Store-operated Ca²⁺ entry (SOCE) is a well-established cellular mechanism involved in the replenishment of sarcoplasmic reticulum (SR) calcium (Ca²⁺) content in smooth muscle cells (SMCs). Reduction of [Ca²⁺] within the SR is sensed by the luminal domain of Stromal interaction molecule 1 (STIM1) which leads to activation of plasmalemmal Calcium release-activated calcium channels (ORai). Ca²⁺ influx into the cytosol via ORai channels can then be sequestered into the SR – restoring Ca²⁺ levels. Many studies have shown that cholinergic-mediated contractions of bladder smooth muscle (Detrusor) rely on Ca²⁺ influx from the extracellular space (~70%) and Ca²⁺ release from the SR (~30%), however, identification of the molecular candidates that facilitate SOCE and SR refilling in the bladder are poorly understood. This purpose of this study was to investigate the contribution of Orai channels to cholinergic-mediated responses of murine detrusor muscle. 

Firstly, transcriptional expression of Orai subtypes was performed on RNA extracted from murine detrusor muscle strips. qRT-PCR experiments reveal that Orai 1, 2 & 3 subtypes are expressed in detrusor muscle, with Orai 3 mRNA levels two-fold higher than Orai1 & 2 (n=4). Isometric tension experiments were utilized to assess the contractility of detrusor muscle strips. SOCE was induced in detrusor muscle via blockade of SR refilling with thapsigargin. SOCE-mediated responses were significantly reduced, in a concentration-dependent manner by the selective Orai channel inhibitor, GSK7975A (ANOVA, p<0.0001, n=6). Next, cholinergic-mediated responses were investigated using the Muscarinic receptor agonist, Carbachol (CCh). In the presence of GSK7975A, CCh-induced contractions of the detrusor were reduced from 8493 ± 616.6 mN to 5065 ± 602.3 mN (40% reduction, p<0.0001, n=6), and CCh-induced Ca²⁺ transients in isolated detrusor SMCs, were ablated in the presence of GSK7975A (n=5). Collectively, these results indicate that ORai channels play a significant role in mediating cholinergic-mediated responses of detrusor smooth muscle.