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FKBP12.6 activates RyR1: investigating the amino acid residues critical for channel modulation.

ABSTRACT: We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12E31Q/D32N/W59F) where the residues Glu(31), Asp(32), and Trp(59) were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12E31Q/D32N/W59F mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12E31Q/D32N/W59F activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu(31), Asp(32), and Trp(59) in FKBP12 and Gln(31), Asn(32), and Phe(59) in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.

SUBMITTER: Venturi E

PROVIDER: S-EPMC3945099 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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FKBP12.6 activates RyR1: investigating the amino acid residues critical for channel modulation.

Venturi Elisa E Galfré Elena E O'Brien Fiona F Pitt Samantha J SJ Bellamy Stuart S Sessions Richard B RB Sitsapesan Rebecca R

Biophysical journal 20140201 4

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produ ...[more]

PMID: 24559985

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Project description:Both non-immune "natural" and antigen-induced "immune" IgM are important for protection against infections and for regulation of immune responses to self-antigens. The roles of its Fc receptor (FcµR) in these IgM effector functions have begun to be explored. In the present study, by taking advantage of the difference in IgM-ligand binding of FcµRs of human (constitutive binding) and mouse (transient binding), we replaced non-conserved amino acid residues of human FcµR with mouse equivalents before establishment of cell lines stably expressing mutant or wild-type (WT) receptors. The resultant eight-different mutant FcµR-bearing cells were compared with WT receptor-bearing cells for cell-surface expression and IgM-binding by flow cytometric assessments using receptor-specific mAbs and IgM paraproteins as ligands. Three sites Asn66, Lys79-Arg83, and Asn109, which are likely in the CDR2, DE loop and CDR3 of the human FcµR Ig-like domain, respectively, were responsible for constitutive IgM binding. Intriguingly, substitution of Glu41 and Met42 in the presumed CDR1 with the corresponding mouse residues Gln and Leu, either single or more prominently in combination, enhanced both the receptor expression and IgM binding. A four-aa stretch of Lys24-Gly27 in the predicted A ß-strand of human FcµR appeared to be essential for maintenance of its proper receptor conformation on plasma membranes because of reduction of both receptor expression and IgM-binding potential when these were mutated. Results from a computational structural modeling analysis were consistent with these mutational data and identified a possible mode of binding of FcµR with IgM involving the loops including Asn66, Arg83 and Asn109 of FcµR interacting principally with the Cµ4 domain including Gln510 and to a lesser extent Cµ3 domain including Glu398, of human IgM. To our knowledge, this is the first experimental report describing the identification of amino acid residues of human FcµR critical for binding to IgM Fc.

Dataset Information

FKBP12.6 activates RyR1: investigating the amino acid residues critical for channel modulation.

Publications

FKBP12.6 activates RyR1: investigating the amino acid residues critical for channel modulation.

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