Orion 81 55

Membrane vesicles were diluted to 50–100 μg protein/ml and incubated at 37 °C in 250 mM HEPES, 80 mM Tris, 10 μM free Ca²⁺, pH 7.2 with 5 nM [³H]ryanodine for 90 min. Following incubation, the samples were diluted with 5 ml of ice-cold buffer and filtered through Whatman GF-B filters. Filters were washed with a further 3 × 5 ml aliquots of buffer and counted in 10 ml aqueous counting scintillant the following day. Non-specific binding was determined from incubations to which 5 μM unlabelled ryanodine had been added. All incubations were performed in triplicate. Adjustments to the ionic strength of the solutions were made as previously described^{19 (link)}. The free [Ca²⁺] and pH of the solutions used during the incubation period were determined at 37 °C using a calcium electrode (Orion 93-20, Thermo Fisher Scientific, UK) and Ross-type pH electrode (Orion 81-55, Thermo Fisher Scientific, UK) as described above for solutions maintained at 22 °C^{18 (link)}. The free [Ca²⁺] of the incubation solutions was maintained at 10 μM in the presence or absence of various ligands by buffering with EGTA and CaCl₂.

Heavy SR membrane vesicles were prepared from sheep cardiac muscle as previously described^{18 (link)}. Sheep hearts (Suffolk breed) were obtained from an abattoir and were 8–10 months old, mixed sex. SR vesicles were frozen rapidly in liquid nitrogen and stored at −80 °C. Vesicles were fused with planar phosphatidylethanolamine lipid bilayers as previously described^{18 (link)}. The vesicles fused in a fixed orientation such that the cis chamber corresponded to the cytosolic space and the trans chamber to the SR lumen. The trans chamber was held at ground and the cis chamber held at potentials relative to ground. After fusion, the cis chamber was perfused with 250 mM HEPES, 125 mM Tris, 10 μM free Ca²⁺ buffered with EGTA and CaCl₂, pH 7.2. The trans chamber was perfused with a solution containing 250 mM glutamic acid, 10 mM HEPES, pH 7.2 with Ca(OH)₂ (free [Ca²⁺] ~ 50 mM). The free [Ca²⁺] and pH of the solutions were measured at 22 °C using a calcium electrode (Orion 93-20, Thermo Fisher Scientific, UK) and Ross-type pH electrode (Orion 81-55, Thermo Fisher Scientific, UK) and were maintained constant as described previously in detail^{18 (link)}. The cytosolic [Ca²⁺] was maintained at 10 µM after additions of PPPi, by buffering with EGTA and CaCl₂ as previously described^{19 (link)}.

Skeletal HSR vesicles were incorporated into planar phospholipid bilayers as previously described (Sitsapesan et al., 1991 (link)). Voltage‐clamp conditions were used to measure the current through the RyR channels, with the trans (luminal) chamber voltage‐clamped at ground. The recording solutions used were 250‐mM HEPES, 80‐mM Tris, 10‐μM free Ca²⁺ and pH 7.2 on the cytosolic side and 250‐mM glutamic acid, 10‐mM HEPES and ≈50‐mM free Ca²⁺ on the luminal side, both at 21°C. The statin compound under investigation was added to the cytosolic or luminal chamber as required. The free [Ca²⁺] was maintained by the addition of EGTA and CaCl₂ solution and measured using a Orion 93‐20 Ca²⁺ electrode (Thermo Fisher Scientific, Horsham and Loughborough, UK) as previously described (Sitsapesan et al., 1991 (link)). The pH of solutions was determined using a Ross‐type pH electrode (Orion 81‐55, Thermo Fisher Scientific, UK) as previously described (Sitsapesan et al., 1991 (link)). To lower free cytosolic [Ca²⁺] below subactivating levels, 1‐mM EGTA was added. This reduced free [Ca²⁺] below measurable levels, and the concentration was calculated using the MaxChelator programme (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/index.html) (Chris Patton, Stanford, USA) to <1 nM.