Dystrophin is a large, submembrane cytoskeletal protein, an absence of which causes Duchenne muscular dystrophy. Utrophin is a dystrophin homologue found in both muscle and brain whose physiological function is unknown. Recordings of single‐channel activity were made from membrane patches on skeletal muscle from mdx, mdx/utrn+/− heterozygotes, and mdx/utrn−/− double knockout mice to investigate the role of these cytoskeletal proteins in mechanosensitive channel gating. We find complex, gene dose‐dependent effects of utophin depletion in dystrophin‐deficient mdx muscle: 1) increased MS channel open probability, 2) a shift of MS chanel gating to larger pressures, 3) appearance of modal gating of MS channels and small conductance channels, and 4) expression of large conductance MS channels. We suggest a physical model in which utrophin acts as a scaffolding protein that stabilizes lipid microdomains and clusters MS channel subunits. Depletion of utrophin disrupts domain composition in a manner that favors open channel area expansion, as well as allowing diffusion and aggregation of addditional MS channel subunits. This article is protected by copyright. All rights reserved