An ankyrinG-binding motif is necessary and sufficient for targeting Nav1.6 sodium channels to axon initial segments and nodes of Ranvier.
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An ankyrinG-binding motif is necessary and sufficient for targeting Nav1.6 sodium channels to axon initial segments and nodes of Ranvier. / Gasser, Andreas; Ho, Tammy Szu-Yu; Cheng, Xiaoyang; Chang, Kae-Jiun; Waxman, Stephen G; Rasband, Matthew N; Dib-Hajj, Sulayman D.
in: J NEUROSCI, Jahrgang 32, Nr. 21, 21, 2012, S. 7232-7243.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - An ankyrinG-binding motif is necessary and sufficient for targeting Nav1.6 sodium channels to axon initial segments and nodes of Ranvier.
AU - Gasser, Andreas
AU - Ho, Tammy Szu-Yu
AU - Cheng, Xiaoyang
AU - Chang, Kae-Jiun
AU - Waxman, Stephen G
AU - Rasband, Matthew N
AU - Dib-Hajj, Sulayman D
PY - 2012
Y1 - 2012
N2 - Neurons are highly polarized cells with functionally distinct axonal and somatodendritic compartments. Voltage-gated sodium channels Na(v)1.2 and Na(v)1.6 are highly enriched at axon initial segments (AISs) and nodes of Ranvier, where they are necessary for generation and propagation of action potentials. Previous studies using reporter proteins in unmyelinated cultured neurons suggest that an ankyrinG-binding motif within intracellular loop 2 (L2) of sodium channels is sufficient for targeting these channels to the AIS, but mechanisms of channel targeting to nodes remain poorly understood. Using a CD4-Na(v)1.2/L2 reporter protein in rat dorsal root ganglion neuron-Schwann cell myelinating cocultures, we show that the ankyrinG-binding motif is sufficient for protein targeting to nodes of Ranvier. However, reporter proteins cannot capture the complexity of full-length channels. To determine how native, full-length sodium channels are clustered in axons, and to show the feasibility of studying these channels in vivo, we constructed fluorescently tagged and functional mouse Na(v)1.6 channels for in vivo analysis using in utero brain electroporation. We show here that wild-type tagged-Na(v)1.6 channels are efficiently clustered at nodes and AISs in vivo. Furthermore, we show that mutation of a single invariant glutamic acid residue (E1100) within the ankyrinG-binding motif blocked Na(v)1.6 targeting in neurons both in vitro and in vivo. Additionally, we show that caseine kinase phosphorylation sites within this motif, while not essential for targeting, can modulate clustering at the AIS. Thus, the ankyrinG-binding motif is both necessary and sufficient for the clustering of sodium channels at nodes of Ranvier and the AIS.
AB - Neurons are highly polarized cells with functionally distinct axonal and somatodendritic compartments. Voltage-gated sodium channels Na(v)1.2 and Na(v)1.6 are highly enriched at axon initial segments (AISs) and nodes of Ranvier, where they are necessary for generation and propagation of action potentials. Previous studies using reporter proteins in unmyelinated cultured neurons suggest that an ankyrinG-binding motif within intracellular loop 2 (L2) of sodium channels is sufficient for targeting these channels to the AIS, but mechanisms of channel targeting to nodes remain poorly understood. Using a CD4-Na(v)1.2/L2 reporter protein in rat dorsal root ganglion neuron-Schwann cell myelinating cocultures, we show that the ankyrinG-binding motif is sufficient for protein targeting to nodes of Ranvier. However, reporter proteins cannot capture the complexity of full-length channels. To determine how native, full-length sodium channels are clustered in axons, and to show the feasibility of studying these channels in vivo, we constructed fluorescently tagged and functional mouse Na(v)1.6 channels for in vivo analysis using in utero brain electroporation. We show here that wild-type tagged-Na(v)1.6 channels are efficiently clustered at nodes and AISs in vivo. Furthermore, we show that mutation of a single invariant glutamic acid residue (E1100) within the ankyrinG-binding motif blocked Na(v)1.6 targeting in neurons both in vitro and in vivo. Additionally, we show that caseine kinase phosphorylation sites within this motif, while not essential for targeting, can modulate clustering at the AIS. Thus, the ankyrinG-binding motif is both necessary and sufficient for the clustering of sodium channels at nodes of Ranvier and the AIS.
KW - Animals
KW - Male
KW - Female
KW - Mice
KW - Mutation
KW - Rats
KW - Rats, Sprague-Dawley
KW - Coculture Techniques
KW - Hippocampus/metabolism
KW - Axons/metabolism
KW - Membrane Potentials/physiology
KW - Ankyrins/genetics/physiology
KW - Ganglia, Spinal/cytology/metabolism
KW - Molecular Imaging/methods
KW - Protein Interaction Domains and Motifs/genetics/physiology
KW - Protein Transport/genetics/physiology
KW - Ranvier's Nodes/metabolism
KW - Sodium Channels/metabolism
KW - Animals
KW - Male
KW - Female
KW - Mice
KW - Mutation
KW - Rats
KW - Rats, Sprague-Dawley
KW - Coculture Techniques
KW - Hippocampus/metabolism
KW - Axons/metabolism
KW - Membrane Potentials/physiology
KW - Ankyrins/genetics/physiology
KW - Ganglia, Spinal/cytology/metabolism
KW - Molecular Imaging/methods
KW - Protein Interaction Domains and Motifs/genetics/physiology
KW - Protein Transport/genetics/physiology
KW - Ranvier's Nodes/metabolism
KW - Sodium Channels/metabolism
M3 - SCORING: Journal article
VL - 32
SP - 7232
EP - 7243
JO - J NEUROSCI
JF - J NEUROSCI
SN - 0270-6474
IS - 21
M1 - 21
ER -
