cells are di- shown to inhibit Na 1 /K 1-ATPase function, providing a
rectly and indirectly maintained by the Na1 /K1-ATPase. possible explanation for the toxicity of these cations (5).
This enzyme utilizes the free energy from ATP hydrolysis
to translocate of Na1 out of the cell and K1 into the cell. THIAMINE TRIPHOSPHATASE. Thiamine in the form of its
Clinically, the Na1 /K 1-ATPase is the target for cardiac gly- diphosphate derivative (thiamine diphosphate, TDP) is an
cosides, such as ouabain, to increase myocardial contractil- essential cofactor for enzymes involved in energy metabo-
ity. These agents increase intracellular Na 1 concentrations lism, such as α-ketoglutarate dehydrogenase, transketolase
within cardiac myocytes indirectly elevating Ca 21 concen- and the pyruvate dehydrogenase complex. Thiamine is
trations. This, in turn, augments the Ca21-dependent force phosphorylated by various kinases yielding mono-(TMP),
of contraction. di-(TDP) and triphosphate (TTP) forms. As its name im-
Electrophorus electrocytes contain relatively massive plies, thiamine triphosphatase hydrolyzes TTP to TDP, the
quantities of Na1 channels and AchRs needed for their spe- cofactor necessary for energy metabolism. Clinically, re-
cialized and exaggerated function, and, therefore, require duced levels of thiamine and its phosphate ester derivatives
proportionally large amounts of Na1 /K1-ATPase to main- have been observed in patients with Alzheimer’s disease
tain ionic gradients. For this reason, electric tissue has been (79). At the cellular level, TTP has been postulated to regu-
used as a tissue source for the purification and subsequent late chloride conductance, since incubation of rat brain ex-
structural study of this enzyme. By measuring Na1-depen- tracts with thiamine and TDP induces a rise in TTP con-
dent ATPase activity and 32P radiolabeling of the protein centrations as well as augmenting chloride permeability
itself as assays, membrane fractions derived from the nonin- (15). Whether this phenomenon represents a direct effect
nervated surface of electrocytes were isolated by differential of thiamine derivatives on chloride conductance rather
and sucrose density gradient centrifugation techniques than an indirect effect through the modulation of energy
(2,9). Further purification was achieved by solubilizing metabolism remains unclear. Nevertheless, because thia-
membranes containing the protein with various detergents mine triphosphatase catalyzes the breakdown of TTP, this
(20). The purified glycosylated 47 kDa β-subunit and the 94 enzyme may be important in regulating membrane excit-
kDa α-subunit could be reconstituted into proteolipisomes ability.
displaying the same functional characteristics as the enzyme The main organ of Electrophorus is exceptionally rich in
Electrophorus—A Model Membrane System 235

FIG. 6. Reaction mechanism for the Na1 /K1-ATPase. Studies using the eel enzyme that contributed to the understanding of
various 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 previous
the characterization of thiamine triphosphatase. In purified section. Studies with this cyclic nucleotide in electric tissue
membrane preparations, the electric eel enzyme binds TTP are ongoing, and will provide interesting insight into the
at high and low affinity sites with Km’s of about 0.5 µM function of this molecule in electrocytes as well as other
and 240 µM, respectively—values close to the physiological excitable cells.
concentration of the substrate in this tissue. Interestingly, Ca21 is another key second-messenger mediating the
the enzyme is activated by monovalent anions (NO32, I2, function of excitable cells. Membrane depolarization in the
and SCN2), and inhibited by 4,4′-diisothiocyanostilbene- form of action potentials is accompanied with a concomi-
2,2′-disulfonic acid (DIDS), an inhibitor of voltage-depen- tant influx of Ca21 from the interstitium or intracellular
dent chloride channels and transporters (13). These investi- stores, which in turn triggers various cellular processes such
gations with the electric eel thiamine triphosphatase, there- as secretion and contraction. Ca 21 also regulates membrane
fore, may provide a link to the increased Cl2 permeability excitability by modulating the activity of membrane recep-
associated with elevated TTP concentrations. Like its mam- tors, ion channels and ATPases by direct interaction with
malian skeletal muscle counterpart, Electrophorus thiamine these proteins and through the activation of Ca21-mediator
triphosphatase appears to be associated with the membrane proteins. Electric tissue of Electrophorus embryonically de-
(14,80), further suggesting a role for the enzyme in mem- velops from skeletal muscle and its membrane properties are
brane electrophysiological function. The exact physiologi- biochemically and functionally similar to that of other ex-
cal role of thiamine triphosphatase in electrocyte function, citable cells (62). For this reason, the binding of Ca21 to
however, remains enigmatic. electric tissue membranes was investigated. Ca21-dependent
ATPase activity is present at the cell surface, particularly
SECOND MESSENGERS AND ASSOCIATED PROTEINS. Elec- in the innervated membrane (135). However, most of the
trocytes are particularly well suited for the purpose of study- Ca21 binding to the plasma membrane fraction did not re-
ing the modulation of membrane ionic currents by second- quire ATP and consisted of two saturable sites (54,130).
messengers, since they are specialized for membrane excit- Ca21 binding sites were localized by electron microscopy,
ability and the biochemical processes involved in mem- and found at discrete sites on invaginations of the in-
brane protein synthesis, targeting, and the regulation of nervated and noninnervated membranes, on cytoplasmic
membrane proteins. Cyclic AMP activation of PKA can microfilaments, and as expected, within mitochondria
modulate numerous ion channels and membrane receptors, (28,103). In 1975, a highly abundant Ca 21 binding protein
236 A. L. Gotter et al.

directed against the N-terminal sequence of the rat brain


isoform of the kinase, and also exhibits Ca21-independent
activity following autophosphorylation similar to its mam-
malian counterpart (48). CaM kinase II phosphorylation
predominates over other modes of second messenger-depen-
dent kinase activity, suggesting a role in the specialized
function of Electrophorus electric tissue (47). Further studies
probing the contribution of calmodulin and CaM kinase II
in electrocyte function will lend to the understanding of
how these proteins operate in excitable membrane electro-
physiology.

