Membrane fusion triggered by Ca²⁺ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes^1-4. For neurotransmitter release, the Ca²⁺ sensitivity is introduced by interactions between the Ca²⁺ sensor synaptotagmin and the SNARE complex⁵, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion⁶. Disruption of Ca²⁺-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis^7-11. Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca²⁺-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca²⁺-binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca²⁺-triggered fusion at physiological Ca²⁺ concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca²⁺-triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.

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