Proteomic analyses of native brain K_V4.2 channel complexes

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_V4 α 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_V4.2-encoded I_A channel complexes. Using anti-K_V4.2 specific antibodies, K_V4.2 channel complexes were immunoprecipitated from adult wild type mouse brain. parallel control experiments were performed on brain samples isolated from (K_V4.2^-/-) mice harboring a targeted disruption of the KCND2 (K_V4.2) 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 _V4 α subunits (K_V4.2, K_V4.3 and K _V4.1) and the K_V4 accessory, KChIp (KChIp1-4) and Dpp (Dpp6 and 10), proteins in native brain K_V4.2 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_Vβ1, are also components of native brain K _V4.2 channel complexes. Additional biochemical and functional approaches will be required to elucidate the physiological roles of these newly identified K_V4 interacting proteins.