Determination of the Junctional Space [Ca2+] Set by Ryanodine Receptor Leak in Slow- and Fast-Twitch Muscle Fibres

Abstract

The tubular (t-) system of skeletal muscle forms a junction with the sarcoplasmic reticulum (SR), with some 12nm between the membranes. In the resting muscle, [Ca2+] within the small volume bound by the junctional membranes will be determined by the leak of Ca2+ through the SR ryanodine receptors (RyRs), the Ca2+ handling ability of the t-system and diffusion of Ca2+ from the junctional space (js). The [Ca2+]js is expected to be higher than [Ca2+]bulk with a standing gradient set between the RyRs and SR Ca2+-pumps. The value of [Ca2+]js is unknown but has implications for signalling cascades initiating in this nanodomain. To determine [Ca2+]js we exploited the fact that t-system Ca2+ uptake activity will be set by [Ca2+]js. T-system Ca2+-uptake activity was tracked with rhod-5N trapped in the t-system of mechanically skinned fibres of rat slow- and fast-twitch muscles on a confocal microscope. Chronic depletion of [Ca2+]SR with caffeine reduced [Ca2+]t-sys to 0.1 mM via chronic activation of store-operated Ca2+ entry. We then exposed Ca2+-depleted preparations to 50-800nM [Ca2+]cyto in 50mM EGTA to allow observation of t-system Ca2+ uptake rates at known [Ca2+]bulk. Experiments were repeated in the presence of 1mM tetracaine to block RyR Ca2+ leak and allow [Ca2+]js to equilibrate with [Ca2+]bulk. Rhod-5N signals and [Ca2+]t-sys were calibrated and t-system Ca2+ fluxes were derived. [Ca2+]bulk and peak t-system Ca2+ fluxes were fitted by Hill curves. Vmax was significantly depressed in slow- compared to fast-twitch fibres. The kD for both fibre types was right-shifted by tetracaine. It followed that at 100nM [Ca2+]bulk, [Ca2+]js was 165 and 220nM in slow and fast-twitch fibres, respectively. These results show that t-system Ca2+ fluxes can be used as a nanodomain sensor of RyR leak.