Clustering and mobility of voltage-dependent sodium channels during myelination

Abstract

In myelinated axons, voltage-dependent sodium channels are segregated at high density at nodes of Ranvier (Rosenbluth, 1976; Waxman and Quick, 1978; Black et al., 1990; Elmer et al., 1990), a distribution that is critical for the saltatory conduction of action potentials (Huxley and Stampfli, 1949). The factors that specifically control the organization and immobilization of sodium channels at nodes are unknown. Recently we have reported that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that continuing axon-glial interactions are necessary to maintain sodium channel distribution during differentiation of myelinated nerve (Joe and Angelides, 1992). The specific recruitment of sodium channels at these early stages of myelination and the conspicuous absence of other axon membrane components suggest that the factors governing sodium channel cluster formation show molecular specificity. However, it is not clear whether these clustered sodium channels originate from a redistribution of preexisting diffusely distributed sodium channels. To determine how Schwann cells might regulate sodium channel distribution during myelination we have examined the lateral mobility of fluorescently labeled sodium channels at defined stages of myelination by fluorescence photobleach recovery using tetramethylrhodamine (TmRhd)-labeled Tityus γ, a sodium channel-specific fluorescent toxin. First, to test whether Schwann cells, in addition to modulating sodium channel distribution, affect the mobility of sodium channels, we cultured dorsal root ganglion neurons in the presence or absence of Schwann cells and monitored sodium channel mobility on cell bodies, axon hillocks, and axons. Even in the absence of Schwann cells, ∼80% of the sodium channels were immobile on the time scale of the fluorescence photobleach recovery measurement (DL ≤ 10-12 cm2/sec), although the remaining fraction of channels are mobile with diffusion coefficients of 5-13 × 10-11 cm2/sec. Most importantly, in contrast to the effects of Schwann cells on altering the distribution of sodium channels, we found that Schwann cells did not alter the rate of lateral mobility or the mobile fraction of axonal sodium channels. Therefore, although sodium channel distribution depends on Schwann cell contact, immobilization of sodium channels is independent of Schwann cell contact. These effects appear to be specific for sodium channels because 45% of the succinyl concanavalin-A receptors on the axon are mobile, a fraction that decreases to 25% in the presence of Schwann cells later in development. To determine how sodium channels might be immobilized other than by Schwann cell contact, TmRhd-Tityus γ-labeled dorsal root neurons were treated with 0.5% Triton X-100. Under these conditions, 17% and 22% of sodium channels were extracted from cell bodies and axons, respectively, suggesting that the immobile sodium channels are linked to the cytoskeleton. Consideration of several mechanisms shows that the results are consistent with the possibility that sodium channel clusters arise by the lateral diffusion and trapping or selective exclusion of the small mobile and homogeneously distributed population of sodium channels and/or by localized insertion of newly synthesized channels that preferentially appear on the axon at sites of Schwann cell contact. These changes in sodium channel distribution are subsequently stabilized and maintained by interactions with the subaxolemmal cytoskeleton. Copyright ©1993 Society for Neuroscience.