Juxtaparanodal K Channels

The first Kv channel subunits to be identified in mammalian myelinated nerves were Kv1.1 and Kv1.2. As such, they are also the channels in myelinated fibers that have been most extensively studied. The most surprising thing about these ion channels is that they are restricted to axolemma beneath the myelin sheath and flanking each node of Ranvier (Figs. 1C and 1D, blue). As a result, the function of these channels is not immediately obvious. It is clear, however, that loss of the myelin sheath exposes this large pool of channels and radically alters the active, voltage-dependent properties of the axon (see later), above and beyond the already dramatic consequences that the loss of the myelin sheath has on the passive electrical properties of the axon.

Based on co-immunoprecipitation and co-localization experiments, juxtaparanodal channels are thought to consist of both Kv1 a and P subunits. Specifically, these experiments suggest that these complexes consist of Kv1.1/1.2/ KvP2, Kv1.1/1.4/KvP2, Kv1.2/Kv1.4/KvP2, and Kv1.1/1.2/ 1.4/KvP2 heteromultimers (Fig. 1D; Wang et al., 1993; Rasband et al., 1999, 2001). For example, immunolabeling optic nerve using Kv1 channel subunit specific antibodies shows that Kv1.1 and Kv1.2 co-localize at nearly every jux-taparanode (Fig. 2A, overlap is yellow). In contrast, relatively few juxtaparanodes have Kv1.4 that co-localizes with Kv1.2 (Fig. 2B, arrow). While it is clear that most juxtaparanodes express a combination of Kv1 channel subunits in varying amounts (Rasband et al., 1998), the significance of the different levels of subunit expression in distinct fibers is unknown. It is possible that these differences reflect unique requirements for membrane excitability and function, and the addition of Kv1.4 a subunits would be predicted to noticeably alter the K+ currents within a myeli-nated fiber. Alternatively, given the fact that subunit composition can affect the surface expression of channels, the addition of Kv1.4 would be predicted to promote surface

Merged

A Kv1.2

B Kv1.2

C PSD-95

Figure 2 Heteromultimeric, juxtaparanodal Kv channels are part of a larger protein complex. (A) Kv1.1 (green) and Kv1.2 (red) co-localize at juxtaparanodes (overlap is yellow) of optic nerve axons. There is a pronounced gap in immunoreactivity corresponding to the paranodal and nodal domains. Note that immunoreactivity is most intense adjacent to the para-nodal zone and decreases towards internodal regions. (B) In the optic nerve, Kv1.4 (green) co-localizes with a minority of the Kv1.2 (red) labeled juxtaparanodes (arrowhead). (C) PSD-95 (red) is present at juxtaparanodes, co-localizes with Kv1 channels (green), and is part of the juxtaparanodal Kv channel protein complex. Scalebars: (A-C) = 10 |m.

Kv1.1

Figure 2 Heteromultimeric, juxtaparanodal Kv channels are part of a larger protein complex. (A) Kv1.1 (green) and Kv1.2 (red) co-localize at juxtaparanodes (overlap is yellow) of optic nerve axons. There is a pronounced gap in immunoreactivity corresponding to the paranodal and nodal domains. Note that immunoreactivity is most intense adjacent to the para-nodal zone and decreases towards internodal regions. (B) In the optic nerve, Kv1.4 (green) co-localizes with a minority of the Kv1.2 (red) labeled juxtaparanodes (arrowhead). (C) PSD-95 (red) is present at juxtaparanodes, co-localizes with Kv1 channels (green), and is part of the juxtaparanodal Kv channel protein complex. Scalebars: (A-C) = 10 |m.

expression of heteromultimers containing this subunit (Manganas et al., 2001).

