Celhdar anti Molecular Neurobiology, Vol. 14. No.

3, 1994

Tetrodotoxin-Resistant Sodium Channels

Shigeru Yoshida ~
Received May 15, 1994, revised June 10, 1994
KEY WORDS: tetrodotoxin; Na channels; neurons; muscles; development.

SUMMARY
1. Tetrodotoxin (TTX) has been widely used as a chemical tool for blocking Na + channels. However, reports are accumulating that some Na channels are resistant to T T X in various tissues and in different animal species. Studying the sensitivity of Na channels to T I ' X may provide us with an insight into the evolution of Na channels. 2. Na channels present in TTX-carrying animals such as pufferfish and some types of shellfish, frogs, salamanders, octopuses, etc., are resistant to TTX. 3. Denervation converts TTX-sensitive Na channels to TTX-resistant ones in skeletal muscle cells, i.e., reverting-back phenomenon. Also, undifferentiated skeletal muscle cells contain TTX-resistant Na channels. Cardiac muscle cells and some types of smooth muscle cells are considerably insensitive to TI'X. 4. TI'X-resistant Na channels have been found in cell bodies of many peripheral nervous system (PNS) neurons in both immature and mature animals. However, TI'X-resistant Na channels have been reported in only a few types of central nervous system (CNS). Axons of PNS and CNS neurons are sensitive to TTX. However, some glial cells have T r X - r e s i s t a n t Na channels. 5. Properties of TTX-sensitive and TTX-resistant Na channels are different. Like Ca 2 channels, TTX-resistant Na channels can be blocked by inorganic (Co z, Mn 2, Ni 2~-, Cd 2+, Zn ~, La 3+) and organic (D-600) Ca Z channel blockers. Usually, TTX-resistant Na channels show smaller single-channel conductance, slower kinetics, and a more positive current-voltage relation than TTX-sensitive ones. 6. Molecular aspects of the TTX-resistant Na channel have been described. The structure of the channel has been revealed, and changing its amino acid(s) alters the sensitivity of the Na channel to TTX.
i Department of Physiology, Fukui Medical School, Fukui 910-11. Japan. 227 0272.434(1/94/06(1~1.11227507.1X)/0 ~) 1994PlenumPublishingCorporation

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7. TTX-sensitive Na channels seem to be used preferentially in differentiated cells and in higher animals instead of TFX-resistant Na channels for rapid and effective processing of information. 8. Possible evolution courses for Na and Ca 2 channels are discussed with regard to ontogenesis and phylogenesis.

INTRODUCTION

Tetrodotoxin (TTX) or "pufferfish toxin" is the most commonly known animal toxin. The pufferfish, called Fugu in Japan, belongs to the genus Tetradon or Fugu, and it contains the toxin in the ovaries, the liver, and occasionally the skin (Kao, 1966; Halstead, 1967). Tetrodotoxin was named in 1894 by the Japanese scientist Tahara; the structure of TTX was decided in 1964 and synthesized in 1970 (see reviews by Kao, 1966; Mosher, 1986). TTX is a very potent toxin, e.g., only 1 mg of TFX can kill as many as 7000 mice, and "ITX is 160,000 times more potent than cocaine (Kao, 1966). Surprisingly, pufferfish are eaten in Asian countries, especially-in Japan. Captain James Cook (1777) described the TI'X poisoning in detail which struck him and his crew in 1774 when they were on their second voyage around the world. They had liver and roe for supper and became poisoned. For a history of TTX poisoning, see the excellent review by Kao (1966). It is also surprising that TTX is not synthesized by pufferfish but by the bacterium Pseudomonas sp., which is present in the red alga and forms part of the pufferfish's diet (Yasumoto et aL, 1986), or Vibrio, in the gut (Noguchi et al., 1986). Therefore cultured pufferfish, which are not fed such contaminated alga, are nontoxic. Since the discovery of the selective blocking action of TFX on Na channels (Kao and Fuhrman, 1963; Narahashi et al., 1964), tetrodotoxin has been widely used as a chemical tool for studying ionic properties of Na channels and other types of channels (for reviews see Kao, 1966; Narahashi, 1974; Ritchie and Rogart, 1977; Catterall, 1980; Yoshida et al., 1980a; Ulbricht, 1981; Kao and Levinson, 1986; Prince, 1988; Numa, 1989). Indeed, the properties of Na and other ionic channels would not have been elucidated without TTX. This review article focuses on TTX and does not mention saxitoxin (STX), which is commonly known as a "paralytic shellfish poison." Both T r x and STX are guanidium toxins and act on Na channels. The major point of this article is to describe voltage-gated and TTX-resistant Na channels in mammalian nerve and muscle cells, especially dorsal root ganglion (DRG) cells, on which extensive study has been carried out. It is generally considered that electrical signals of mammalian excitable ceils are fundamentally dependent on voltage-gated Na channels which are sensitive to T r x . However, evidence has been accumulating

