reviews-
Ion channelorganizationof the myelinatedfiber Joel A. Black, Jeffery D. Kocsis and Stephen G. W a x m a n
JoelA. Black,Jeffet7 D. KocsisandStephen G Waxmanare at the Departmentof Neurology, Yale UniversitySchoolof Medicine, New Haven, CT06510, USAand the Center for Neuroscience Research,VAMedical Center, WestHaven, CT06516, USA.
The myelinated axon provides a model in which it is possible to examine how various types of ion channels are incorporated into a membrane to form an excitable neuronal process. The available evidence now indicates that mammalian myelinated fibers contain a repertoire of physiologically active membrane molecules including at least four types of ion channels and an electrogenic Na+,K+-pump. Physiological properties of myelinated fibers reflect the distribution of these various types of channels and pumps, as well as interactions with myelinating Schwann cells in the P N S or oligodendrocytes in the CNS. A growing body of data also suggests a role for astrocytes and Schwann cells at nodes of Ranvier. This article reviews the current understanding of the ion channel organization of the mammalian myelinated fiber. Ion channels in the mammalian myelinated axon have a complex organization. It is widely appreciated that the axon membrane in myelinated fibers is spatially heterogeneous, with a non-uniform distribution of voltage-dependent sodium (Na ÷) channels and 4-aminopyridine-sensitive potassium (K +) channels 1. Recently, however, it has become apparent that the mammalian myelinated fiber is even more complex than previously thought. V o l t a g e - s e n s i t i v e Na + c h a n n e l s
Voltage-dependent Na + channels are present in high density in the axon membrane in the node of Ranvier, and in lower densities in the internodal axon membrane under the myelin sheath 1. Cytochemical studies z'3 provided early morphological evidence for this heterogeneous distribution of Na + channels along the myelinated axon membrane. More recently, axon membrane ultrastructure has been studied using a variety of other methods, including freeze-fracture and immunoelectron microscopy. The axon membrane of most non-myelinated axons shows a uniform structure when viewed by freezefracture4; intramembranous particles, which represent integral membrane protein or glycoprotein molecules, are distributed evenly throughout the membrane in freeze-fracture replicas. This uniform pattern of membrane structure presumably reflects the homogeneous distribution of Na ÷ channels along the non-myelinated axon, and provides a morphological correlate for continuous conduction in most nonmyelinated axons. Myelinated axons, on the other hand, display a nonuniform axon membrane structure. The axon membrane at nodes of Ranvier exhibits a high density (~1300/~m 2) of external leaflet (E-face) intramembranous particles, with a relatively high percentage of large diameter (> 10 nm) particles. The internodal axon membrane has a lower density (75-150/~m 2) of E-face particles ~-7. Rosenbluth5 has suggested that some large intramembranous particles within the nodal axon membrane may be the morphological correlate of voltage-dependent Na + channels. 48
Subsequent to these early morphological studies, immunocytochemical techniques have been developed that permit the immunolocalization of Na ÷ channels, at the electron microscopic level, in mammalian CNS 8'9. As shown in Fig. 1, incubation of rat optic nerve with polyclonal antibody 7493 (directed against the 260 kDa oc-subunit of Na + channels purified from mammalian brain l°) yields dense immunoreactivity that is specifically associated with the axon membrane at the node of Ranvier. In contrast, the internodal axon membrane does not stain with 7493 despite its accessibility to a variety of antibodies such as those directed against neurofilaments8. These observations provide an immuno-ultrastructural demonstration of the localization of Na ÷ channels within nodal membrane in the mammalian CNS. Saxitoxin (STX)-binding studies provide a numerical estimate of Na ÷ channel density n. The early STXbinding studies suggested a Na + channel density of ~-12000/~m 2 in the axon membrane at the node of Ranvier, and a much lower Na ÷ channel density (30 ms and reaches a steady level over the next 100200 ms. Baker et al. 32 used electrotonus measurements to study the inward rectifier in rat spinal root myelinated axons, and showed that it is abolished by caesium (Cs +) but not by barium (Ba2+). Eng et al. a5 found that in rat optic nerve, low concentrations of Cs + completely blocked the inward rectification, and Ba 2+ induced a partial block. Ion substitution experiments demonstrate that both Na + and K + are responsible for inward current associated with inward rectification in optic nerve fibersaS. Interestingly, stimulus-response curves obtained during the hyperpolarization pulse, before and during inward rectification, indicate that excitability is increased (probably as a result of membrane potential moving to a less hyperpolarized level) during inward rectification. This, along with intra-axonal recordings showing inward rectification, demonstrates that the origin of the inward rectification is axonal and not gliala5. The fast and slow K ÷ conductances and the inward rectifier appear to be present in human axons. Human sensory axons have been shown to