Spontaneous and voltage‐activated Ca2+ release in adult mouse skeletal muscle fibres expressing the type 3 ryanodine receptor

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Abstract
The physiological properties and role of the type 3 ryanodine receptor (RyR3), a calcium release channel expressed in a wide variety of cell types, remain mysterious. We forced, in vivo, the expression of RyR3 in adult mouse skeletal muscle fibres using a GFP‐RyR3 DNA construct. GFP fluorescence was found within spatially restricted regions of muscle fibres where it exhibited a sarcomere‐related banded pattern consistent with a localization within or near the junctional sarcoplasmic reticulum membrane. Immunostaining confirmed the presence of RyR3 together with RyR1 within the GFP‐positive areas. In ∼90% of RyR3‐positive fibres microinjected with the calcium indicator fluo‐3, we detected repetitive spontaneous transient elevations of intracellular Ca2+ that persisted when fibres were voltage‐clamped at −80 mV. These Ca2+ transients remained essentially confined to the RyR3 expression region. They ranged from wide local events to propagating Ca2+ waves and were in some cases associated with local contractile activity. When voltage‐clamp depolarizations were applied while fluo‐3 or rhod‐2 fluorescence was measured within the RyR3‐expressing region, no voltage‐evoked ‘spark‐like’ elementary Ca2+ release event could be detected. Still global voltage‐activated Ca2+ release exhibited a prominent early peak within the RyR3‐expressing regions. Measurements were also taken from muscles fibres expressing a GFP‐RyR1 construct; positive fibres also yielded a local banded pattern of GFP fluorescence but exhibited no spontaneous Ca2+ release. Results demonstrate that RyR3 is a very potent source of voltage‐independent Ca2+ release activity. Conversely we find no evidence that it could contribute to the production of discrete voltage‐activated Ca2+ release events in differentiated mammalian skeletal muscle.