CONCLUDING REMARKS
Electrocytes of Electrophorus provide an excellent model sys-
FIG. 7. Immunofluorescent localization of calmodulin
within main organ electrocytes. Paraffin-embedded 4 mm tem for excitable membranes. First, they supply an appro-
sections were probed with anti-calmodulin sheep antibodies. priate system that embryonically develops from skeletal
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 quite
mm.
similar to mammalian neurons and myocytes. As exempli-
fied by numerous studies utilizing electric tissue as a source
was isolated from Electrophorus electric organ (23). This cal- of excitable cell proteins for structure, function and pharma-
cium-dependent regulator protein, later termed calmodulin, cological analysis cited in this review, the biochemistry of
has been found in nearly every eukaryotic cell including the electric organ shows considerable conservation with
plants, and has been found to mediate the effects of Ca21 mammalian species. Second, electrocytes are specialized for
in numerous cell functions including cell division, myocyte membrane excitability. These cells are deficient in proteins
contraction, exocytosis, and the regulation of membrane re- involved in processes such as secretion or contraction. Sub-
ceptors, ATP-ases and ionic channels (55,83,84,118). Elec- cellular organelles that do exist are localized near the mem-
troporus calmodulin shows sequence and immunological ho- brane, presumably dedicated to the production of proteins
mology to the mammalian protein, as expected since only and ATP needed for membrane function. Finally, electro-
limited amino acid substitutions are observed between the cytes provide an exaggerated system in which membrane
protein from plants, invertebrates and mammals (21,90). receptors, channels and ATPases are abundant. Macro-
Roughly 2% of electric organ protein is calmodulin—the molecules important for the function of excitable cell
highest concentration of any tissue reported (91). Immuno- membrane processes may be scarce in their original tis-
fluorescent localization of calmodulin within electric tissue sues such as muscle and nerve, but are found in vast
demonstrates its abundance within electrocytes (Fig. 7). quantities in Electrophorus electric tissue. This is indeed the
Fluorescence is observed throughout the cytoplasm. How- case for proteins such as the Na1 channel, the AchR,
ever, calmodulin appears concentrated subjacent to both in- AchE, Na1 /K1-ATPase, and calmodulin.
nervated and noninnervated membranes, suggesting a role The electrophysiological function of Electrophorus elec-
in membrane function. trocytes is well characterized, as are the membrane proteins
In an effort to determine the role of calmodulin in elec- responsible for these potential changes. The focus of mem-
trocyte function, and therefore excitable membranes in gen- brane electrophysiology continues to shift toward the regu-
eral, calmodulin target proteins have been isolated and lation of ion channels, membrane receptors and ATPases.
characterized. Among numerous calmodulin-binding pro- Electrocytes of the eel now provide a specialized system in
teins, Kaetzel and Dedman (60) isolated an abundant and which to examine these signal transduction pathways. For
novel 55 kDa protein that bound to calmodulin-Sepharose example, the Electrophorus Na1 channel has been shown to
with high affinity. This protein was also found in skeletal be regulated by PKA. The electrocyte is also an ideal model
muscle, albeit in lesser amounts, suggesting a role for this to probe the role of Ca21, calmodulin, and CaM kinase II
protein in skeletal muscle electrophysiology. Another cal- in electrocyte function due to their high activities. The reg-
modulin-target protein isolated in these studies was the 50 ulatory mechanisms discovered in Electrophorus electrocytes
kDa calmodulin-dependent protein kinase II (CaM kinase can be evaluated in reconstituted systems such as Xenopus
II). This kinase, implicated in neuron and myocyte func- oocytes, similar to what has been done with the electric
tion, was purified from electric tissue and found to have eel Na1 channel and AchR. Such experiments will lead to
properties similar to that of CaM kinase II from mammalian investigations that determine the role of second messenger-
brain. The electric eel protein cross reacts with antibodies dependent proteins, such as PKA, CaM kinase II or novel
Electrophorus—A Model Membrane System 237

proteins yet to be discovered, in the electrophysiology of organ of Electrophorus electricus: Substrate-enzyme interac-
isolated myocytes and neurons. The contribution of these tions. J. Neurochem. 53:738–746;1989.
14. Bettendorff, L.; Longrée, I.; Wins, P.; Schoffeniels, E. Solubi-
proteins to muscle and nervous system physiology can then lization of thiamine triphosphatase from the electric organ
be determined by genetically disrupting these pathways in of Electrophorus electricus. Biochim. Biophys. Acta 1073:69–
transgenic mice. 76;1991.
The electric eel has proven to be of timeless value in 15. Bettendorff, L.; Peeters, M.; Wins, P.; Schoffeniels, E. Me-
providing a model to understand not only early concepts tabolism of thiamine triphosphate in rat brain: Correlation
with chloride permeability. J. Neurochem. 60:423–434;
of electricity, but also to resolve the molecular function of 1993.
membrane proteins. Electrocytes will continue to provide 16. Biesecker, G. Molecular properties of the cholinergic recep-
insight into the physiology and disease of excitable tissues. tor purified from Electrophorus electricus. Biochemistry 12:
4403–4409;1973.
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