Sodium channels develop a tyrosine phosphatase ...

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Michael W. Salter and Yu Tian Wang. A new study shows that sodium channels interact with receptor-like tyrosine phosphatase β, which increases sodium.

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can switch from making long movements along microtubule highways to making short ones—sometimes stopping altogether—along actin backroads 13,14 , a process also detected in extracts of squid axoplasm 15. The saltatory movements observed in axons could be due to pausing at actin-rich side roads or rest stops along the microtubule highway. Fortunately, predictions such as these will be relatively easy to test with technologies now available; for example, one can compare the distributions of actin and actin motors with that of the prototerminals. The work of Ahmari and colleagues dramatically changes how we should think about neuronal polarity, and it suggests many further experiments. The results

also illustrate that the first technology of cell biologists, merely looking at cells, remains one of the best; this is especially true now that we can follow a single type of fluorescent protein in a living cell. 1. Ahmari, S. E., Buchanan, J. & Smith, S. J. Nat. Neurosci. 3, 445–451 (2000). 2. Ledesma, M. D., Brugger, B., Bunning, C., Wieland, F. T. & Dotti, C. G. EMBO J. 18, 1761–1771 (1999). 3. Roos, J. & Kelly, R. B. Curr. Biol. 9, 1411–1444 (1999). 4. Hirokawa, N. Science 279, 519–526 (1998). 5. Kraszewski, K. et al. J. Neurosci. 15, 4328–4342 (1995). 6. Zakharenko, S., Chang, S., O’Donoghue, M. & Popov, S. V. J. Cell Biol. 144, 507–518 (1999).

Sodium channels develop a tyrosine phosphatase complex Michael W. Salter and Yu Tian Wang A new study shows that sodium channels interact with receptor-like tyrosine phosphatase β, which increases sodium currents and may also help localize the channels. Phosphorylation is a ubiquitous posttranslational mechanism for rapid, reversible modulation of proteins, increasingly recognized as a principal means for regulating the function, expression and localization of ion channels1. Phosphorylation at a particular site is determined by the balance between phosphate addition by protein kinases and its removal by phosphatases. Protein kinases and phosphatases fall into two main types: those that act on serine and threonine residues and those that act on tyrosine residues. We have an extensive understanding of ion channel regulation by numerous serine/threonine protein kinases and phosphatases1. Tyrosine phosphorylation2 also regulates both ligand3 and voltage-gated4 channels. Implicit in these studies is the opposing modulation of ion channel The authors are in the Programme in Brain and Behaviour, University of Toronto, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. e-mail: [email protected] or [email protected] nature neuroscience • volume 3 no 5 • may 2000

function by phosphotyrosine phosphatases (PTPs), but the identity of specific enzymes has been elusive. Thus the report by Catterall and colleagues5 fills an important void in our understanding by identifying the first example of a PTP regulating an ion channel. The authors show that voltage-gated sodium channels are associated with and regulated by receptorlike tyrosine phosphatase β (RPTPβ). PTPs comprise a large family of enzymes6, of which more than 80 have been identified7. PTPs are grouped primarily into receptor-like, or transmembrane, PTPs and cytoplasmic PTPs. Receptor-like PTPs have a single membrane-spanning domain, and most contain tandem intracellular PTP domains, with the one closer to the membrane typically having greater or sometimes all the catalytic activity. Extracellular domains are variable in the PTP family, but most contain structural motifs suggesting roles in cell–cell or cell–matrix adhesion. RPTPβ (also known as PTPζ8) is unique in the PTP family in that three forms are generated by alternative splicing: a long

7. Strasser, J. F., Arribas, M., Blagoveshchenskaya, A. D. & Cuter, D. F. Mol. Biol. Cell 10, 2619–2630 (1999). 8. Faundez, V., Horng, J. T. & Kelly, R. B. Cell 93, 423–432 (1998). 9. Shi, G., Faundez, V., Roos, J., Dell’Angelica, E. C. & Kelly, R. B. J. Cell Biol. 143, 947–955 (1998). 10. Grindstaff, K. K. et al. Cell 93, 731–740 (1998). 11. Hazuka, C. D. et al. J. Neurosci. 19, 1324–1334 (1999). 12. Uchida, N., Honjo, Y., Johnson, K. R., Wheelock, M. J. & Takeichi, M. J. Cell Biol. 135, 767–779 (1996). 13. Rodionov, V. I., Hope, A. J., Svitkina, T. M. & Borixy, G. G. Curr. Biol. 8, 165–168 (1998). 14. Rogers, S. L. & Gelfand, V. I. Curr. Biol. 8, 161–165 (1998). 15. Kuznetsov, S. L., Langford, G. M. & Weiss, D. G. Nature 356, 722–725 (1992).