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that some such cells are insensitive to TTX. Data on nonmammalian cells are also mentioned to consider the development of TTX-resistant Na channels.

Na CHANNELS

IN TTX-CARRYING

ANIMALS

TI'X is found not only in pufferfish and its related fishes but also in other species of animals such as Costa Rican frogs, Californian salamanders (Taricha), Australian blue-ringed octopuses, shellfish, goby, and some crabs, starfish, and angelfish (Russel, 1965; Kao, 1966; Halstead, 1967; Mosher and Fuhrman, 1984; Yasumoto et aL, 1986; Prince, 1988). These animals use TTX for self-defense or attacking other animals, e.g., the Australian blue-ringed octopus contains T-I'X in its salivary venom glands and uses the toxin for paralyzing its prey, including humans (Mosher and Fuhrman, 1984). It seems reasonable that the voltage-gated Na channels of these animals, carrying TTX in their tissues, need to be insensitive to TTX to survive (Mosher et aL, 1964; Hagiwara and Takahashi, 1967; Kidokoro et al., 1974).

TI'X-RESISTANT Na + C H A N N E L S IN MUSCLE CELLS

S k e l e t a l M u s d e Cells Denervation converts q-TX-sensitive Na + channels in skeletal muscle cells to TTX-resistant ones, i.e., reverting-back phenomenon (Harris and Thesleff, 1971; Redfern and Thesleff, 1971; P~tppone, 1980). The kinetics of converted Na channels became slower (Pappone, 1980). TI'X-resistant Na + channels have also been found in undifferentiated or developing muscle cells (Kidokoro, 1973, 1975; Kano, 1975). It is suggested that Na channels in undifferentiated cells do not possess sensitivity to q'TX. Cardiac M u s c l e Cells Usually, cardiac Na + channels are much less sensitive to TTX than those in most neurons and skeletal muscle cells (Ritchie and Rogart, 1977; CatteraU, 1980; Brown et aL, 1981; Satin et al., 1992). When seen from a point of cellular development, a fraction of Na channels which were resistant to TTX in embryonic chicken heart cells became sensitive to T r x in the later stages (Shigenobu and Sperelakis, 1971; McDonald et al., 1973). However, acquired sensitivity to TTX is not rigid. In some cases, for example, in chick heart cell aggregates, Na channels easily changed their TTX sensitivity (McDonald et al., 1973). Even in older chick hearts, the TrX-sensitive fast Na channels reverted back to the TTX-resistant slow Na + channels when placed in culture (Sperelakis and Shigenobu, 1972).

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Smooth Muscle Cells Some types of smooth muscle cells are reported to have TTX-resistant Na + channels. An example is the smooth muscle isolated from stomach fundus of the rat (Muraki et al., 1991). This TTX-resistant Na channel showed a more negative current-to-voltage ( l - V ) relation than the qTX-sensitive Na channel present in smooth muscle such as that in guinea pig ureter.

