Remote ischemic postconditioning attenuates cerebral ischemia–reperfusion injury by preserving the astrocytic Na+/K+-ATPase α1–Src complex and preventing ERK-driven GLT-1 surface loss

Background Remote ischemic postconditioning (RIPC) protects against cerebral ischemia-reperfusion (I/R) injury, yet its molecular mechanism is not fully elucidated. Na + /K + -ATPase α1 (NKAα1) forms a signaling complex with Src kinase in cardiac and renal systems, but direct biochemical evidence for this signalosome in the brain has not been discovered. Whether ERK1/2 drives loss of surface GLT-1 during cerebral I/R and whether RIPC restores GLT-1 by reactivating NKAα1 have also not been determined. Methods Rat MCAO/R and primary cortical neuron-astrocyte oxygen-glucose deprivation/reoxygenation models were combined with pharmacological inhibition, adenovirus-mediated NKAα1 overexpression, molecular docking, human transcriptomic analysis (GSE58294), two-sample Mendelian randomization (MR), and Bayesian colocalization. Results We provide the first direct biochemical evidence by forward and reverse co-immunoprecipitation for an NKAα1–Src interaction in brain astrocytes. Cerebral I/R disrupted this complex, releasing Src from NKAα1-mediated tonic inhibition, which in turn triggered Src (Tyr419) and ERK1/2 hyperphosphorylation and drove GLT-1 membrane loss. RIPC restored NKA activity and NKAα1 expression. This reinforced NKAα1–Src binding and attenuated the downstream Src–ERK cascade, rescuing GLT-1 membrane localization and astrocytic glutamate clearance. RIPC also reduced infarct volume and improved neurological outcomes. NKAα1 overexpression independently reproduced these neuroprotective effects. In brain astrocytes, Src release from NKA drove ERK-dependent GLT-1 loss and excitotoxic injury—contrasting with the pro-survival role reported in cardiac cells. In stroke patients, peripheral blood transcriptomic co-expression supported pathway coordination ( n = 92). MR identified a causal effect of ATP1A1 on stroke risk (β = 1.362, p < 0.0001), and colocalization showed suggestive evidence for a shared causal variant (PP.H4 = 0.643). Conclusion This study reveals the NKAα1–Src–ERK–GLT-1 axis as a newly identified brain pathway linking energy failure to excitotoxicity and provides MR support for ATP1A1 as a candidate causal gene in ischemic stroke.