be sensitive to 4-AP a6. Moreover, human axons recorded in vivo possess a slowly activating K ÷ channel and inward rectificationaT. The data from human axons suggest that, as in the rat, slow K ÷ channels play a role in accommodation. The working hypothesis that has emerged from the above work is that myelinated axons display a variety of rectifying channels that have important functional roles in the activity of axonal action potentials (Table I). The highest density of Na t channels is found at the node of Ranvier, where they are required for the depolarization phase of the action potential. The 4-AP-sensitive K ÷ channel, which has a significant representation in the paranodal/intemodal axon membrane, is kinetically fast and, when activated, may contribute to rapid repolarization after the action potential. The kinetically slower TEA-sensitive K + channel is present at the node of Ranvier, and its activation during repetitive spike activity leads to a pronounced afterhyperpolarization. This conductance TINS, Vol. 13, No. 2, 1990
may play a role in accommodation and in the regulation of multiple spike discharge. The functional role of the inward rectifier is less clear, but it may provide for repolarization of the axon membrane during periods of intense action potential activity marked by hyperpolarization. More work to delineate the precise role of these channels, and to determine whether any other types of channels are present in mammalian myelinated axons, is certainly warranted. An important focus for future work will be the development of specific ligands that permit the ultrastructural localization of the various types of K+channels.
TABLE I. Ion channels in mammalian myelinated axons
Channel type
Blocked by
Probable function
Primary location
Na +
TTX, STX
Depolarization phase of action potential
High density at node; low density in internode
K+ (fast)
4-AP
Rapid repolarization after action potential
Internode
K+(slow)
TEA, Ba2+
Modulate repetitive firing; Node, internode repolarization in response to prolonged depolarization
Inward rectifier (mixed K+, Na + conductance)
Cs+ (reduced by Ba2+)
May modulate excitability; attenuate hyperpolarization
L o w d e n s i t i e s of Na ÷ c h a n n e l s c a n support c o n d u c t i o n in s m a l l a x o n s While Na + channel densities in the internodal axon membrane may be insufficient for action potential conduction under normal circumstances, recent results suggest that conduction may be supported by low densities of Na + channels in some small-diameter axons. As part of an effort to examine the basis for conduction in fibers that lack myelin, together with J. M. Ritchie, we have studied Na + channel density and action potential electrogenesis in the neonatal rat optic nerve 38. While no myelin is present in the neonatal optic nerve, all of the fibers are destined to become myelinated as development proceeds 29. The density of Na ÷ channels in premyelinated optic nerve axons was estimated from measurements of the binding of [3H]STX. Surprisingly, maximum saturable binding capacities corresponded to a high-affinity (KD=0.88 riM) STX-binding site density of ~2/[zm2 within premyelinated axon membrane of the neonatal optic nerve (Fig. 4A). This low Na + channel density was especially interesting in view of previous results 29 that demonstrated conduction of action potentials (conduction velocity = 0.2 m/s at 37°C) in neonatal optic nerve. These earlier studies demonstrated that the action potential is abolished by 1 ~M tetrodotoxin, and by replacement of bath Na + with the impermeant cation TRIS. Moreover, there is no evidence for propagated Ca 2+ responses along the premyelinated axon trunks in neonatal rat optic nerve 29. In view of the low density of saxitoxin-binding sites, it was important to determine whether STXinsensitive channels might contribute to action potential electrogenesis. We therefore examined the effects of low concentrations of STX on conduction, and demonstrated that concentrations as low as 5 nM STX reversibly abolished the neonatal optic nerve action potentialas (Fig. 4B). This is close to the value predicted to block conduction, on the basis of the dissociation constant for STX observed in our binding experiments (0.88 riM). Taken together with the STX binding results, these observations indicate that low densities of Na + channels can support action potential conduction in axons of neonatal optic nerve. The Na + channel densities in neonatal rat optic nerve are substantially below those predicted to maximize conduction velocities in the squid giant axon39'4°. However, there is a precedent for action TINS, VoL 13, No. 2, 1990
Node, internode
potential conduction in some non-myelinated axons with low Na + channel densities. Table II summarizes estimates of Na + channel density in non-myelinated and premyelinated axon membrane, derived from [uH]STX-binding studies. In olfactory nerve from garfish and bullfrog (Rana catesbeiana), and in optic nerve from Necturus, Na ÷ channel densities of
Ion channelorganizationof the myelinatedfiber Joel A. Black, Jeffery D. Kocsis and Stephen G. W a x m a n
JoelA. Black,Jeffet7 D. KocsisandStephen G Waxmanare at the Departmentof Neurology, Yale UniversitySchoolof Medicine, New Haven, CT06510, USAand the Center for Neuroscience Research,VAMedical Center, WestHaven, CT06516, USA.
