Background

RyRs are intracellular Ca²⁺ channels located on sarcoplasmic and endoplasmic reticula and are present in nearly all cell types, including excitable cells such as neurons and muscles. In skeletal and cardiac muscles, excitation-contraction coupling takes place by activation of RyRs via dihydropyridine receptors on the T-tubular membrane (Samsó, 2017). Since RyRs are membrane-embedded proteins, their purification requires solubilization in detergent (e.g., CHAPS, Digitonin, Tween-20) within aqueous buffers; however, excessive or insufficient detergent can denature or leave the channels only partially solubilized, respectively. The FK506-binding proteins of 12 and 12.6 kDa (FKBP12 and FKBP12.6) are important RyR modulators, which bind to RyR with high affinity. Hence, detergent-solubilized RyR can be purified using GST-FKBP (or SBP-FKBP) affinity chromatography. However, FKBP exhibits reduced binding to RyR in vitro (Zissimopoulos et al., 2012), resulting in a lower than expected yield of pure RyR. Considering that RyR is the largest known ion channel, with a molecular weight of ~2.3 MDa (a tetramer of ~565 kDa monomers), sucrose gradient centrifugation and gel filtration are suitable for its purification, in which RyR elutes as a peak from the denser fraction (sucrose gradient) or as the first peak (gel filtration). In recent years, structures have been solved at near-atomic resolution for both RyR1 and RyR2, which were purified from skeletal or cardiac muscle (Gong et al., 2020). More than 300 identified RyR mutations cause a number of debilitating or fatal disorders, most of which result in a gain-of-function phenotype; therefore, their structure is of significant interest. However, transgenic animals carrying RyR mutations have a lower or zero survival rate, and obtaining homozygous animals for severe RyR mutations is exceptionally difficult or impossible; thus, purification of RyR from cells is an attractive solution. Among the different cell types, bacteria lack the endoplasmic reticulum compartment where RyR resides and are therefore unsuitable for RyR expression. Among eukaryotic systems, the most widely used are mammalian cells such as HEK293 cells, which can easily accommodate the 15,000-bp cDNA and possess the mammalian machinery for post-translational modifications. Ideally, the cells are stably transfected with a plasmid containing an inducible promoter, such as pcDNA5/FRT/TO, which was designed for use with the Flp-In-T-Rex system in our case. The main limitation of cell culture is the low mass yield as compared with that of muscle tissue. Other limitations include the requirement for high quantities of expensive supplies such as cell culture media and the time required for cell culture (2-3 weeks) as well as for the sucrose gradient centrifugation-based protocol (~3 days). We were the first to purify recombinant RyR at a high yield from HEK293 cells using sucrose gradient centrifugation and ion-exchange chromatography (Iyer et al., 2020). In the same work, we also used a one-step affinity chromatography that, although resulting in a lower yield, was faster and allowed complete purification in one day. Hence, purifying recombinant RyR from HEK293 cells using either of these two protocols is suitable for structural and functional studies that require high quantities of pure mutant RyR. Additionally, this protocol may serve as the starting point for purification of other membrane proteins of similar size, although the detergent solubilization conditions will need to be customized for the membrane protein of interest.