The cytoplasmic KvP subunits of Kv1 channels associate with a subunits during early channel biosynthesis and are thought to promote channel surface expression (Shi et al., 1996). Juxtaparanodal Kv1 channel complexes include KvP2 subunits, although other KvP subunits associate with Kv1 channels at various locations throughout the nervous system (Rhodes et al., 1997; Rasband et al., 1998). The crystal structure of the KvP subunit has shown that there is significant structural similarity to aldo-keto reductases, including the conservation of key catalytic and cofactor binding sites (Gulbis et al., 1999). However, mutation of the catalytic site has no effect on the intracellular trafficking of channels, suggesting that any role for the enzymatic activity of the KvP-subunit likely lies outside of its function in promoting the surface expression of channels (Campomanes et al., 2002). It is somewhat surprising, then, that mice lacking KvP2 apparently have no change in either the surface expression of Kv1 channels, or in the localization of channels to juxtaparanodes (McCormack et al., 2002). However, whether other kinds of KvP subunits compensate for the lack of KvP2 has not been addressed. Furthermore, these results are unable to account for the fact that KvP2-null mice have seizures, reduced life span, and cold-swim induced tremors, a phenotype that is quite similar to that observed for Kv1.1-deficient mice (Zhou et al., 1998b). Thus, additional experiments will be needed to determine the consequence of the KvP2 deletion and its precise role with respect to juxtaparanodal Kv1 channels.

As shown in Fig. 1C, Kv1 channels (blue) are localized to very discrete domains and do not overlap with paranodal proteins. This high degree of regulation in channel localization implies that these channels play an important role in nervous system function. However, while the role of these channels during early development suggests they are important for stabilizing nodal excitability (Vabnick et al., 1999), their function in normal adult myelinated fibers remains an enigma. An in-depth discussion of the normal function of these juxtaparanodal channels is provided by Chiu (see Chapter 5); the function of these channels subsequent to demyelination and during remyelination is discussed later.

1. Juxtaparanodal K+ Channels Are Part of a Larger Protein Complex

Besides Kv1 a and P subunits, other proteins have now been recognized at juxtaparanodes. Together with Kv1 channels, these form a large protein complex consisting of cell adhesion molecules, cytoskeletal, and scaffolding proteins. The first component of juxtaparanodes, besides K+ channel subunits, was identified by Poliak et al. (1999) and named Caspr2 based on its similarity to the paranodal axoglial junction component contactin-associated protein (Caspr; also known as paranodin or NCP1; Einheber et al., 1997; Menegoz et al., 1997; Peles et al., 1997). Caspr2 is a 180 kD cell adhesion molecule clustered at juxtaparanodes. That it interacts with the Kv1 channel was demonstrated through the reciprocal co-immunoprecipitation of Kv1 a and P subunits and Caspr2 (Poliak et al., 1999). Despite being in the same protein complex, the interaction between Kv1 channels and Caspr2 is likely indirect, because mutation of the C-terminal PDZ (PSD-95, discs-large, zonula occludens) binding motifs of either Kv1 channels or Caspr2 abolishes their interaction (Poliak et al., 1999). Caspr2 deficient mice have a dramatic reduction in jux-taparanodal clustering of channels, without an overall reduction in the total number of channels (Poliak et al., 2003). These results indicate that Caspr2 is required for the localization and/or maintenance of Kv1 channels at juxtaparanodes.

When Caspr2 was first identified, it was proposed to be involved in the localization of Kv1 channels to juxtapara-nodes through interaction with a glial binding partner (Poliak et al., 1999). This hypothesis is based, in part, on the fact that dysmyelination, hypomyelination, and demyelina-tion result in the loss of clustered Kv1 channels (Wang et al., 1995; Rasband et al., 1998; Baba et al., 1999). Caspr2's binding partner has now been identified as the glyco-

