Somatodendritic A-type (IA) 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 KV4 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 KV4.2-encoded IA channel complexes. Using anti-KV4.2 specific antibodies, KV4.2 channel complexes were immunoprecipitated from adult wild type mouse brain. parallel control experiments were performed on brain samples isolated from (KV4.2-/-) mice harboring a targeted disruption of the KCND2 (KV4.2) 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 (KV4.2, KV4.3 and K V4.1) and the KV4 accessory, KChIp (KChIp1-4) and Dpp (Dpp6 and 10), proteins in native brain KV4.2 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 KV channel accessory subunit KVβ1, 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 KV4 interacting proteins.
- Accessory subunits
- Mass spectrometric identification