FASEB journal : official publication of the Federation of American Societies for Experimental Biology
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In this study, the principles of surface sensing of translation (SUnSET) were used to develop a nonradioactive method for ex vivo and in vivo measurements of protein synthesis (PS). Compared with controls, we first demonstrate excellent agreement between SUnSET and a [(3)H]phenylalanine method when detecting synergist ablation-induced increases in skeletal muscle PS ex vivo. We then show that SUnSET can detect the same synergist ablation-induced increase in PS when used in vivo (IV-SUnSET). ⋯ Furthermore, transfection with Ras homolog enriched in brain (Rheb) revealed that a PKB-independent activation of mammalian target of rapamycin is also sufficient to induce an increase in skeletal muscle PS. Finally, IV-IHC-SUnSET exposed the existence of fiber type-dependent differences in skeletal muscle PS, with PS in type 2B and 2X fibers being significantly lower than that in type 2A fibers within the same muscle. Thus, our nonradioactive method allowed us to accurately visualize and quantify PS under various ex vivo and in vivo conditions and revealed novel insights into the regulation of PS in skeletal muscle.
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TMEM16A (anoctamin 1, Ano1), a member of a family of 10 homologous proteins, has been shown to form an essential component of Ca(2+)-activated Cl(-) channels. TMEM16A-null mice exhibit severe defects in epithelial transport along with tracheomalacia and death within 1 mo after birth. Despite its outstanding physiological significance, the mechanisms for activation of TMEM16A remain obscure. ⋯ In contrast, the Ca(2+) binding protein calmodulin appears indispensable and interacts physically with TMEM16A. Openers of small- and intermediate-conductance Ca(2+)-activated potassium channels known to interact with calmodulin, such as 1-EBIO, DCEBIO, or riluzole, also activated TMEM16A. These results reinforce the use of these compounds for activation of electrolyte secretion in diseases such as cystic fibrosis.