and short transmembrane form, both of which contain the catalytic domains, and a secreted form of the protein, which lacks the transmembrane and intracellular portions. The secreted form and the long catalytic splice form have a large extracellular insert rich in glycosaminoglycan side chains, rendering both proteins chrondroitin sulfate proteoglycans. For clarity, only splice variants containing phosphatase domains are referred to as RPTPβ; the secreted form is commonly known as phosphacan, named as an abundant brain proteoglycan9. Voltage-gated sodium channels are fundamental for neural functioning, as they mediate the sodium current responsible for the rapid rising phase of action potentials10. In the brain, sodium channels are composed of three glycoprotein subunits: the α subunit, which forms the channel pore and is sufficient to produce a functional channel on its own, and the β1 and β2 subunits, which modulate channel gating. Like other ion channels, sodium channel function is modulated by serine/threonine11 and tyrosine kinases12. In this issue5, Catterall and colleagues5 show that sodium channels isolated from rat brain interact with RPTPβ extracellularly and intracellularly. The extracellular portion of RPTPβ has two prominent amino (N)-terminal domains: a carbonic anhydrase-homology (CAH) domain and a fibronectin type III (FN) repeat. Catterall and colleagues found in experiments with fragments of the extracellular region of RPTPβ that the CAH domain but not the FN domain binds to the extracellular region of the sodium channel. Thus, sodium channels may act as an extracellular ligand for the receptor por417

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Fig. 1. Regulation of the function of voltage-gated sodium channels by the balance of tyrosine phosphorylation and tyrosine dephosphorylation of the α subunit. (a) In developing neurons, the α subunit may be dephosphorylated by RPTPβ associated with the channel complex and might be phosphorylated through growth factor receptor signaling pathways. (b) In the adult, the non-catalytic splice variant, phosphacan, associates with the sodium channel complex and may displace RPTPβ.

tion of RPTPβ. Intracellularly, the first PTP domain of RPTPβ was found to bind to the α and β1 subunits. The intracellular binding to the α subunit is likely substrate trapping (see below) whereas binding to the β1 subunit is non-catalytic, and may serve to bring the phosphatase domain near the phosphorylation sites on the intracellular domain of the sodium channel. To examine the effects of RPTPβ on sodium channel function, Catterall and colleagues expressed the catalytic domains together with the sodium channel α subunit in a heterologous cell line. The RPTPβ catalytic domain produces two changes in sodium channel inactivation that would result in increased sodium currents: inactivation requires more membrane depolarization, and the rate of inactivation is slowed. These effects of RPTPβ are reversed by pharmacologically blocking the enzyme, and moreover, they oppose the reported inhibition of sodium currents by tyrosine kinases in neurons12. Tyrosine phosphorylation produced by stimulating receptor tyrosine kinases (RTKs), including trkA, EGF and PDGF receptors, inhibits sodium channel function via a signaling cascade involving non-receptor tyrosine kinases of the Src family. At a typical resting membrane potential of –60 mV, about 50% of sodium channels are inactivated. Thus, all other things being equal, tyrosine phosphorylation would tend to depress excitability, whereas dephosphorylation would have the opposite effect. 418

Catterall and colleagues5 found that, when expressed heterologously, the sodium channel α subunit is phosphorylated on tyrosine, and that phosphorylation is increased by PTP inhibition. Thus, the simplest model is that in situ tyrosine phosphorylation of the α subunit, by RTKs and/or src kinases, and dephosphorylation by RPTPβ, provide opposing regulation of sodium channel function (Fig. 1a). This type of acute regulation, which occurs over seconds to minutes, is separate from the increased expression of sodium channel subunit proteins produced by stimulating RTKs, which requires hours13. Expression of the receptor forms of RPTPβ and of phosphacan is developmentally regulated. The RPTPβ forms are expressed prenatally, and the level of the short form is maintained after birth, whereas phosphacan expression increases dramatically in the postnatal period8. Catterall and colleagues 5 found that at postnatal day 1 (P1) the short, but not the long, receptor form associates with sodium channels. In contrast, at P16 only phosphacan was detected in association with the channels. One interpretation is that phosphacan may compete with the CAH domain of RPTPβ and displace it from the sodium channel. Although this remains to be tested directly, the results do raise the intriguing possibility that the interaction between the extracellular domain of RPTPβ and that of the sodium channel is required to localize the enzyme to its target.

