FKBP12 Binding Modulates Ryanodine Receptor Channel Gating

The ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum of skeletal muscle is comprised of four 565,000-dalton RyR1s, each of which binds one FK506 binding protein (FKBP12). RyR1 is required for excitation-contraction coupling in skeletal muscle. FKBP12, a cis-transpeptidyl-prolyl isomerase, is required for the normal gating of the RyR1 channel. In the absence of FKBP12, RyR1 channels exhibit increased gating frequency, suggesting that FKBP12 “stabilizes” the channel in the open and closed states. We now show that substitution of a Gly, Glu, or Ile for Val2461 in RyR1 prevents FKBP12 binding to RyR1, resulting in channels with increased gating frequency. In the case of the V2461I mutant RyR1, normal channel function can be restored by adding FKBP12.6, an isoform of FKBP12. These data identify Val2461 as a critical residue required for FKBP12 binding to RyR1 and demonstrate the functional role for FKBP12 in the RyR1 channel complex. The ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum of skeletal muscle is comprised of four 565,000-dalton RyR1s, each of which binds one FK506 binding protein (FKBP12). RyR1 is required for excitation-contraction coupling in skeletal muscle. FKBP12, a cis-transpeptidyl-prolyl isomerase, is required for the normal gating of the RyR1 channel. In the absence of FKBP12, RyR1 channels exhibit increased gating frequency, suggesting that FKBP12 “stabilizes” the channel in the open and closed states. We now show that substitution of a Gly, Glu, or Ile for Val2461 in RyR1 prevents FKBP12 binding to RyR1, resulting in channels with increased gating frequency. In the case of the V2461I mutant RyR1, normal channel function can be restored by adding FKBP12.6, an isoform of FKBP12. These data identify Val2461 as a critical residue required for FKBP12 binding to RyR1 and demonstrate the functional role for FKBP12 in the RyR1 channel complex. ryanodine receptor FK506-binding protein The ryanodine receptor (RyR)1-1/Ca2+release channel on the sarcoplasmic reticulum of skeletal muscle is a tetrameric channel with a molecular mass of ∼2.3 million daltons. The channel is comprised of a signaling complex that includes four molecules of FKBP12, originally identified as KC7, a peptide that co-purified with RyR1 (1Marks A.R. Tempst P. Hwang K.S. Taubman M.B. Inui M. Chadwick C. Fleischer S. Nadal-Ginard B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8683-8687Crossref PubMed Scopus (170) Google Scholar), and kinases and phosphatases that regulate the channel function in particular by modulating the binding of the immunophilin subunit to the channel (2Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar). FKBP12 is required for the normal gating of the channel and for coupled gating between neighboring channels (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar, 4Marx S.O. Ondrias K. Marks A.R. Science. 1998; 281: 818-821Crossref PubMed Scopus (354) Google Scholar). Binding of FKBP12 to RyR1 in skeletal muscle and the closely related isoform FKBP12.6 to RyR2 in cardiac muscle modulates channel gating. We have shown previously that dissociation of FKBP12 from RyR1 or FKBP12.6 from RyR2 channels increases the gating frequency of the channel manifested as reduced open and closed dwell times (2Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar, 5Kaftan E. Marks A.R. Ehrlich B.E. Circ. Res. 1996; 78: 990-997Crossref PubMed Scopus (169) Google Scholar). Others have confirmed a role for FKBP12/12.6 in RyR1/RyR2 function (6Timerman A.P. Ogunbumni E. Freund E. Wiederrecht G. Marks A.R. Fleischer S. J. Biol. Chem. 1993; 268: 22992-22999Abstract Full Text PDF PubMed Google Scholar, 7Chen S.R. Zhang L. