Project Summary Maintenance of muscle mass is known to be beneficial in the prevention of obesity and obesity-related diseases such as diabetes and heart disease, in addition to promoting overall health of our aging population. Skeletal muscle atrophy is associated with cancer (cachexia), heart failure, chronic corticosteroid use, paralysis or denervation (disuse atrophy), aging, contributing to poor metabolic health, and increased mortality. Accordingly, a deeper understanding of the molecular mechanisms that regulate skeletal muscle maintenance, growth and function is critical for human health. We recently showed that, in skeletal muscle, SWELL1 is required for maintaining AKT-mTOR signaling, normal muscle fiber size, exercise capacity, force generation, adiposity and systemic glycemia, thereby revealing a novel role for a SWELL1-AKT-mTOR signaling axis in skeletal muscle physiology. Furthermore, our group now has published and unpublished biochemical, patch-clamp and imaging evidence that SWELL1 channel complexes are also expressed and functional in lysosomes (Lyso-SWELL1) – a notion also supported by a recent unbiased CRISPR screen. Given that lysosomes are signaling hubs that integrate nutrient sensing and AKT-mTOR signaling, we hypothesize that SWELL1-LRRC8 channels co-regulate plasma membrane PI3K-AKT signaling and lysosome centered nutrient-mTOR signaling. To test this hypothesis, we combine our unique reagents and expertise in SWELL1 signaling with the those of the Diwan (lysosomal signaling), Xu (lysosomal patch-clamp), and Meyer (skeletal muscle physiology) laboratories. Our objective is to understand the dual mechanisms of plasma membrane SWELL1 signaling and lysosomal SWELL1 (Lyso-SWELL1) nutrient sensing in skeletal muscle and its contribution to skeletal muscle growth and function. The rationale for these studies is that delineating the molecular mechanisms of skeletal muscle SWELL1-AKT-mTOR signaling will advance our understanding of novel, fundamental mechano-signaling and nutrient sensing mechanisms that regulate skeletal muscle growth and function. We propose the following AIMs: · AIM#1: Delineate the mechanisms of plasma membrane versus lysosomal SWELL1 signaling to AKT- mTOR signaling in skeletal muscle cells. These studies will test a novel paradigm for cellular nutrient sensing by a lysosomal ion channel signaling complex in vitro, setting the stage for in vivo experiments. · AIM#2: Examine the contribution of SWELL1 signaling to aerobic capacity, skeletal muscle growth, and force generation in vivo with training and with aging. These studies will define the contributions of SWELL1 signaling to skeletal muscle growth and signaling in vivo, and with aging.