The increase in sodium channel function by RPTPβ likely occurs only during development when the short receptor form associates with the channel. Over the course of postnatal development, expression of many tyrosine kinases decreases; thus one potential outcome is that sodium channels might not be regulated by tyrosine phosphorylation later in development (Fig. 1b). Alternatively, if the action of PTKs is unopposed by RPTPβ, then adult channels may show sustained phosphorylation. However, it is also conceivable that a different PTP substitutes for RPTPβ in the adult. Distinguishing between these possibilities will require determination of the level of tyrosine phosphorylation of sodium channel proteins and its dynamics in the adult. Perhaps the most surprising aspect of the work by Catterall and colleagues5 is that the PTP associated with voltage-gated sodium channels is RPTPβ. This is unexpected because of the abundant evidence that RPTPβ is overwhelmingly expressed by glia7,8, although expression of RPTPβ mRNA has been reported in subpopulations of neurons14. The present findings5 should motivate efforts to determine the extent of neuronal expression of RPTPβ. Another surprising aspect of this work is the dual binding interaction between RPTPβ and the sodium channel. The dual extracellular/intracellular binding interaction is in contrast to the general rule for association of intracellular signaling complexes with other ion channels, where, even for membrane-spanning proteins, the dominant interaction is mediated by motifs in intracellular domains of the channel that bind directly, or by means of adaptor proteins, to the relevant enzymes15. Given that RPTPβ interacts with many extracellular ligands and extracellular matrix molecules, are there other potential functions of the RPTPβ /phosphacan association with sodium channels? In speculating about this, Catterall and colleagues suggest that RPTPβ/phosphacan could be involved in anchoring or regulating the location of sodium channels, which are clustered at the initial segment of the axon and at nodes of Ranvier. How could this come about? One possibility is through interactions of the extracellular domains of RPTPβ/phosphacan with celladhesion molecules, as well as with sodium channels. RPTPβ and phosphacan bind multiple extracellular ligands, and a variety of potentially interacting partners have been identified8. Such molecules in turn may link to cytoskeletal elements that nature neuroscience • volume 3 no 5 • may 2000

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accumulate at the nodes and the initial segment, such as ankryin, causing sodium channels to cluster there. An even more intriguing possibility is that the extracellular domain of the sodium channel might interact with the CAH domain of RPTPβ expressed by glia. The extracellular region of RPTPβ is expected to be sufficiently long and flexible to allow for such interaction, which could form a physical bridge bringing the membrane of neurons and glia into close apposition. By producing a localized increase in the concentration of RPTPβ molecules, such interaction could enhance dephosphorylation of glial proteins, and thereby modulate intracellular signaling cascades, at sites opposing sodium channel clusters in neurons. The findings of Catterall and colleagues5 are an important step in the quest to understand the regulation of ion channels by tyrosine phosphorylation. Clearly a

number of issues remain to be resolved, but many new questions are now opened up. For example, it remains to be determined whether tyrosine phosphorylation on the α subunit is necessary and sufficient for the alteration in sodium channel function. If so, what tyrosine residues are responsible, and what is the biophysical basis for the change in channel function? With the large number of PTPs likely encoded in the genome and the diversity of ion channels regulated by tyrosine phosphorylation and dephosphorylation, we expect RPTPβ is but the first in a line of PTPs that control ion channel function. 1. Levitan, I. B. Adv. Second Mess. Phosphoprotein Res. 33, 3–22 (1999). 2. Hopfield, J. F., Tank, D. W., Greengard, P. & Huganir, R. L. Nature 336, 677–680 (1988). 3. Yu, X. M., Askalan, R., Keil, G. J. & Salter, M. W. Science 275, 674–678 (1997).

Locating an error correction signal for adult birdsong Catherine Carr A recent report in Nature suggests that the stability of adult birdsong is maintained by error correction via an auditory feedback pathway involved in juvenile song learning.

