The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle, and the lack of dystrophin in Duchenne muscular dystrophy results in a reduction of DAPC components such as syntrophins and in fiber necrosis. By anchoring various molecules, the syntrophins may confer a role in cell signaling to the DAPC. Calcium disorders and abnormally elevated cation influx in dystrophic muscle cells have suggested that the DAPC regulates some sarcolemmal cationic channels. We demonstrated previously that mini-dystrophin and alpha1-syntrophin restore normal cation entry in dystrophin-deficient myotubes and that sarcolemmal TRPC1 channels associate with dystrophin and the bound PDZ domain of alpha1-syntrophin. This study shows that small interfering RNA (siRNA) silencing of alpha1-syntrophin dysregulated cation influx in myotubes. Moreover, deletion of the PDZ-containing domain prevented restoration of normal cation entry by alpha1-syntrophin transfection in dystrophin-deficient myotubes. TRPC1 and TRPC4 channels are expressed at the sarcolemma of muscle cells; forced expression or siRNA silencing showed that cation influx regulated by alpha1-syntrophin is supported by TRPC1 and TRPC4. A molecular association was found between TRPC1 and TRPC4 channels and the alpha1-syntrophin-dystrophin complex. TRPC1 and TRPC4 channels may form sarcolemmal channels anchored to the DAPC, and alpha1-syntrophin is necessary to maintain the normal regulation of TRPC-supported cation entry in skeletal muscle. Cation channels with DAPC form a signaling complex that modulates cation entry and may be crucial for normal calcium homeostasis in skeletal muscles.