TY - JOUR
T1 - Proteomic analyses of native brain KV4.2 channel complexes
AU - Marionneau, Céline
AU - LeDuc, Richard D.
AU - Rohrs, Henry W.
AU - Link, Andrew J.
AU - Townsend, R. Reid
AU - Nerbonne, Jeanne M.
N1 - Funding Information:
Nerbonne, R01-GM064779 to Andrew J. Link), the National Center for Research Resources (NIH P41RR000954), the NIH Neuroscience Blueprint Center Core Grant (P30-NS057105), the W.M. Keck Foundation, and the Heartland Affiliate of the American Heart Association (Postdoctoral Fellowship to Céline Marionneau) is gratefully acknowledged.
Funding Information:
The financial support provided by the Washington University— Pfizer Biomedical Research Program (to Jeanne M. Nerbonne), the National Institutes of Health (R01-HL034161 to Jeanne M.
Funding Information:
Antibodies and western blot analyses. The brain KV4.2 protein was detected using an anti-KV4.2 mouse monoclonal antibody (mαKV4.2, K57/1), developed by and obtained from UC Davis/NIH NeuroMab Facility (supported by NIH grant U24NS050606 and maintained by the University of California, Davis, CA 95616). Bound primary antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (Pierce). Protein signals were visualized using the SuperSignal West Dura Extended Duration substrate (Pierce).
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
KW - Accessory subunits
KW - I
KW - Mass spectrometric identification
UR - http://www.scopus.com/inward/record.url?scp=68949147692&partnerID=8YFLogxK
U2 - 10.4161/chan.3.4.9553
DO - 10.4161/chan.3.4.9553
M3 - Article
C2 - 19713751
AN - SCOPUS:68949147692
VL - 3
JO - Channels
JF - Channels
SN - 1933-6950
IS - 4
ER -