The Acidic Helix Plays a Crucial Role in Regulating KCC2 Activity in Response to Osmotic Stress

Background: Electroneutral K-Cl cotransporters (KCCs) are membrane proteins essential for regulating cell volume, ion transport, and GABA neurotransmission. Their activity is modulated by phosphorylation and dephosphorylation mechanisms. Among KCCs, KCC2 is critical for neuronal development, as it regulates intracellular chloride concentration, which is essential for neuronal excitability and synaptic inhibition. Consequently, dysregulation of KCC2 has been implicated in neurological disorders, including epilepsy and autism. The recent application of cryogenic electron microscopy (cryo-EM) has provided detailed insights into the ultrastructure of KCC2, identifying key functional domains, such as the acidic helix, which is located in the N-terminal region and comprises four glutamate residues. It is postulated that this domain interacts with the C-terminal region to facilitate cation attraction, contributing to KCC2 activity. The present study aimed to investigate the impact of osmotic stress on the phosphorylation status of the acidic helix in human KCC2a by introducing mutations. Methods: Four mutant clones targeting the acidic helix were generated using wild-type (WT) KCC2a and overexpressed in Xenopus laevis oocytes and HEK293 cells. The mutants were stimulated under isotonic and hypotonic condition, and their phosphorylation status was evaluated using Western blot with specific antibodies. Phosphorylation of SPAK kinase (pS373) was analyzed to validate osmotic stimulation, while β-actin was used as loading control. Results: Total expression of the mutated clones demonstrated proper translational processing, comparable to that of the wild-type clone. Since KCC2 activation is regulated by dephosphorylation in response to increased cell volume, its activity was assessed using phospho-specific antibodies targeting threonine residues T929 and T1030. During the osmotic stimulation assay, a significant decrease in phosphorylation was observed under hypotonic conditions in the single mutant (E100A) and the double mutant (2MHx: E100/102A). This response was comparable to that in the WT KCC2 clone, suggesting that these mutants retained the ability to activate the cotransporter upon cell swelling, facilitating ion outflow to reduce cell volume. In contrast, when three (3MHx: E100/102/103A) or all four glutamate residues (4MHx: E100/102/103/105A) in the acidic helix were mutated to alanine, protein expression levels increased under both isotonic and hypotonic conditions. However, phosphorylation levels at threonine T929 and T1030 did not decrease under hypotonic conditions. Instead, they remained similar to those observed under isotonic conditions, suggesting a disruption in the regulatory response of the cotransporter to cell swelling. This finding indicates that the acidic helix plays a crucial role in the proper function of KCC2 in response to osmotic changes. Conclusions: 3MHx and 4MHx mutants disrupt the phosphorylation dynamics of the canonical regulatory sites (T929 and T1030) following cell swelling. This alteration in phosphorylation status likely impacts the functional activity of the KCC2 cotransporter. To further validate this hypothesis, ongoing studies are focused on conducting functional assays to directly assess the impact of these mutations on KCC2 activity. AM: CONAHCyT CF-2023-I532 and INCar 22-1345. PdlH: Papiit IN228123. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.