Organelle Transport Of Ion Channels In Excitable Cells

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The purpose of this project is to investigate the mechanisms which underlie nerve degeneration. A characteristic sequence of events occur following nerve injury, collectively termed Wallerian degeneration, such as dissolution of the axonal cytoskeleton, degeneration of the myelin sheath, invasion of the nerve by macrophages, and Schwann cell proliferation. These processes are usually complete 24-48 hr following transection. Degeneration is extraordinarily delayed in a mutant mouse model, the C57BL/Ola strain. In these otherwise normal appearing mice, axons retain their ability to conduct action potentials up to 2 weeks after transection, and they remain structurally intact for as long as 1 month. We have been using transected giant axons, in vivo, from squid as a model system to understand this delayed version of Wallerian degeneration. These axons retain their ability to conduct action potentials 2 days following transection, and their cytoskeleton also remains intact. However, we have noticed striking changes in fast axonal traffic measured with video enhanced contrast, differential interference contrast microscopy (VEC-DIC). Control axons contain a large number of small particles moving primarily in the orthograde direction, which we have previously demonstrated are transport vesicles 30 nm in diameter. A small fraction of the vesicles contain the axonal, voltage-gated potassium channel. The other vesicles presumably contain other protein cargo also destined for the axolemma. All of the vesicles contain one copy of the microtubule based motor protein, kinesin, as well as one copy of the actin based motor protein, myosin-II. The sole locus of the latter appears to be these transport vesicles. VEC-DIC recordings from transected axons reveal an almost complete loss of orthograde traffic, with little or no alteration of traffic in the retrograde direction. Moreover, myosin-II and potassium channels were not observed in the axoplasm of transected axons using immunological techniques (immunoblots and immunocytochemistry). We have concluded that the loss of transport vesicles appears to be the first step underlying delayed Wallerian degeneration.