title: Kinesin I transports tetramerized Kv3 channels through the axon initial segment via direct binding.
abstract: Precise targeting of various voltage-gated ion channels to proper membrane domains is crucial for their distinct roles in neuronal excitability and synaptic transmission. How each channel protein is transported within the cytoplasm is poorly understood. Here, we report that KIF5/kinesin I transports Kv3.1 voltage-gated K(+) (Kv) channels through the axon initial segment (AIS) via direct binding. First, we have identified a novel interaction between Kv3.1 and KIF5, confirmed by immunoprecipitation from mouse brain lysates and by pull-down assays with exogenously expressed proteins. The interaction is mediated by a direct binding between the Kv3.1 N-terminal T1 domain and a conserved region in KIF5 tail domains, in which proper T1 tetramerization is crucial. Overexpression of this region of KIF5B markedly reduces axonal levels of Kv3.1bHA. In mature hippocampal neurons, endogenous Kv3.1b and KIF5 colocalize. Suppressing the endogenous KIF5B level by RNA interference significantly reduces the Kv3.1b axonal level. Furthermore, mutating the Zn(2+)-binding site within T1 markedly decreases channel axonal targeting and forward trafficking, likely through disrupting T1 tetramerization and hence eliminating the binding to KIF5 tail. The mutation also alters channel activity. Interestingly, coexpression of the YFP (yellow fluorescent protein)-tagged KIF5B assists dendritic Kv3.1a and even mutants with a faulty axonal targeting motif to penetrate the AIS. Finally, fluorescently tagged Kv3.1 channels colocalize and comove with KIF5B along axons revealed by two-color time-lapse imaging. Our findings suggest that the binding to KIF5 ensures properly assembled and functioning Kv3.1 channels to be transported into axons.
authors.affiliation: Department of Neuroscience and Center for Molecular Neurobiology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Mingxuan
authors.initials: M
authors.lastname: Xu
authors.forename: Yuanzheng
authors.initials: Y
authors.lastname: Gu
authors.forename: Joshua
authors.initials: J
authors.lastname: Barry
authors.forename: Chen
authors.initials: C
authors.lastname: Gu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001369:Axons / Q000378:metabolism*; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D051670:Shaw Potassium Channels / Q000737:chemistry / Q000378:metabolism*
chemical_list: C489597:KCNC1 protein, human; C488101:KIF5B protein, human; D051670:Shaw Potassium Channels; D016547:Kinesins