sylphosphatidyl-inositol (GPI) anchored Transient Axonal Glycoprotein, or TAG-1. While other functions unrelated to myelinated nerve fibers have been ascribed to this molecule (Furley et al., 1990), TAG-1 is also present at juxtapara-nodes and is expressed by myelinating glia (Traka et al., 2002). More recently, both Poliak et al. (2003) and Traka et al. (2003) have shown that TAG-1 and Caspr2 form a protein complex in the brain, as they can reciprocally co-immunoprecipitate one another. The molecular basis of this interaction was determined by transfecting COS-7 cells with Caspr2 or TAG-1, or co-transfecting cells with both TAG-1 and Caspr2. In these transfected cells, TAG-1 Fc fusion proteins bound only to cells transfected with TAG-1 or TAG-1 and Caspr2 (Poliak et al., 2003; Traka et al., 2003). Together, these results indicate that the interaction between axonal Caspr2 and glial TAG-1 is not direct, but requires the cis-interaction of Caspr2 and TAG-1 in the same membrane. Thus, axonal cis-interacting TAG-1 and Caspr2 bind in trans with glial TAG-1. The significance of this interaction for juxtaparanodal Kv1 channel localization was shown in TAG-1-null mice as these animals have a phenotype identical to the Caspr2-null mouse: reduced juxtaparanodal Kv1 channels without any reduction in Kv1 channel subunit protein levels (Poliak et al., 2003; Traka et al., 2003). Since TAG-1 is mislocalized in Caspr2-null mice, a reciprocal interaction must organize the glial juxtaparanodal domains. Together, these results suggest that neuroglial interactions regulate the reciprocal subcellular differentiation of juxta-paranodal axolemma and myelin membrane.

An important component of the juxtaparanodal protein complex not yet identified is a protein linking Kv1 channels and the Caspr2/TAG-1 complex. This protein likely consists of multiple PDZ domains, as mutation of the PDZ binding motifs in either Caspr2 or Kv1 channel subunits abolishes their interaction (Poliak et al., 1999). Multi-PDZ-domain scaffolding proteins, such as PSD-95 have been shown to cluster Kv1 channels in vitro (Kim et al., 1995). As a result, it is reasonable to postulate that this type of protein might link and cluster Kv1 channels and Caspr2. Indeed PSD-95 is present at juxtaparanodes (Fig. 2C, the co-localization of Kv1.1 and PSD-95 appears yellow) and forms a macromo-lecular protein complex with Kv1 channels as demonstrated by co-immunoprecipitation (Rasband et al., 2002; Menon etal., 2003). Surprisingly, however, loss of PSD-95 from juxtaparanodes disrupts neither the localization of Kv1 channels and Caspr2 nor their biochemical interaction (Rasband et al., 2002). Although PSD-95 is present at juxtaparanodes, either its function lies outside of mediating the interaction between Caspr2 and Kv1 channels or there is another multi-PDZ domain protein that can link Caspr2 and Kv1 channel sub-units and fully compensate for loss of PSD-95.

Membrane proteins are often anchored to the cytoskele-ton through adaptor and/or scaffolding proteins. Both Caspr and Caspr2 have an intracellular, c-terminal motif similar to the erythrocyte protein 4.1 binding motif found in gly-cophorin C (Denisenko-Nehrbass et al., 2003). Among the 4.1 proteins, type II or 4.1B has been described at both paranodes and juxtaparanodes (Ohara et al., 2000; Poliak et al., 2001). Pull-down assays have shown that 4.1B binds the intracellular GNP (glycophorin C, Neurexin, Paranodin/ Caspr) motif of both Caspr and Caspr2 (Denisenko-Nehrbass et al., 2003). During development 4.1B is detected at both paranodes and juxtaparanodes, but is detected at these sites after Caspr and Caspr2, suggesting that the primary role for 4.1B may be to anchor and maintain Kv1 channel/Caspr2 protein complexes in their respective domains rather than initiating clustering.

Besides TAG-1, the only other glial juxtaparanodal protein to have been described is Connexin29 (Cx29; Altevogt et al., 2002). Cx29 is expressed in myelinating glia and co-localizes with Kv1 channels at juxtaparanodes in the PNS and in small diameter myelinated fibers in the CNS. However, no adjacent axolemmal Connexin has been found, bringing into question whether Cx29 forms functional gap junctions at these sites. Thus, the role(s) of Cx29 at juxtaparanodes is unknown, although Altevogt et al. (2002) speculate that Cx29 may form functional hemichannels involved in the removal of K+ from the periaxonal space.