TTX-RESISTANT Na + CHANNELS IN NEURONS

Peripheral Nervous System (PNS) Neurons: Dorsal Root Ganglion Cells Since the discovery of TTX-resistant Na + channels using the intracellularrecording method in mammalian neurons, e.g., mouse dorsal root ganglion ( D R G ) cells (Matsuda et aL, 1976), extensive studies have been carried out on D R G cells because of ease of access and the spherical shape of the cell body. In recent years, moreover, there has been renewed interest in D R G cells using whole-cell and single-channel recording techniques for analysis in detail. This section, therefore, outlines the studies on D R G cells. TTX-resistant Na + channels were found first in cultured D R G cells taken from fetal mice (Matsuda et aL, 1976, 1978). Most D R G cells generated a TTX-resistant spike with Na + and Ca 2+ components, i.e., Na + channels were insensitive to qTX. TrX-resistant Na channels were also detected in D R G cells of the adult mouse but in a smaller number (Yoshida et al., 1978). According to morphological, physiological, and pharmacological properties, D R G cells have been classified into subgroups by many researchers. Our classification of mouse D R G cells is described here as an example. We classified D R G cells into three major groups: (1) large or medium-sized neurons which elicit a q~X-sensitive Na soma spike with a myelinated axon and a fast conduction velocity, (2) small neurons which generate a TTX-resistant Na + soma spike with an unmyelinated axon and a slow conduction velocity, and (3) small (occasionally large) neurons with a generally unmyelinated axon which produce a TTX-resistant soma spike with Na + and Ca 2+ components (Matsuda et al., 1978; Yoshida et al., 1978; Yoshida and Matsuda, 1979). Even in cells in which the contribution of Ca 2+ channels to the action potential was negligible under physiological conditions, raising the concentration of a divalent cation (Ca 2+, Sr z+, or Ba 2) led to action potentials, indicating that Ca 2+ channels are present in most D R G cells in varying proportions (Yoshida and Matsuda, 1980; Fedutova et al., 1991). Although there is some debate on how to classify neurons into subgroups, similar results have been obtained from D R G cells of other animal species such as frog (Morita and Katayama, 1989; Campbell, 1992), rat (Lawson et al., 1984; Harper and Lawson, 1985a, b; Caffrey et aL, 1992; Ogata and Tatebayashi, 1992a, 1993), and guinea pig (Fukuda and Kameyama, 1980). It is to be noted, however, that no appreciable TI'X-resistant Na + channels have

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been detected in chick D R G cells (Dichter and Fischbach, 1977; Petersen, et al., 1987). As for axons, spike conduction along the axon was blocked by TTX in all types of mouse D R G cells in physiological solution (Yoshida and Matsuda, 1979). However, propagating Ba 2 or Sr 2 action potentials were observed in a part of the unmyelinated and myelinated axons, indicating that voltage-gated Ca 2 channels are also present in the axon (Yoshida and Matsuda, 1980; Yoshida et aL, 1980b). These findings were supported by a study using a patch-pipette on the large growth cone membrane, i.e., 13 fiber terminal, of the cultured rat D R G cells (Yamaguchi, 1992). The growth cone had voltage-gated Na , Ca 2, and K channels, and the Na channels were sensitive to TTX.

Other Peripheral Nervous System Neurons

TTX-resistant Na channels have been found in other PNS neurons such as bullfrog sympathetic (Jones, 1987) and parasympathetic neurons (Clark et al., 1990), trigeminal and ciliary ganglion cells of quail embryos (Schlichter et al., 1991), rat and rabbit nodose ganglion cells (Bossu and Feltz, 1984; Baccaglini and Cooper, 1982; Ikeda et al., 1986; Ikeda and Schofield, 1987; Stansfeld and Wallis, 1985), rat and cat petrous ganglion cells (Bossu and Feltz, 1984; Gallego, 1983), and rat superior cervical neurons (Schofield and Ikeda, 1988). Results similar to those with D R G cells have been obtained from these preparations. For example, TTX-resistant Na channels in adult rat nodose ganglion cells were blocked by Ca 2 channel blockers (Cd 2, Co 2, and D600) and exhibited a slower time course than TTX-sensitive ones (Bossu and Feltz, 1984; Ikeda et aL, 1986; Ikeda and Schofield, 1987; Clark et al., 1990).

Central Nervous System Neurons

Intensive study has been carried out on central nervous system (CNS) neurons. However, not many TI'X-resistant Na channels have been reported compared with PNS neurons. Rather, TTX is used to block Na + currents to work on other types of channels in CNS neurons. A chain of segmental ganglia forms the CNS of the leech, and action potentials of the Retzius ganglion neuron depend on TTX-resistant Na channels (Kleinhaus and Prichard, 1976). As for mammals, TTX-resistant voltage-gated Na channels were activated by neurotensin in dopaminergic neurons of the rat mesencephalon (Mercuri et al., 1993) and by oxytocin in vagal neurons of the rat brainstem (Raggenbass and Dreifuss, 1992). Moreover, approximately half of the neurons in the rat ventral tegmental area showed only an ordinary TTX-sensitive Na + current, but the remaining neurons exhibited a Tl'X-resistant Na current as well (Ogata and Tatebayashi, 1992c). Furthermore, acutely dissociated neurons from the medial entorhinal cortex of the rat exhibited a TI'X-resistant Na current in addition to the TTX-sensitive Na current (White et al., 1993). The TTX-resistant Na + current was more sensitive to inorganic Ca 2 channel blockers ( C d 2+, La 3+, and Z n 2+) than the TTX-sensitive one.

