RyR1-specific requirement for depolarization-induced Ca²⁺ sparks in urinary bladder smooth muscle

Ryanodine receptor subtype 1 (RyR1) has been primarily characterized in skeletal muscle but several studies have revealed its expression in smooth muscle. Here, we used Ryr1-null mice to investigate the role of this isoform in Ca²⁺ signaling in urinary bladder smooth muscle. We show that RyR1 is required for depolarization-induced Ca²⁺ sparks, whereas RyR2 and RyR3 are sufficient for spontaneous or caffeine-induced Ca²⁺ sparks. Immunostaining revealed specific subcellular localization of RyR1 in the superficial sarcoplasmic reticulum; by contrast, RyR2 and RyR3 are mainly expressed in the deep sarcoplasmic reticulum. Paradoxically, lack of depolarization-induced Ca²⁺ sparks in Ryr1^{-/ -} myocytes was accompanied by an increased number of cells displaying spontaneous or depolarization-induced Ca²⁺ waves. Investigation of protein expression showed that FK506-binding protein (FKBP) 12 and FKBP12.6 (both of which are RyR-associated proteins) are downregulated in Ryr1^-/- myocytes, whereas expression of RyR2 and RyR3 are unchanged. Moreover, treatment with rapamycin, which uncouples FKBPs from RyR, led to an increase of RyR-dependent Ca²⁺ signaling in wild-type urinary bladder myocytes but not in Ryr1^-/- myocytes. In conclusion, although decreased amounts of FKBP increase Ca²⁺ signals in Ryr1^-/- urinary bladder myocytes the depolarization-induced Ca²⁺ sparks are specifically lost, demonstrating that RyR1 is required for depolarization-induced Ca²⁺ sparks and suggesting that the intracellular localization of RyR1 fine-tunes Ca²⁺ signals in smooth muscle.