Abstract
Precise localization of axonal ion channels is crucial for proper electrical and chemical functions of axons. In myelinated axons, Kv1 (Shaker) voltage-gated potassium (Kv) channels are clustered in the juxtaparanodal regions flanking the node of Ranvier. The clustering can be disrupted by deletion of various proteins in mice, including contactin-associated protein-like 2 (Caspr2) and transient axonal glycoprotein-1 (TAG-1), a glycosylphosphatidylinositol-anchored cell adhesion molecule. However, the mechanism and function of Kv1 juxtaparanodal clustering remain unclear. Here, using a new myelin coculture of hippocampal neurons and oligodendrocytes, we report that tyrosine phosphorylation plays a critical role in TAG-1-mediated clustering of axonal Kv1.2 channels. In the coculture, myelin specifically ensheathed axons but not dendrites of hippocampal neurons and clustered endogenous axonal Kv1.2 into internodes. The trans-homophilic interaction of TAG-1 was sufficient to position Kv1.2 clusters on axonal membranes in a neuron/HEK293 coculture. Mutating a tyrosine residue (Tyr⁴⁵⁸) in the Kv1.2 C terminus or blocking tyrosine phosphorylation disrupted myelin- and TAG-1-mediated clustering of axonal Kv1.2. Furthermore, Kv1.2 voltage dependence and activation threshold were reduced by TAG-1 coexpression. This effect was eliminated by the Tyr⁴⁵⁸ mutation or by cholesterol depletion. Taken together, our studies suggest that myelin regulates both trafficking and activity of Kv1 channels along hippocampal axons through TAG-1.
Highlights
(CNS) and Schwann cells in the peripheral nervous system
Based on the findings that the JXP targeting of Kv1 channels was largely eliminated in both contactin-associated protein-like 2 (Caspr2) and transient axonal glycoprotein-1 (TAG-1) knock-out mice, it was hypothesized that Caspr2 interacts with TAG-1 and clusters Kv1 channels via a PDZ domain-containing protein [18, 19]
A New Myelin Coculture of Hippocampal Neurons and Oligodendrocytes—To carry out molecular studies for understanding of how myelin clusters Kv1 channels along axons, we established a new myelin coculture system consisting of hippocampal neurons and oligodendrocytes (Fig. 1A)
Summary
Myelin Coculture of Hippocampal Neurons and Oligodendrocytes—Hippocampal neuron cultures were prepared as described [1]. The culture medium was replenished twice a week by replacing half its volume These neurons were usually transfected at 5–7 days in vitro (DIV) and assayed several days later or cocultured with oligodendrocytes at 14 DIV. Ma. For transient transfection, neurons in culture at 5–7 DIV or cell lines were incubated in Opti-MEM containing 0.8 g of cDNA plasmid and 1.5 l of Lipofectamine 2000 (Invitrogen) for 30 min at 37 °C. Whole-cell Patch Clamp Recording—The experimental procedure of whole-cell recording of HEK293 cells expressing Kv channel constructs was described previously [5]. Transfected HEK293 cells were identified by fluorescence from cotransfected YFP or mCherry inserted into the N-terminal region of TAG-1 and recorded in Hanks’ buffer (150 mM NaCl, 4 mM KCl, 1.2 mM MgCl2, 10 mg/ml glucose, 1 mM CaCl2, 20 mM HEPES, pH 7.4).
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