Although many animals communicate via elaborate sounds, few of them must learn to produce these sounds. Humans are superb vocal learners, but no other primates and only a small number of other mammals learn their vocalizations. In contrast, many thousands of songbird species, as well as parrots and hummingbirds, acquire their vocal repertoires by learning. There are striking parallels between human speech and birdsong learning1. Most importantly, both humans and songbirds learn their vocal motor behavior early in life, and to do so, they must be able to hear both the adults that they will imitate and their own attempts at vocalization. In addition, both humans and songbirds have evolved a complex hierarchy of specialized brain areas essential for vocal control2,3. It has long been Catherine Carr is in the Department of Biology, University of Maryland, College Park, Maryland 20742-4415, USA. e-mail: [email protected] nature neuroscience • volume 3 no 5 • may 2000

recognized that this combination of a discrete learned vocal behavior and dedicated neural circuitry makes the song system a very useful model for the study of sensory and motor learning, with particular relevance to speech. Until recently, the song of most adult birds was thought to have become entirely independent of auditory feedback, perhaps being driven solely by a central pattern generator4. However, it is now clear that although the dependence of vocal behavior on hearing is reduced in adulthood, adult song in some species does require continued auditory feedback, as does human speech. The songs of adult zebra finches slowly deteriorate after deafening5, or even after playback of delayed or altered versions of the bird’s voice during singing6. Recently in Nature, Brainard and Doupe7 showed that this deterioration is prevented when deafening is paired with a lesion of a specialized basal ganglia circuit within the song system, the anterior fore-

4. Holmes, T. C., Fadool, D. A., Ren, R. & Levitan, I. B. Science 274, 2089–2091 (1996). 5. Ratcliffe, C. F. et al. Nat. Neurosci. 3, 437–444 (2000). 6. Tonks, N. K. & Neel, B. G. Cell 87, 365–368 (1996). 7. Stoker, A. & Dutta, R. Bioessays 20, 463–472 (1998). 8. Peles, E., Schlessinger, J. & Grumet, M. Trends Biochem. Sci. 23, 121–124 (1998). 9. Maurel, P., Rauch, U., Flad, M., Margolis, R. K. & Margolis, R. U. Proc. Natl. Acad. Sci. USA 91, 2512–2516 (1994). 10. Armstrong, C. M. & Hille, B. Neuron 20, 371–380 (1998). 11. Cantrell, A. R., Ma, J. Y., Scheuer, T. & Catterall, W. A. Neuron 16, 1019–1026 (1996). 12. Hilborn, M. D., Vaillancourt, R. R. & Rane, S. G. J. Neurosci. 18, 590–600 (1998). 13. Mandel, G., Cooperman, S. S., Maue, R. A., Goodman, R. H. & Brehm, P. Proc. Natl. Acad. Sci.USA 85, 924–928 (1988). 14. Snyder, S. E., Li, J., Schauwecker, P. E., McNeill, T. H. & Salton, S. R. Mol. Brain Res. 40, 79–96 (1996). 15. Fanning, A. S. & Anderson, J. M. Curr. Opin. Cell Biol. 11, 432–439 (1999).

brain pathway. Thus a second insult (the lesion) counters the effects of the first insult (deafening), apparently because the lesion has removed an error signal that is produced by or routed through this pathway. The result is particularly exciting because it has allowed Brainard and Doupe to extend the developmental model of song learning to explain how song remains stable during adult life. Moreover, because anterior forebrain pathway lesions in normal adult birds singing stable song have no effect8, this result reminds us that finding no effect of lesioning (or knocking out) a brain area on a previously learned behavior does not rule out a continuing function for the same area in modification of that behavior. Finally, it supports a role for basal ganglia–cortical circuitry in vocal learning that may have relevance to speech, and to motor learning in general. The strong dependence of song learning on hearing or auditory feedback suggests that birdsong and speech develop in similar ways. Neither human speech nor song develops normally in deaf individuals, and hearing loss or experimental alteration of auditory feedback in adults can lead to dramatic changes in vocal production. Like human speech, birdsong is a vocal behavior that is learned during early life. In 1965, Konishi showed that the development of song behavior is a twostage process4. Young white-crowned sparrows exposed to a particular song in one season do not produce a copy of that song until the next year, suggesting that they 419

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