title: Proteomic analyses of native brain K(V)4.2 channel complexes.
abstract: Somatodendritic A-type (I(A)) voltage-gated K(+) (K(V)) channels are key regulators of neuronal excitability, functioning to control action potential waveforms, repetitive firing and the responses to synaptic inputs. Rapidly activating and inactivating somatodendritic I(A) channels are encoded by K(V)4 alpha subunits and accumulating evidence suggests that these channels function as components of macromolecular protein complexes. Mass spectrometry (MS)-based proteomic approaches were developed and exploited here to identify potential components and regulators of native brain K(V)4.2-encoded I(A) channel complexes. Using anti-K(V)4.2 specific antibodies, K(V)4.2 channel complexes were immunoprecipitated from adult wild type mouse brain. Parallel control experiments were performed on brain samples isolated from (K(V)4.2(-/-)) mice harboring a targeted disruption of the KCND2 (K(V)4.2) locus. Three proteomic strategies were employed: an in-gel approach, coupled to one-dimensional liquid chromatography-tandem MS (1D-LC-MS/MS), and two in-solution approaches, followed by 1D- or 2D-LC-MS/MS. The targeted in-gel 1D-LC-MS/MS analyses demonstrated the presence of the K(V)4 alpha subunits (K(V)4.2, K(V)4.3 and K(V)4.1) and the K(V)4 accessory, KChIP (KChIP1-4) and DPP (DPP6 and 10), proteins in native brain K(V)4.2 channel complexes. The more comprehensive, in-solution approach, coupled to 2D-LC-MS/MS, also called Multidimensional Protein Identification Technology (MudPIT), revealed that additional regulatory proteins, including the K(V) channel accessory subunit K(V)beta1, are also components of native brain K(V)4.2 channel complexes. Additional biochemical and functional approaches will be required to elucidate the physiological roles of these newly identified K(V)4 interacting proteins.
authors.affiliation: Department of Developmental Biology, Washington University, St. Louis, MO, USA.
authors.forename: Céline
authors.initials: C
authors.lastname: Marionneau
authors.forename: Richard D
authors.initials: RD
authors.lastname: LeDuc
authors.forename: Henry W
authors.initials: HW
authors.lastname: Rohrs
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Link
authors.forename: R Reid
authors.initials: RR
authors.lastname: Townsend
authors.forename: Jeanne M
authors.initials: JM
authors.lastname: Nerbonne
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002853:Chromatography, Liquid; D051605:Kv Channel-Interacting Proteins / Q000032:analysis / Q000302:isolation & purification*; D051379:Mice; D008817:Mice, Mutant Strains; D040901:Proteomics / Q000379:methods*; D051672:Shal Potassium Channels / Q000172:deficiency / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D051605:Kv Channel-Interacting Proteins; D051672:Shal Potassium Channels