SCN5A encodes the alpha subunit of the cardiac muscle and intestinal smooth muscle mechanosensitive Na(+) channel. Mechanosensitivity in the intestine requires an intact cytoskeleton. We report, using laser capture microdissection, single cell PCR, and immunohistochemistry, that syntrophins, scaffolding proteins, were expressed in human intestinal smooth muscle cells. The distribution of syntrophin gamma 2 was similar to that of SCN5A. Yeast two-hybrid and glutathione S-transferase pull-down experiments show that SCN5A and syntrophin gamma 2 co-express and that the PDZ domain of syntrophin gamma 2 directly interacts with the C terminus of SCN5A. In native cells, disruption of the C terminus-syntrophin gamma 2 PDZ domain interaction using peptides directed against either region result in loss of mechanosensitivity. Co-transfection of syntrophin gamma 2 with SCN5A in HEK293 cells markedly shifts the activation kinetics of SCN5A and reduces the availability of Na(+) current. We propose that syntrophin gamma 2 is an essential Na(+) channel-interacting protein required for the full expression of the Na(+) current and that the SCN5A-syntrophin gamma 2 interaction determines mechanosensitivity and current availability.