Muscle protein wasting (cachexia) arising from metabolic acidosis is a serious problem in illnesses such as renal failure, and may be reversed by therapeutic exercise. It is therefore important to understand how physiological stimuli such as acidosis and exercise signal to protein metabolism in muscle. We have shown previously that acidosis inhibits the SNAT2 L-glutamine (L-Gln) transporter, the dominant determinant of amino acid levels in skeletal muscle cells. This transporter may also be up-regulated by exercise (King 1994). Intracellular amino acid concentration is sensed by mammalian target of Rapamycin (mTOR), a key regulator of protein synthesis. Aims: To investigate the effect of SNAT2 inhibition during acidosis on Rapamycin-sensitive signalling through mTOR to protein synthesis in L6 skeletal muscle cells. Methods: Cells were cultured with 2mM L-Gln and 2% serum or 100nM insulin. Protein synthesis was determined from incorporation of ³H-L-Phe. Phosphorylation of targets of mTOR which regulate translation was determined by immunoblotting with phosphospecific antibodies. Results: Lowering extracellular pH to 7.1 to model acidosis led to a sustained (48h) impairment of protein synthesis (13.7 ± 0.6 nmol ³H-L-Phe/mg protein/4h v 16.0 ± 0.9 in control cultures at pH 7.4 (P<0.05)). Blockade of mTOR signalling with 10nM Rapamycin abolished this effect (12.3 ± 0.6 at pH 7.1 v 13.0 ± 0.6 at pH 7.4, NS), as did removal of extracellular L-Gln (11.6 ± 0.7 at pH 7.1 v 11.9 ± 0.6 at pH 7.4, NS). Competitive inhibition of SNAT2 at pH 7.4 with its selective substrate MeAIB also impaired protein synthesis (12.9 ± 0.3 v 15.4 ± 0.4 in controls, P<0.05) as did selective silencing of SNAT2 expression with siRNA (11.9 ± 0.9 v 13.9 ± 0.3 in controls, P<0.05). Within 2h acidosis or MeAIB impaired signalling through mTOR, leading to reduced phosphorylation of ribosomal protein (rp)S6, rpS6 kinase and 4E-BP1, assessed by densitometry of immunoblots (e.g. 52 ± 8 % inhibition of rpS6 phosphorylation with MeAIB, P<0.05). Similar impairment of rpS6 phosphorylation (78 ± 9 % inhibition, P<0.05) was induced by silencing SNAT2 with siRNA. Conclusions: Acidosis signals to protein synthesis by inhibiting SNAT2 and depleting intracellular amino acids thereby inhibiting mTOR. SNAT2 is therefore a key regulator of mTOR and, in view of its strong responsiveness to factors such as acidosis and exercise, is of central importance in regulating protein metabolism and muscle cell growth.