The myelinated axon provides a model in which it is possible to examine how various types of ion channels are incorporated into a membrane to form an excitable neuronal process. The available evidence now indicates that mammalian myelinated fibers contain a repertoire of physiologically active membrane molecules including at least four types of ion channels and an electrogenic Na+,K+-pump. Physiological properties of myelinated fibers reflect the distribution of these various types of channels and pumps, as well as interactions with myelinating Schwann cells in the P N S or oligodendrocytes in the CNS. A growing body of data also suggests a role for astrocytes and Schwann cells at nodes of Ranvier. This article reviews the current understanding of the ion channel organization of the mammalian myelinated fiber. Ion channels in the mammalian myelinated axon have a complex organization. It is widely appreciated that the axon membrane in myelinated fibers is spatially heterogeneous, with a non-uniform distribution of voltage-dependent sodium (Na ÷) channels and 4-aminopyridine-sensitive potassium (K +) channels 1. Recently, however, it has become apparent that the mammalian myelinated fiber is even more complex than previously thought. V o l t a g e - s e n s i t i v e Na + c h a n n e l s
Voltage-dependent Na + channels are present in high density in the axon membrane in the node of Ranvier, and in lower densities in the internodal axon membrane under the myelin sheath 1. Cytochemical studies z'3 provided early morphological evidence for this heterogeneous distribution of Na + channels along the myelinated axon membrane. More recently, axon membrane ultrastructure has been studied using a variety of other methods, including freeze-fracture and immunoelectron microscopy. The axon membrane of most non-myelinated axons shows a uniform structure when viewed by freezefracture4; intramembranous particles, which represent integral membrane protein or glycoprotein molecules, are distributed evenly throughout the membrane in freeze-fracture replicas. This uniform pattern of membrane structure presumably reflects the homogeneous distribution of Na ÷ channels along the non-myelinated axon, and provides a morphological correlate for continuous conduction in most nonmyelinated axons. Myelinated axons, on the other hand, display a nonuniform axon membrane structure. The axon membrane at nodes of Ranvier exhibits a high density (~1300/~m 2) of external leaflet (E-face) intramembranous particles, with a relatively high percentage of large diameter (> 10 nm) particles. The internodal axon membrane has a lower density (75-150/~m 2) of E-face particles ~-7. Rosenbluth5 has suggested that some large intramembranous particles within the nodal axon membrane may be the morphological correlate of voltage-dependent Na + channels. 48
Subsequent to these early morphological studies, immunocytochemical techniques have been developed that permit the immunolocalization of Na ÷ channels, at the electron microscopic level, in mammalian CNS 8'9. As shown in Fig. 1, incubation of rat optic nerve with polyclonal antibody 7493 (directed against the 260 kDa oc-subunit of Na + channels purified from mammalian brain l°) yields dense immunoreactivity that is specifically associated with the axon membrane at the node of Ranvier. In contrast, the internodal axon membrane does not stain with 7493 despite its accessibility to a variety of antibodies such as those directed against neurofilaments8. These observations provide an immuno-ultrastructural demonstration of the localization of Na ÷ channels within nodal membrane in the mammalian CNS. Saxitoxin (STX)-binding studies provide a numerical estimate of Na ÷ channel density n. The early STXbinding studies suggested a Na + channel density of ~-12000/~m 2 in the axon membrane at the node of Ranvier, and a much lower Na ÷ channel density (30 ms and reaches a steady level over the next 100200 ms. Baker et al. 32 used electrotonus measurements to study the inward rectifier in rat spinal root myelinated axons, and showed that it is abolished by caesium (Cs +) but not by barium (Ba2+). Eng et al. a5 found that in rat optic nerve, low concentrations of Cs + completely blocked the inward rectification, and Ba 2+ induced a partial block. Ion substitution experiments demonstrate that both Na + and K + are responsible for inward current associated with inward rectification in optic nerve fibersaS. Interestingly, stimulus-response curves obtained during the hyperpolarization pulse, before and during inward rectification, indicate that excitability is increased (probably as a result of membrane potential moving to a less hyperpolarized level) during inward rectification. This, along with intra-axonal recordings showing inward rectification, demonstrates that the origin of the inward rectification is axonal and not gliala5. The fast and slow K ÷ conductances and the inward rectifier appear to be present in human axons. Human sensory axons have been shown to be