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TTX-RESISTANT Na + CHANNELS IN GLIAL CELLS Vertebrate glial cells have voltage-gated Na channels (see review by Barres et al., 1990). Different results have been obtained for the TFX sensitivity of Na channels in these cells. For example, poor sensitivity of Na + channels to TTX was reported for cultured rat astrocytes (Bevan et aL, 1985) and acutely isolated Schwann ceils in adult rabbits (Chiu, 1987). On the other hand, Na channels were highly sensitive to TTX in acutely dissociated astrocytes of retinal ganglion (Barres et aL, 1989) and Schwann cells in culture (Shrager et al., 1985). Furthermore, expression of TTX-sensitive and TTX-resistant Na channels was confined to morphologically distinguishable subtypes of astrocytes and their kinetics were differently controlled by protein kinase C, indicating that TI'X sensitivity of the Na channel is related to the function of these cells (Thio and Sontheimer, 1993).

DIFFERENCES

IN P R O P E R T I E S OF T T X - R E S I S T A N T A N D TTXS E N S I T I V E Na C H A N N E L S

Sensitivity to Ca 2+ Channel Blockers

Usually TTX-resistant Na channels are blocked by both inorganic (Co 2, Mn 2+, Ni 2, Cd 2+, Zn ~+, La 3+) and organic (D-600) Ca ~+ channel blockers (Kostyuk et al., 1981; Ikeda and Schofield, 1987; Roy and Narahashi, 1992; White et al., 1993). However, TrX-resistant Na channels in adult rat nodose ganglion cells were less sensitive to inorganic Ca 2+ channel blockers than Ca 2+ channels present in the same preparation (Ikeda and Schofield, 1987).
Channel Kinetics

Electrophysiological properties of TI'X-resistant Na + channels in DRG cells have been investigated in more detail using the whole-cell and single-channel recording techniques, and it has been shown that the kinetics of TI'X-resistant Na + channels are usually different from TTX-sensitive Na channels. In comparison with TTX-sensitive Na channels, "l-TX-resistant Na channels show a lower single-channel conductance, slower activation and inactivation processes, and a shift in a more depolarized direction of the current-voltage relation, the conductance-voltage curve, and the steady-state inactivation curve in both immature and adult D R G neurons (Pappone, 1980; Kostyuk et al., 1981; McLean et al., 1988; Omri and Meiri, 1990; Schwartz et al., 1990; Fedulova et al., 1991; Roy and Narahashi, 1992; Ogata and Tatebayashi, 1992b, 1993; Caffrey et al., 1992; Campbell, 1993; Elliott and Elliott, 1993), sensory cranial ganglion cells (Bossu and Feltz, 1984), and rat skeletal muscle cells (Weiss and Horn, 1986). In contrast, no significant differences or even reversal in the polarity of differences in the current-voltage relationship were reported in the frog node of Ranvier (Benoit et aL, 1985), guinea pig DRG ceils (Petersen et al., 1987), bullfrog

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sympathetic (Jones, 1987), and parasympathetic (Clark et aL, 1990) neurons, and a human neuroblastoma cell line LA-N-5 (Weiss and Sidell, 1991). In connection with this, it should be remembered that a single-point mutation in the amino acid sequence of the rat brain Na channel decreased its sensitivity to TFX and induced a hyperpolarizing shift in the current-voltage relation (Noda et al., 1989). As for CNS neurons, TTX-resistant Na channels are kinetically indistinguishable from TTX-sensitive ones in neurons of the rat medial entorhinal cortex (White et aL, 1993) and the rat ventral tegmental area (Ogata and Tatebayashi, 1992c). It is interesting that only TI'X-resistant Na channels were found when adult rat D R G cells were cultured in the absence of serum and nerve growth factor (Aguayo et al., 1991). The major effect of nerve growth factor was concluded to be an acceleration in kinetics and acquisition of sensitivity to TFX of Na channels (Omri and Meiri, 1990). In addition, the proportion of Na channels of fast and slow kinetics was dependent on temperature in the frog node of Ranvier, and it was suggested that forms of the Na channel were related to different lipid environments of the protein constituting the channel (Benoit et aL, 1985).

