Quantitative proteomics of the Cav2 channel nano-environments in the mammalian brain

Abstract

Local Ca 2+ signaling occurring within nanometers of voltage-gated Ca 2+ (Cav) channels is crucial for CNS function, yet the molecular composition of Cav channel nano-environments is largely unresolved. Here, we used a proteomic strategy combining knockoutcontrolled multiepitope affinity purifications with high-resolution quantitative MS for comprehensive analysis of the molecular nano-environments of the Cav2 channel family in the whole rodent brain. The analysis shows that Cav2 channels, composed of poreforming α1 and auxiliary β subunits, are embedded into protein networks that may be assembled from a pool of ∼200 proteins with distinct abundance, stability of assembly, and preference for the three Cav2 subtypes. The majority of these proteins have not previously been linked to Cav channels; about two-thirds are dedicated to the control of intracellular Ca 2+ concentration, including G proteincoupled receptor-mediated signaling, to activity-dependent cytoskeleton remodeling or Ca 2+ -dependent effector systems that comprise a high portion of the priming and release machinery of synaptic vesicles. The identified protein networks reflect the cellular processes that can be initiated by Cav2 channel activity and define the molecular framework for organization and operation of local Ca 2+ signaling by Cav2 channels in the brain.calcium channel | Ca 2+ signaling | proteome | biochemistry | mass spectrometry

Age of quantitative proteomics hits voltage-gated calcium channels

“…When the stargazer mouse was identified to have a mutation in a protein that was a homolog of γ1 (termed stargazin or γ2), it was classified as a neuronal calcium channel γ-subunit (15). However, in the study by Müller et al (6), no interactions with any γ-subunits were found, which agrees with the predominantly held view that these proteins are transmembrane AMPA receptor interacting proteins (16).…”

“…Müller et al (6) are dealing with ion channels that are primarily presynaptic, and this is confirmed by the absence of key PSD proteins from the interactome. In this study, complexes of Ca V 2 channels and associated β-subunits from brain have been isolated using affinity purification steps, and MS has been used to identify the copurified proteins.…”

Calcium influx through L-type CaV1.2 Ca2+ channels regulates mandibular development

“…Regulation of Ca 2+ influx is the best-characterized role for Ca V 1.2, but voltage-gated Ca 2+ channels anchor large macromolecular complexes (19) and could therefore control cellular processes independent of Ca 2+ influx. Indeed, the Ca 2+ channel auxiliary subunit β4 controls epiboly in zebrafish through a Ca 2+ -independent manner (20).…”

Ca 2+ channel nanodomains boost local Ca 2+ amplitude

“…Alternatively, the region of amplification could extend beyond the Ca V itself, if augmented by diffusion-impeding assemblies of associated components. In this regard, it is interesting to note that Ca V subtypes interact with a tremendous variety of proteins in different cellular contexts (52,53). Indeed, the very same molecules that aggregate near Ca V channels to convey downstream signals could also act to restrict diffusion, thereby increasing the gain between Ca 2+ flux and biochemical response.…”