Biophysical Society Thematic Meeting | Bucharest 2026

Biophysics of Membrane Reactions in Brian

Tuesday Speaker Abstracts

STRUCTURAL MECHANISMS OF MEMBRANE TRANSPORT AND ALLOSTERIC REGULATION IN KCC2, A KEY REGULATOR OF NEURONAL INHIBITION Katharina L Duerr University of Oxford, Chemistry, Oxford, United Kingdom The neuronal K ⁺ –Cl ⁻ cotransporter KCC2 establishes the chloride gradient required for hyperpolarizing inhibitory neurotransmission. Disrupted KCC2 function is linked to epilepsy, neuropathic pain, and neurodevelopmental disorders. Although phosphorylation and cellular metabolites are known to influence KCC2, the structural mechanisms coupling regulatory inputs to transport activity remain poorly defined. Here we delineate conformational and allosteric mechanisms that control KCC2 using hydrogen–deuterium exchange mass spectrometry (HDX MS), native mass spectrometry, cryo-EM, and functional chloride-transport assays. HDX-MS resolved state-dependent dynamics across the transporter and identified coupling between the cytoplasmic C-terminal domain (CTD) and the transmembrane transport core. ATP binding to a conserved pocket in the CTD stabilized the inner lobe while increasing dynamics in transmembrane helices that form the ion-translocation pathway, consistent with reduced energetic barriers toward outward-open states. Native MS enabled direct quantification of ATP binding and showed that mutations within the CTD nucleotide pocket tune ATP affinity, rationalizing effects of naturally occurring and disease-associated variants. Cryo-EM structures in inward-open, outward-open, and outward-occluded conformations revealed pronounced dimer rearrangements during the transport cycle, including ~60° rotation of the CTD relative to the membrane domain. These transitions involve reorganization of a conserved region near the phosphoregulatory site T906 and propagate to the N-terminal autoinhibitory segment, defining a structural pathway by which phosphorylation and nucleotide binding modulate activity. Small molecules that compete with ATP reduced transport and attenuated ATP-dependent conformational priming, supporting a positive allosteric role of nucleotide binding. Together, these data define a mechanistic framework in which cytoplasmic regulatory domains, metabolite sensing, and large-scale conformational changes are tightly coupled to membrane transport. This work provides a structural basis for understanding how neuronal inhibition is tuned at the membrane level and identifies regulatory sites that can be leveraged to modulate KCC2 activity.

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