Molecular architecture of potassium chloride co-transporter KCC2
KCC2 is a neuron-specific potassium-chloride co-transporter that plays a crucial role in maintaining neuronal chloride balance and is implicated in several neurological disorders, including traumatic brain injury, epilepsy, autism, and schizophrenia. Although much is known about its biological and electrophysiological properties, the structural basis of its function remains largely unclear. In this study, we successfully solubilized and purified wild-type, non-aggregated, and homogeneous KCC2 using calixarene detergent. Surface plasmon resonance (SPR) confirmed specific binding of the inhibitor VU0463271. Mass spectrometry analysis identified expected post-translational modifications, including glycosylation and phosphorylation, confirming the functionality of the purified protein. Electron microscopy revealed that KCC2 exists in both monomeric and dimeric forms in solution. Monomers feature distinct “head” and “core” domains connected by a flexible “linker” region, while dimers adopt an asymmetric, bent S-shaped conformation composed of four domains and a flexible dimerization interface. Chemical crosslinking experiments under reducing conditions indicate that disulfide bonds contribute to dimer formation. Additionally, we found that tagging the C-terminus impairs KCC2 function, suggesting that the conserved C-terminal region plays a critical role in dimerization. Overall, these findings provide the first detailed insights into KCC2’s flexible, multi-domain architecture and highlight the structural features essential for its function.