PE-22-28 is a synthetic heptapeptide analog derived from proteolytic fragments of the sortilin propeptide spadin (PE12–28) that functions as a potent and selective antagonist of the TREK-1 (KCNK2) two-pore domain potassium channel. Whole-cell patch-clamp electrophysiology in HEK293 cells stably expressing human TREK-1 demonstrates that PE-22-28 inhibits channel currents with an IC₅₀ of 0.12 nM, conferring an approximately 300- to 500-fold increase in potency relative to spadin (IC₅₀ 40–60 nM) while exhibiting no detectable activity on the closely related K₂P channels TREK-2, TRAAK, TRESK, or TASK-1, nor on hERG. N- or C-terminal modifications of PE-22-28, including glycine-to-alanine substitution or biotinylation, preserve or further enhance this affinity profile. Pharmacokinetic assessments in rodent systems reveal markedly improved in vivo stability, with duration of action extended to approximately 23 h compared with 7 h for spadin. In primary cultures of mouse cortical neurons, PE-22-28 upregulates expression of the synaptogenic markers PSD-95 and synapsin, indicating enhanced synaptogenesis. Subchronic administration in mice stimulates hippocampal neurogenesis, as quantified by increased BrdU-positive cell counts after 4 days of treatment, and elevates BDNF mRNA and protein levels. In murine focal cerebral ischemia models, the peptide displays biphasic modulation of TREK-1 conductance—facilitating channel activity at low nanomolar doses while blocking it at higher concentrations—coincident with reduced neuronal apoptosis, attenuated dopaminergic degeneration in the substantia nigra, and augmented post-ischemic neurogenesis and synaptogenesis.