title: Brain-derived neurotrophic factor modulation of Kv1.3 channel is disregulated by adaptor proteins Grb10 and nShc.
abstract: BACKGROUND
  Neurotrophins are important regulators of growth and regeneration, and acutely, they can modulate the activity of voltage-gated ion channels. Previously we have shown that acute brain-derived neurotrophic factor (BDNF) activation of neurotrophin receptor tyrosine kinase B (TrkB) suppresses the Shaker voltage-gated potassium channel (Kv1.3) via phosphorylation of multiple tyrosine residues in the N and C terminal aspects of the channel protein. It is not known how adaptor proteins, which lack catalytic activity, but interact with members of the neurotrophic signaling pathway, might scaffold with ion channels or modulate channel activity.
  
  
  RESULTS
  We report the co-localization of two adaptor proteins, neuronal Src homology and collagen (nShc) and growth factor receptor-binding protein 10 (Grb10), with Kv1.3 channel as demonstrated through immunocytochemical approaches in the olfactory bulb (OB) neural lamina. To further explore the specificity and functional ramification of adaptor/channel co-localization, we performed immunoprecipitation and Western analysis of channel, kinase, and adaptor transfected human embryonic kidney 293 cells (HEK 293). nShc formed a direct protein-protein interaction with Kv1.3 that was independent of BDNF-induced phosphorylation of Kv1.3, whereas Grb10 did not complex with Kv1.3 in HEK 293 cells. Both adaptors, however, co-immunoprecipitated with Kv1.3 in native OB. Grb10 was interestingly able to decrease the total expression of Kv1.3, particularly at the membrane surface, and subsequently eliminated the BDNF-induced phosphorylation of Kv1.3. To examine the possibility that the Src homology 2 (SH2) domains of Grb10 were directly binding to basally phosphorylated tyrosines in Kv1.3, we utilized point mutations to substitute multiple tyrosine residues with phenylalanine. Removal of the tyrosines 111-113 and 449 prevented Grb10 from decreasing Kv1.3 expression. In the absence of either adaptor protein, channel co-expression reciprocally down-regulated expression and tyrosine phosphorylation of TrkB kinase and related insulin receptor kinase. Finally, through patch-clamp electrophysiology, we found that the BDNF-induced current suppression of the channel was prevented by both nShc and Grb10.
  
  
  CONCLUSIONS
  We report that adaptor protein alteration of kinase-induced Kv1.3 channel modulation is related to the degree of direct protein-protein association and that the channel itself can reciprocally modulate receptor-linked tyrosine kinase expression and activity.
authors.affiliation: Department of Biological Science, Programs in Neuroscience and Molecular Biophysics, The Florida State University, Tallahassee, Florida, USA. bscolley@vcu.edu
authors.forename: Beverly S
authors.initials: BS
authors.lastname: Colley
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Cavallin
authors.forename: Kc
authors.initials: K
authors.lastname: Biju
authors.forename: David R
authors.initials: DR
authors.lastname: Marks
authors.forename: Debra A
authors.initials: DA
authors.lastname: Fadool
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D051383:GRB10 Adaptor Protein / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D051664:Kv1.3 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009830:Olfactory Bulb / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000502:physiology; D017354:Point Mutation; D011485:Protein Binding / Q000502:physiology; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000378:metabolism*
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D051664:Kv1.3 Potassium Channel; D055765:Shc Signaling Adaptor Proteins; D051383:GRB10 Adaptor Protein; D020794:Receptor Protein-Tyrosine Kinases