M O L E C U L A R A S P E C T S OF T H E T T X - R E S I S T A N T Na + C H A N N E L

The Na channel purified from the electric eel is a glycoprotein with a molecular weight of approximately 215,000 and is comprised of approximately 2000 amino acids, 500 sugar residues, and 50 fatty acid chains. However, Na + channels isolated from mammalian skeletal muscle and brain have more complex subunit structures consisting of a- and/3-subunits (Noda et al., 1984, 1986; Numa, 1989). Most of the functional properties, including the sensitivity to TTX, of the Na channel reside in the large a-subunit (Noda et al., 1986; Catterall, 1988; Barchi, 1988; Trimmer and Agnew, 1989; Mandel, 1992). TTX is a membrane impermeant and positively charged guanidium toxin and it blocks Na channels from the extracellular side. It is thought that the positively charged guanidium moiety of TTX interacts with the negatively charged amino acids present near or within the mouth of the channel. Indeed, when the amino acids which form the negatively charged lining at the mouth of the Na channel were replaced by positively charged same amino acids or by some other amino acids, the sensitivity of the Na channel to T r X was completely abolished (Terlau et al., 1991). Thus, a TFX molecule physically occludes the channel pore by plugging the mouth of the Na + channel with the aid of electrical force. This plugging scheme indicates that TTX blocks a flow of ions through the Na channel without interfering with the movement of the gate of the channel (Armstrong and Bezanilla, 1974). It is commonly assumed that one T'I'X molecule suffices to block one Na + channel, although it has been proposed that two TTX molecules are necessary to block the channel (Benoit et al., 1985). Interestingly, TrX-sensitive Na + channels can be easily rendered insensitive to the toxin by treatment with trimethyloxonium (TMO), which methylates carboxylic acids of the TTX receptor. The single-channel conductance of such

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converted Na channels has been shown to be reduced (Sigworth and Spalding, 1980; Spalding, 1980; Weiss and Horn, 1986). Three distinct mRNAs (types I, II, and III) encoding Na channels have been isolated from the rat brain, and only the type I Na channel lacked T-FX sensitivity (Noda et al., 1986). Seen from a structural point of view, glutamate residue 387 seems to be an essential amino acid in the TTX-binding site of the Na channel because replacing residue 387 with the uncharged amino acid glutamine greatly reduced the sensitivity of the Na channel (Noda et al., 1989). As for the cardiac Na channel, the amino acid at position 374 controlled the sensitivity of the channel to TTX and Cd 2 (Satin et al., 1992). In normal skeletal muscle, only m R N A encoding TTX-sensitive Na channels is expressed. However, expression of m R N A encoding the TTXresistant Na channels increased after denervation and declined as reinnervation appeared (Yang et al., 1991). Moreover, a Na channel subtype, microI, isolated from a denervated rat skeletal muscle cDNA library, was not expressed in brain or heart but was found at low levels in neonatal skeletal muscle and increased to maximum levels in adult tissue, paralleling the expression of TFX-sensitive Na channels (Trimmer et aL, 1990). Surprisingly, denervation of adult muscle was also followed by a rise in microI mRNA, at a time when Tl~X-resistant Na channels reappear. Furthermore, SkM2 mRNA, an m R N A encoding the c~subunit of the TTX-resistant Na channel in denervated rat skeletal muscle, was not detectable in normally innervated adult skeletal muscle but was abundant in rat cardiac muscle (Kallen et aL, 1990). The SkM2 m R N A level was highest in early development, when TTX-resistant Na channels predominate, but declined rapidly with the age of the animal. The cardiac TTX-resistant Na channels have been proved to be identical to those in denervated skeletal muscles (Kallen et al., 1990; White et al., 1991). It is puzzling that Na channels expressed in X e n o p u s oocytes by injecting m R N A from mammalian heart cells, which are considerably resistant to TTX, became highly sensitive to TTX (Sutton et aL, 1988) and that transfection of the ras oncogene into a cell line derived from a mouse anterior pituitary tumor converted TTX-resistant Na channels to TTX-sensitive ones (Flamm et aL, 1990).

