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 Ca(2+) 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.