Isolation and Characterization of Multi-Protein Complexes Enriched in the K-Cl Co-transporter 2 From Brain Plasma Membranes.

Frontiers in Molecular Neuroscience
J. L. SmalleyStephen J Moss

Abstract

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. ...Continue Reading

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Citations

Feb 8, 2021·Neuroscience and Biobehavioral Reviews·Carlijn Peerboom, Corette J Wierenga
Feb 5, 2021·The Journal of Biological Chemistry·Georgina KontouJoshua L Smalley
Sep 29, 2021·Open Biology·Angeliki DitsiouGeorgios Giamas

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Datasets Mentioned

BETA
PXD021368

Methods Mentioned

BETA
immunoprecipitation
PCA
glycosylation
electrophoresis
genotyping

Software Mentioned

R
MSnbase package
Fiji
Vennerable
MSGF
Multiple Sequence Alignment ( MSA )
ggfortify
igraph
mygene
STRINGdb

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