The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer) 4 (FKBP12) 4 and (RyR2 protomer) 4 (FKBP12.6) 4 , respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35 S-labeled FKBP12 and 35 S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12.6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBPstripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.FK506 is a potent immunosuppressive drug that binds to a family of related intracellular receptors termed FK506-binding proteins, varying in size from 12 to 54 kDa. Among these FKBPs, 1 FKBP12 is the most abundant and is involved in mediating the immunosuppressive action of FK506 in T lymphocytes. All of the known FKBP family members display cis-trans peptidyl-prolyl isomerase (PPIase) activity that is inhibited by FK506 and a structurally related compound, rapamycin (1). However, neither the immunosuppressive nor toxic side effects (including severe neuro-and nephrotoxicity) associated with FK506 therapy result from the inhibition of PPIase activity. Rather, the action of FK506 results from the specific inhibition of calcineurin by the FKBP12⅐drug complex. Calcineurin is a calcium-dependent protein phosphatase involved in the activation of T-lymphocytes (2). FKBP12 is a mere bystander protein during T-cell activation that becomes involved in blocking activation after it binds FK506. To date, the only physiological function directly associated with FKBP12 is its tight binding to (3, 4) and modulation of the ryanodine receptor (RyR-1) or calcium release channel of skeletal muscle SR (5-10). The purified SkM CRC 2 isolated from the CHAPS-solubilized TC is a hetero-oligomeric complex, with the structural formula of (RyR-1 protomer) 4 (FKBP12) 4 (5). Both FK506 and rapamycin bind to and dissociate FKBP12 from the SkM CRC (5). Both the native and FKBP-stripped CRCs have similar unitary conductance and sensitivity to ruthenium red (6). However, in co...

Individual cell heterogeneity within a population can be critical to its peculiar function and fate. Subpopulations studies with mixed mutants and wild types may not be as informative regarding which cell responds to which drugs or clinical treatments. Cell to cell differences in RNA transcripts and protein expression can be key to answering questions in cancer, neurobiology, stem cell biology, immunology, and developmental biology. Conventional cell-based assays mainly analyze the average responses from a population of cells, without regarding individual cell phenotypes. To better understand the variations from cell to cell, scientists need to use single cell analyses to provide more detailed information for therapeutic decision making in precision medicine. In this review, we focus on the recent developments in single cell isolation and analysis, which include technologies, analyses and main applications. Here, we summarize the historical background, limitations, applications, and potential of single cell isolation technologies.

FK506 binding proteins 12 and 12.6 (FKBP12 and FKBP12.6) are intracellular receptors for the immunosuppressant drug FK506 (ref. 1). The skeletal muscle ryanodine receptor (RyR1) is isolated as a hetero-oligomer with FKBP12 (ref. 2), whereas the cardiac ryanodine receptor (RyR2) more selectively associates with FKBP12.6 (refs 3, 4, 5). FKBP12 modulates Ca2+ release from the sarcoplasmic reticulum in skeletal muscle and developmental cardiac defects have been reported in FKBP12-deficient mice, but the role of FKBP12.6 in cardiac excitation-contraction coupling remains unclear. Here we show that disruption of the FKBP12.6 gene in mice results in cardiac hypertrophy in male mice, but not in females. Female hearts are normal, despite the fact that male and female knockout mice display similar dysregulation of Ca2+ release, seen as increases in the amplitude and duration of Ca2+ sparks and calcium-induced calcium release gain. Female FKBP12.6-null mice treated with tamoxifen, an oestrogen receptor antagonist, develop cardiac hypertrophy similar to that of male mice. We conclude that FKBP12.6 modulates cardiac excitation-contraction coupling and that oestrogen plays a protective role in the hypertrophic response of the heart to Ca2+ dysregulation.

Isolated skeletal muscle ryanodine receptors (RyRs) complexed with the modulatory ligands, calmodulin (CaM) or 12-kDa FK506-binding protein (FKBP12), have been characterized by electron cryomicroscopy and three-dimensional reconstruction. RyRs are composed of 4 large subunits (molecular mass 565 kDa) that assemble to form a 4-fold symmetric complex that, architecturally, comprises two major substructures, a large (Ϸ80% of the total mass) cytoplasmic assembly and a smaller transmembrane assembly. Both CaM and FKBP12 bind to the cytoplasmic assembly at sites that are 10 and 12 nm, respectively, from the putative entrance to the transmembrane ion channel. FKBP12 binds along the edge of the square-shaped cytoplasmic assembly near the face that interacts in vivo with the sarcolemma/transverse tubule membrane system, whereas CaM binds within a cleft that faces the junctional face of the sarcoplasmic reticulum membrane at the triad junction. Both ligands interact with a domain that connects directly to a cytoplasmic extension of the transmembrane assembly of the receptor, and thus might cause structural changes in the domain which in turn modulate channel gating.