F U N C T I O N A L SIGNIFICANCE OF T H E 'ITX-RESISTANT N a
CHANNEL

Ogata and Tatebayashi (1992c, 1993) proposed that the slow inactivation process of TTX-resistant Na channels plays an important role in deciding cellular function, i.e., the regulation of cellular long-term excitability such as firing pattern and adaptation. Elliott and Elliott (1993) suggest that TTX-resistant Na channels rapidly recover from inactivation and are suited to firing long trains of action potentials in response to a depolarizing stimulus. Thus, distinct types of Na channels are suggested to contribute to different types of sensory information. We should, however, consider that the D R G cell is unipolar (or pseudounipolar) and the cell body does not directly take part in impulse conduction.

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Indeed, Na channels of both A and C nerve fibers of mouse D R G cells have been shown to be sensitive to TTX and suitable for rapid impulse conduction (Yoshida and Matsuda, 1979; Yamaguchi, 1992).
Comparison of CNS and PNS Neurons

Differences in ionic mechanisms of action potentials are observed in CNS and PNS neurons. Action potentials of spinal cord neurons are dependent exclusively on TTX-sensitive Na channels, whereas TTX-resistant Na and Ca 2 channels also contribute to action potentials in D R G cells of the mouse (Ransom and Holz, 1977; Heyer and MacDonald, 1982). Therefore, Ca 2+ enters during an action potential in D R G cells and the slow time course of TI'X-resistant Na channels aids influx of Ca 2 by causing a sustained depolarization of the membrane (Fedulova et al., 1991). Interestingly, influx of Ca 2 has been shown to be toxic to CNS neurons but not to PNS neurons (Yoshida et al., 1979). Indeed, the conductance of Na channels was reduced by intracellular Ca 2 (Heyer and MacDonald, 1982). It is possible that the function of CNS neurons is so delicately controlled by the intracellular concentration of Ca 2 that it is necessary to avoid exposure to a large Ca 2 influx from outside. Kinetics of TTX-resistant Na channels have been demonstrated to be slower than those of TTX-sensitive ones in PNS neurons (Kostyuk et al., 1981; Fedulova et al., 1991; Ogata and Tatebayashi, 1993; Elliott and Elliott, 1993). However, most Na channels in cell bodies of CNS neurons are sensitive to TTX, and both TTX-sensitive and T-l'X-resistant Na channels exhibit similar kinetics in CNS neurons (Ogata and Tatebayashi, 1992c; White et aL, 1993). It has been speculated that the kinetics of all Na channels must be fast in CNS neurons for rapid and effective information processing. The advantages of using Na channels, especially TTX-sensitive Na channels which have a high single-channel conductance and the fastest kinetics among ionic channels, for phylogenetically higher animals and ontogenetically differentiated cells are (1) fast conduction of action potentials along the axon, (2) rapid processing of information in neurons, and (3) accurate and undisturbed information processing, because using Na separates the process from other many cellular functions which are dependent on Ca 2. TTX-carrying animals seem to utilize the toxin for protecting themselves from enemies whose movements are faster but are dependent on the activity of TTX-sensitive Na channels.

Na + CHANNELS DURING DEVELOPMENT

Ionic channels change their numbers and properties during development. In the mouse, action potentials could be evoked in ovarian and ovulated oocytes when inactivation of ionic channels was removed by hyperpolarizing the membrane potential (Okamoto et al., 1977; Yoshida 1982, 1983a, b). This action

