Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons

Abstract

Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are many published protocols to isolate protein complexes in cell lines, isolation in neurons has not been well established. Here we introduce a method that combines lentiviral protein expression with tandem affinity purification followed by mass-spectrometry (TAP-MS) to identify protein complexes in neurons. This protocol can also be used to identify post-translational modifications (PTMs) of synaptic proteins. We used the A-type voltage-gated K+ channel subunit Kv4.2 as the target protein. Kv4.2 is highly expressed in the hippocampus where it contributes to learning and memory through its regulation of neuronal excitability and synaptic plasticity. We tagged Kv4.2 with the calmodulin-binding-peptide (CBP) and streptavidin-binding-peptide (SBP) at its C-terminus and expressed it in neurons via lentivirus. Kv4.2 was purified by two-step TAP and samples were analyzed by MS. MS identified two prominently known Kv4.2 interacting proteins [dipeptidyl peptidase like (DPPs) and Kv channel-interacting proteins (KChIPs)] in addition to novel synaptic proteins including glutamate receptors, a calcium channel, and anchoring proteins. Co-immunoprecipitation and colocalization experiments validated the association of Kv4.2 with glutamate receptors. In addition to protein complex identification, we used TAP-MS to identify Kv4.2 phosphorylation sites. Several known and unknown phosphorylation sites were identified. These findings provide a novel path to identify protein-protein interactions and PTMs in neurons and shed light on mechanisms of neuronal signaling potentially involved in the pathology of neurological diseases.

Ion Channel and Transporter Involvement in Chemotherapy-Induced Peripheral Neurotoxicity

“…This specific neuronal distribution allows I A not only to regulate the integration and the propagation of the excitatory synaptic potentials, but also to modulate the back-propagating potentials in dendritic branches [ 60 , 61 ]. Among the five A-channels in mammals, recently, several authors have focused their attention on two specific subunits: Kv4.2 and Kv4.3 [ 43 , 55 , 62 , 63 ]. The latter is often associated with different β subunits and multiple K + channel interacting proteins, such as KChIPs, which can modulate Kv4 current properties, neuronal trafficking and, in turn, its functions [ 63 , 64 ].…”

“…The latter is often associated with different β subunits and multiple K + channel interacting proteins, such as KChIPs, which can modulate Kv4 current properties, neuronal trafficking and, in turn, its functions [ 63 , 64 ]. Kv4.2 subunits are specifically localized in the dendritic membrane, fine-tuning the back-propagation of signals and dendritic excitability, whereas Kv4.3 resides principally on the soma [ 43 , 62 ]. Highlighting Kv4’s fundamental functions, different defects in trafficking, expression or kinetics are observed in several disorders, such as memory deficits or peripheral neuropathic pain [ 55 , 57 , 62 ].…”

“…Kv4.2 subunits are specifically localized in the dendritic membrane, fine-tuning the back-propagation of signals and dendritic excitability, whereas Kv4.3 resides principally on the soma [ 43 , 62 ]. Highlighting Kv4’s fundamental functions, different defects in trafficking, expression or kinetics are observed in several disorders, such as memory deficits or peripheral neuropathic pain [ 55 , 57 , 62 ].…”