Disruption of cytoskeletal integrity impairs G_i-mediated signaling due to displacement of G_i proteins

β1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in β1 integrin deficient (−/−) embryonic stem cells. Muscarinic inhibition of the L-type Ca²⁺ current (I_Ca) and activation of the acetylcholine-activated K⁺ current (I_K,ACh) was found to be absent in β1 integrin^−/− cardiomyocytes. Conversely, β adrenoceptor-mediated modulation of I_Ca was unaffected by the absence of β1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that G_i exhibited an atypical subcellular distribution in the β1 integrin^−/− cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced G_i and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of G_i-associated signaling microdomains.