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potential was dependent on the opening of Ca'-* channels which were insensitive to TTX. No Na channels were detected. However, Na could pass through oocyte Ca 2 channels when the extracellular Ca 2 concentration was very low. Similarly, action potentials were exclusively dependent on the activity of Ca 2 channels in cleaving embryos of the mouse and the density of Ca 2 channels was reduced with development (Yoshida, 1983b, 1985, 1986b; Mitani, 1985). The next stages of development have been studied in dorsal root ganglion (DRG) cells from fetal to adult mice. The cell body of the fetal mouse DRG had a high proportion of TTX-resistant Na channels. Na channels increased in number and sensitivity to TTX with the maturation of cells and the myelination of axons in the adult mouse (Matsuda et al., 1978; Yoshida et aI., 1978, 1980a). This replacement of TI'X-resistant Na channels by TFX-sensitive ones has been reported in various other tissues such as skeletal muscle cells (Gonoi et al., 1985; Weiss and Horn, 1986), human neuroblastoma cells (Weiss and Sidell, 1991), and mouse brain (Unsworth and Hafemann, 1975). In contrast, Ca 2 channels acquired sensitivity to an organic Ca -,+ channel blocker diltiazem (Yoshida, 1986a, b) while reducing their number during development from oocytes to adults (Matsuda et al., 1978; Yoshida et al., 1978, 1980a; Yoshida, 1983b, 1985; Mitani, 1985; Kostyuk et al., 1993). Interestingly, TTX-resistant Na + channels of rat DRG cells were inhibited by Ca 2 channel blockers, but the selectivity of these channels did not significantly differ from TFX-sensitive Na channels (Kostyuk et al., 1981; Roy and Narahashi, 1992), indicating that the T-FX-resistant Na channel combines some features of Ca 2 and Na channels. Also, TFX-resistant Na channels in embryonic chick heart share some characteristics with the Ca 2+ channel and the T-FX-sensitive Na channel (Bkaily et al., 1991). In contrast, Ca 2 conducting Na + channels have been reported in rat hippocampal neurons (Akaike and Takahashi, 1992; Takahashi et al., 1992). This Ca 2 conducting Na channel was sensitive to TTX and blocked by Ca : antagonists. Also, the TTX-resistant Na channel in entorhinal cortex neurons of the rat was suppressed by inorganic Ca 2 channel blockers, although the channel kinetics were similar to those of a T-I'X-sensitive Na current (White et aL, 1993). In contrast to the cell body, the axon of mouse DRG produced conducting spikes chiefly dependent on TTX-sensitive Na + channels (Yoshida and Matsuda, 1979). Hence it is possible that (1) the axon develops faster than the cell body with respect to spike-generation mechanisms because impulse conduction occurs along the axon and the cell body does not directly take part in it, and (2) Na channels in the cell body whose axon participates in primitive information (e.g., conveying dull pain along the unmyelinated C fiber) remains undifferentiated, i.e., somatic Na + channels are resistant to TTX and slow in time course.

Lower Animals

Ionic channel types appear to be different in lower animals. Na + channels are present as early as in oocytes in the tunicate (Okamoto et al., 1976), starfish

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(Miyazaki et al., 1975), flatworm (Miyazawa et al., 1987), and frog (Schlichter, 1983). These Na channels are all resistant to TTX. Also, in the blastomere of the tunicate embryo, Na channels in differentiating neurons were highly resistant to TTX and the channel subtypes showed distinct kinetic properties at the single-channel level (Okamura and Shidara, 1990).

Major Developmental Courses of Ionic Channels In summary, developmental changes of major ionic channels contributing to action potentials under physiological conditions seems to take either one of the two courses. One course is that the inward current, which is responsible for generating action potentials, is initially dependent on Ca 2+ channels, then on Na + and Ca 2+ channels, and finally, on Na + channels, as was observed in mouse DRG cells (Matsuda et aL, 1978; Yoshida et aL, 1978, 1980a; Yoshida, 1985). There have been similar findings in chick (Kano, 1975) and rat (Kidokoro, 1975) skeletal muscle cells and in amphibian neurons (Spitzer, 1979). The alternative course is that the number of Ca 2+ channels increases, whereas that of the Na channels decreases with age as seen in phylogenetically lower animals, e.g., tunicate muscle cells (Miyazaki et al., 1974).