sensitive to 4-AP a6. Moreover, human axons recorded in vivo possess a slowly activating K ÷ channel and inward rectificationaT. The data from human axons suggest that, as in the rat, slow K ÷ channels play a role in accommodation. The working hypothesis that has emerged from the above work is that myelinated axons display a variety of rectifying channels that have important functional roles in the activity of axonal action potentials (Table I). The highest density of Na t channels is found at the node of Ranvier, where they are required for the depolarization phase of the action potential. The 4-AP-sensitive K ÷ channel, which has a significant representation in the paranodal/intemodal axon membrane, is kinetically fast and, when activated, may contribute to rapid repolarization after the action potential. The kinetically slower TEA-sensitive K + channel is present at the node of Ranvier, and its activation during repetitive spike activity leads to a pronounced afterhyperpolarization. This conductance TINS, Vol. 13, No. 2, 1990
may play a role in accommodation and in the regulation of multiple spike discharge. The functional role of the inward rectifier is less clear, but it may provide for repolarization of the axon membrane during periods of intense action potential activity marked by hyperpolarization. More work to delineate the precise role of these channels, and to determine whether any other types of channels are present in mammalian myelinated axons, is certainly warranted. An important focus for future work will be the development of specific ligands that permit the ultrastructural localization of the various types of K+channels.
TABLE I. Ion channels in mammalian myelinated axons
Channel type
Blocked by
Probable function
Primary location
Na +
TTX, STX
Depolarization phase of action potential
High density at node; low density in internode
K+ (fast)
4-AP
Rapid repolarization after action potential
Internode
K+(slow)
TEA, Ba2+
Modulate repetitive firing; Node, internode repolarization in response to prolonged depolarization
Inward rectifier (mixed K+, Na + conductance)
Cs+ (reduced by Ba2+)
May modulate excitability; attenuate hyperpolarization
L o w d e n s i t i e s of Na ÷ c h a n n e l s c a n support c o n d u c t i o n in s m a l l a x o n s While Na + channel densities in the internodal axon membrane may be insufficient for action potential conduction under normal circumstances, recent results suggest that conduction may be supported by low densities of Na + channels in some small-diameter axons. As part of an effort to examine the basis for conduction in fibers that lack myelin, together with J. M. Ritchie, we have studied Na + channel density and action potential electrogenesis in the neonatal rat optic nerve 38. While no myelin is present in the neonatal optic nerve, all of the fibers are destined to become myelinated as development proceeds 29. The density of Na ÷ channels in premyelinated optic nerve axons was estimated from measurements of the binding of [3H]STX. Surprisingly, maximum saturable binding capacities corresponded to a high-affinity (KD=0.88 riM) STX-binding site density of ~2/[zm2 within premyelinated axon membrane of the neonatal optic nerve (Fig. 4A). This low Na + channel density was especially interesting in view of previous results 29 that demonstrated conduction of action potentials (conduction velocity = 0.2 m/s at 37°C) in neonatal optic nerve. These earlier studies demonstrated that the action potential is abolished by 1 ~M tetrodotoxin, and by replacement of bath Na + with the impermeant cation TRIS. Moreover, there is no evidence for propagated Ca 2+ responses along the premyelinated axon trunks in neonatal rat optic nerve 29. In view of the low density of saxitoxin-binding sites, it was important to determine whether STXinsensitive channels might contribute to action potential electrogenesis. We therefore examined the effects of low concentrations of STX on conduction, and demonstrated that concentrations as low as 5 nM STX reversibly abolished the neonatal optic nerve action potentialas (Fig. 4B). This is close to the value predicted to block conduction, on the basis of the dissociation constant for STX observed in our binding experiments (0.88 riM). Taken together with the STX binding results, these observations indicate that low densities of Na + channels can support action potential conduction in axons of neonatal optic nerve. The Na + channel densities in neonatal rat optic nerve are substantially below those predicted to maximize conduction velocities in the squid giant axon39'4°. However, there is a precedent for action TINS, VoL 13, No. 2, 1990
Node, internode
potential conduction in some non-myelinated axons with low Na + channel densities. Table II summarizes estimates of Na + channel density in non-myelinated and premyelinated axon membrane, derived from [uH]STX-binding studies. In olfactory nerve from garfish and bullfrog (Rana catesbeiana), and in optic nerve from Necturus, Na ÷ channel densities of
