Ryanodine Receptor Activity Regulates the Levels of Ca2+ Extrusion and Store-Operated Ca2+ Entry in Skeletal Muscle

Abstract

The molecular units for store-operated Ca2+ entry (SOCE) and tubular (t-) system Ca2+ extrusion are uniformly distributed across the junctional membranes of skeletal muscle and can be rapidly activated upon Ca2+ release, intimately linking these processes with ryanodine receptor activity. However, the physiological relationship between Ca2+ extrusion, SOCE and RyR activity in skeletal muscle remains poorly defined. To examine this we tracked Ca2+ extrusion and entry into muscle fibres by imaging a Ca2+-sensitive dye trapped in the t-system of skinned fibres (Launikonis et al 2003, PNAS). Application of an internal solution containing 50 mM EGTA, caffeine and low Mg2+ caused depletion of the SR and rapid activation of SOCE. We found that reintroducing [Ca2+]cyto in the broad physiological range caused the t-system to take up Ca2+ at rates dependent on [Ca2+]cyto and reach steady levels in the mM range and this was partially inhibited by RyR leak blockers. Thus t-system Ca2+-handling was sensitive to RyR Ca2+ leak and we found that RyR Ca2+ leak increased non-linearly with [Ca2+]cyto. By lowering [Mg2+]cyto to 0.13mM to increase RyR leak further than physiological levels in the presence of a broad range of [Ca2+]cyto, t-system Ca2+ uptake rate and steady state [Ca2+]t-sys was reduced to below resting conditions but above that where SOCE was activated in the absence of Ca2+. We conclude that increasing RyR Ca2+ leak activates SOCE, allowing SOCE to simultaneously act as a counter-current against Ca2+ being extruded from the fibre. Thus SOCE holds Ca2+ within the fibre rather than sequentially recovering Ca2+ previously lost from the fibre, as commonly presumed.