Finally, one other protein has been reported to be present in the juxtaparanodal K+ channel protein complex. Recently, Nie et al. (2003) reported that oligodendroglial Nogo-A, Caspr, and Kv1 channels reciprocally co-precipitated each other from brain membranes and transfected cells (suggesting that these proteins form a complex and that Caspr and Kv1 channels interact directly). It was also reported that in adult animals Nogo-A is present in paranodal domains (Nie et al., 2003). These results are surprising since Kv1 channels are normally excluded from paranodal domains. Subsequent experiments attempting to repeat these controversial results have not met with success (Rasband, 2004). Thus, while Nogo-A appears to be localized at paranodes in adult animals, more experiments are needed to determine whether a macromolecular protein complex exists that includes both paranodal and juxtaparanodal components.

2. Cellular Mechanisms of Juxtaparanodal K+ Channel Localization

As described previously, a variety of proteins (most notably cell adhesion molecules) interact with and participate in the localization of juxtaparanodal Kv1 channels. This fact is clearly demonstrated in Caspr2 and TAG-1 deficient mice where Kv1 channels are no longer clustered in their appropriate domains (Poliak et al., 2003; Traka et al., 2003). In addition to these molecular interactions, cellular mechanisms also play important roles in organizing and restricting the localization of Kv1 channels. In particular, axoglial junctions participate in channel localization. The idea that sites of axoglial contact regulate the localization of channels was first suggested after careful analysis of myeli-

nated nerve fibers by freeze-fracture and electron microscopic methods. These studies showed a high density of intramembranous particles confined to regions between adjacent axoglial junctions (Rosenbluth, 1988). Later, using immunofluorescence methods, it was found that during development Kv1 channels were often present in distinct bands that were oriented parallel to the direction of axoglial junctions. Double-immunostaining using markers for para-nodal junctions showed that the Kv1 channel bands were confined between axoglial junctions (Rasband et al., 1999; Vabnick et al., 1999). A careful analysis of the internodal localization of Caspr and Kv1 channels in the PNS showed that a thin strand of Caspr runs adjacent to the inner mesaxon and is flanked on each side by a double strand of Kv1 channels (Arroyo et al., 1999). Together, these results point to a role for myelinating glial cells as primary determinants of channel localization.

The conclusion that axoglial junctions restrict the lateral diffusion of Kv1 channels in the axolemma and confine them to juxtaparanodal domains has been confirmed by several important studies examining mutant animals with altered paranodal junctions. For example, mice lacking paranodal components such as Caspr (Bhat et al., 2001) or con-tactin (Boyle et al., 2001), or lacking key myelin galactolipids such as Galc and sulfatide (Dupree et al., 1998, 1999) or sulfatide alone (Ishibashi et al., 2002), do not have the transverse bands that are a hallmark of mature, well-defined axoglial junctions. These mice all have conduction deficits, tremors, reduced densities of Nav channels at nodes of Ranvier, and in many cases everted paranodal loops that point away from the axolemma. In these animals, there is a dramatic redistribution and invasion of Kv1 channels from the juxtaparanode into the paranode. Together, these results show that paranodal junctions are essential to restrict Kv1 channels to juxtaparanodes and that these junctions provide a diffusion barrier within the axonal membrane.

In contrast to the juxtaparanodal Kv1 channels, we do not yet have any information about the cellular or molecular mechanisms of either Kv3.1b or KCNQ2 channel trafficking, targeting, and clustering at nodes of Ranvier. Since both Kv3.1b and KCNQ2 can co-immunoprecipitate ankyrinG (Devaux et al., 2003, 2004), and ankyrinG is thought to be a primary determinant of Nav channel clustering (Zhou et al., 1998a), the mechanisms of nodal ion channel localization and clustering may be common for KCNQ2, Kv3.1b, and Nav channels.

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