EVOLUTION OF Na CHANNELS

Evolution of Ionic Channels Seen at the Gene Level The homology of amino acid sequences of Na channels in various tissues and in animals is high, suggesting that these Na channels are derived from the same gene. Moreover, the structures of voltage-gated Ca 2 and K channels have been shown to be similar to the voltage-gated Na channel (Catterall, 1988; Trimmer and Agnew, 1989; Neumcke, 1990; Mandel, 1992). This suggests therefore, that these voltage-gated channels, i.e., Na , Ca 2 and K channels, all differentiated from the same ancestor gene encoding a prototype of these channels during development. It is to be noted that replacement of only one amino acid of the Na channel structure has been demonstrated to change its ionic selectivity such that not only Na but also K and Ca 2 can easily pass through the Na channel, i.e., the Na channel became like the nonselective cation channel. In addition, when two amino acids were replaced, the Na channel became more permeable to Ca 2 than to Na (Heinemann et al., 1992). These observations indicate that Na , Ca 2, and K channels utilize a very similar structure for their base, suggesting that these types of channels derived from a prototype channel. Furthermore, it should be noted that TTX-resistant Na channels resemble Ca 2 channels in their slow time course, TTX insensitivity and sensitivity to inorganic divalent cations, and organic Ca 2 channel blockers (Kostyuk et aL 1981; Roy and Narahashi, 1992). These findings suggest a close developmental relationship between Na and Ca 2 channels.

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%"

( St0m

.=l )

Fig. 1. Schematicdiagram of presumed evolution of Na channels. Na +, Ca2+, and nonselective cation channels probably derived from the same "stem channel." Ca2 channels in the circle and square denote that Ca2 channels are diverse in type in various types of cells and animal species. In contrast, Na channels are much the same. Therefore, the primitive Na channel must have appeared later than the primitive Ca~- channel. Both Tl'X-sensitive and TTX-insensitive Na channels, which originated from the primitive Na channel, are present in the tissues named in the diagram. Dashed lines indicate the speculative connections. CNS and PNS, central and peripheral nervous systems.

Evolutional Hypothesis of Ionic Channels

D e v e l o p m e n t of ionic channels should be considered not only ontogenetically but also phylogenetically, and hypotheses on evolution of channels have been p r o p o s e d (see review by Hille, 1992). I have p r o p o s e d here an evolutional hypothesis of ionic channels on the basis of o u r results on the m o u s e and the data obtained by other researchers (see Fig. 1). Voltage-gated Ca 2 channels are present in various types of cells in m a n y animals, especially at early stages of development. Also, properties of Ca 2 channels are considerably diverse between cell types and animal species. Thus, Ca 2 channels m a y be assumed to be both ontogenetically and phylogenetically old and to have undergone alterations during a long period of evolution. In contrast, Na channels s e e m to be incorporated into muscles, neurons, glial cells etc. at later stages of ontogenesis and phylogenesis (Fig. 1). Therefore, the properties of Na channels are m o r e conserved a m o n g different tissues and animals. As m e n t i o n e d earlier, TTX-sensitive Na channels with fast kinetics are m o r e frequently observed in differentiated cells and in higher animals. If we take the sensitivity of Na channels to T I ' X as an indication of a higher evolutional stage, we can conclude that axons are m o r e developed than cell bodies of

"l'TX-Resistant Na + Channels

239

neurons, CNS neurons more developed than PNS neurons, and skeletal muscle cells more developed than cardiac and smooth muscle cells (Fig. 1). The fact that Na channels in axons are resistant to "ITX in lower animals such as snail (Wald, 1972), clams and shellfish (Twarog et al., 1972) is a phylogenetical example of less developed Na channels. Ionic selectivity of channels should also be considered here. Ca 2 channels could be made permeable to Na when the concentration of Ca 2 was low (Yoshida, 1982, 1983a, 1985). In addition, TI'X-resistant Na channels could be inhibited by Ca 2 channel blockers but the ionic selectivity was not significantly different from that of TrX-sensitive Na channels (Kostyuk et aL, 1981; Roy and Narahashi, 1992; White et al., 1993). Hence TrX-resistant Na channels combine features of both Ca 2 and Na channels. Furthermore, Ca 2 conducting Na channels which are sensitive to TI'X but are blocked by Ca 2 antagonists have been reported (Akaike and Takahashi, 1992; Takahashi et al., 1992). When ionic channels are seen from a molecular aspect, it is possible that there is a "stem channel" from which Ca 2 and Na channels derived (Hille, 1992). This stem channel may explain the evolution of ionic channels, i.e., nonselective cation channels and primitive forms of Ca 2 and Na channels may have originated from stem channels (Fig. 1). As indicated by dashed lines in Fig. 1, the evolutional interactions of these channels still remain to be elucidated.

ACKNOWLEDGMENTS
I appreciate Dr. S. Macmillan for her suggestions on the manuscript, Professors M. Niwa, Y. Matsuda, K. Taniyama, and H. Oka for their encouragement.

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