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11953-11957Crossref PubMed Scopus (53) Google Scholar, 8Ahern G.P. Junankar P.R. Dulhunty A.F. FEBS Lett. 1994; 352: 369-374Crossref PubMed Scopus (130) Google Scholar, 9Ahern G.P. Junankar P.R. Dulhunty A.F. Biophys. J. 1997; 72: 146-162Abstract Full Text PDF PubMed Scopus (117) Google Scholar), whereas one group has reported no functional role for FKBP12.6 in RyR2 (10Timerman A.P. Onoue H. Xin H.B. Barg S. Copello J. Wiederrecht G. Fleischer S. J. Biol. Chem. 1996; 271: 20385-20391Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). However, this same group has now reported that cardiomyocytes from an FKBP12.6 null mouse exhibit defects in Ca2+signaling (11Xin H.B. Senbonmatsu T. Wang Y.-X. Copello J. Ji G.-J. Collier M.L. Deng K.-Y. Jeyakumar L. Sutherland M. Magnuson M. Inagami T. Kotilikoff M. Fleischer S. Biophys. J. 2001; 80 (abstr.): 579Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), consistent with a role for FKBP12.6 in regulating RyR2 as we showed originally (2Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 5Kaftan E. Marks A.R. Ehrlich B.E. Circ. Res. 1996; 78: 990-997Crossref PubMed Scopus (169) Google Scholar). Protein kinase A phosphorylation of RyR2 dissociates FKBP12.6 from the channel, resulting in increased Ca2+ sensitivity for activation (2Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar). We have proposed that this is a physiological pathway (part of the “fight or flight” response) that regulates excitation-contraction coupling and specifically increases excitation-contraction coupling gain by increasing the amount of Ca2+ released for a given trigger (2Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 12Marks A.R. Circ. Res. 2000; 87: 8-11Crossref PubMed Scopus (194) Google Scholar). In failing hearts this pathway is over-stimulated and becomes maladaptive, resulting in protein kinase A hyperphosphorylation of the RyR2 channels (2Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar). Protein kinase A-hyperphosphorylated RyR2 channels in failing hearts are depleted of FKBP12.6 and exhibit single-channel properties (2Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar) similar to those observed in RyR1 channels in the absence of FKBP12 (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar). A motif that can bind FKBP12 was suggested by studies that identified the ideal substrate for prolyl isomerization (13Albers M.W. Walsh C.T. Schreiber S.L. J. Org. Chem. 1990; 55: 4984-4986Crossref Scopus (128) Google Scholar). In these studies Schreiber and co-workers (13Albers M.W. Walsh C.T. Schreiber S.L. J. Org. Chem. 1990; 55: 4984-4986Crossref Scopus (128) Google Scholar) concluded that FKBP12 was binding to a twisted-amide transition state intermediate in the peptidyl-prolyl bond of its substrates. They subsequently showed that FK506 and rapamycin, immunosuppressant drugs that bind to FKBP12, are twisted amide surrogates. The binding of FKBP12 to the hydrophobic motif identified as the optimal substrate for peptidyl-prolyl isomerization present in the inositol 1,4,5-triphosphate receptor (an intracellular Ca2+ release channel with structural homology to RyR channels) was examined subsequently using a yeast two-hybrid interaction screen (14Cameron A.M. Nucifora Jr., F.C. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The present study is the first to establish the precise location of an FKBP12 binding site on the intact full-length RyR1 channel. We show that Val2461 is a key residue mediating FKBP12 binding to RyR1 and that mutations at this site that inhibit FKBP12 binding to RyR1 result in significant defects in single-channel function, which is consistent with altered channel gating because of the absence of FKBP12. To demonstrate that the defects in single-channel properties are caused by lack of FKBP12 binding as opposed to some other structural change in the channel, we generated a mutant V2461I RyR1 channel that does not bind FKBP12 but does bind FKBP12.6. Addition of FKBP12.6 to the V2461I mutant RyR1 restores normal channel function. Mutagenesis of RyR1 was performed using a cassette containing nucleotides 6597–11766. The sequences of the primers used for mutagenesis (including conservative substitutions to generate new restriction sites) were as follows: V2461H, c cgc gcc atc ctt cgc agt ctt cat ccc ctg gac gac c; V2461E, c ctt cgc tcc ctc gag ccc ctg gac gac c; V2461G, c ctt cgc tcc ctc ggg ccc ctg gac gac c; and V2461I, cc atc ctt cgc tca tta att ccc ctg gac gac c. The Morph site-specific plasmid DNA mutagenesis kit (5 Prime → 3 Prime, Boulder, CO) was used according to the manufacturer's instructions. The corresponding mutant fragments were digested withNsiI and SapI (at nucleotides 7453 and 7647, respectively) and subcloned into an expression vector (pCMV5) containing a full-length RyR1 clone cut with the same enzymes. HEK293 cells were maintained in T175 flasks in minimum Eagle's media containing 10% fetal bovine serum and were passed every 3–4 days. One T175 flask (50% confluent) was transfected with 20.0 μg of DNA using the Ca2+ phosphate precipitation method. Forty-eight h post-transfection, the cells were washed twice, scraped into phosphate-buffered saline, and pelleted by centrifugation at 2500 × g for 5 min at 4 °C. After resuspending the pellet in 0.5 ml of 20 mm HEPES-NaOH, pH 7.5, containing protease inhibitors (complete EDTA-free inhibitors from Roche Molecular Biochemicals), the cells were allowed to swell for 30 min on ice before lysis by 20 strokes of a Dounce homogenizer. Cell homogenates were diluted with an equal volume of ice-cold medium containing 500 mm sucrose and 10 mm HEPES, pH 7.2, and centrifuged at 10,000 × g for 15 min. Supernatants were recovered and centrifuged at 100,000 × g for 45 min. Pellets were resuspended in a buffer containing 250 mmsucrose, and the protein concentration of the microsomes was determined by Bradford assay. Aliquots were stored at −80 °C. Approximately 200 μg of wild-type or mutant RyR1 containing microsomes were diluted to a total of 500 μl with immunoprecipitation buffer (50 mm Tris-HCl, pH 7.4, 0.25% sodium deoxycholate, 150 mm NaCl, 1.0 mmEDTA, 1.0 mm NaF, 1.0 mmNa3VO4, and 0.25% Triton X-100) containing protease inhibitors. These samples were incubated with 2.0 μl of anti-RyR antibody for 1 h at 4 °C followed by the addition of 40 μl of protein A-Sepharose (1:1 in phosphate-buffered saline) for 1 h at 4 °C. After the beads were washed four times for 5 min in immunoprecipitation buffer, the proteins were eluted with SDS sample buffer, heated to 95 °C for 4 min, and assayed for both RyR and FKBP by Western analysis. Recombinant RyR1 microsomes were washed in 0.5 ml of 10 mm HEPES-NaOH, pH 7.5, containing protease inhibitors and were centrifuged at 100,000 × g for 45 min. Pellets were resuspended in a buffer containing 10 mm HEPES, 300 mm sucrose, and 0.9% NaCl. Microsomes (200 μg) were added to 0.2 ml of imidazole buffer (5 mm imidazole, pH 7.4, and 0.3 m sucrose) and incubated for 1 h at 37 °C with varying concentrations of rapamycin. Samples were centrifuged at 95,000 × g for 10 min, and the supernatants were collected. Pellets were washed two times in 0.2 ml of imidazole buffer and centrifuged at 95,000 × g for 10 min. The final pellet was resuspended in 0.2 ml of imidazole the pellet and were by and for FKBP12 by Western Recombinant RyR1 containing microsomes was resuspended in 250 μl of imidazole buffer (5 mm imidazole, pH 7.4, and sucrose) and incubated with 5 FKBP12.6 for 4 h at 4 °C. Samples were centrifuged at 95,000 × g for 10 min, and the were washed and resuspended in 500 ml of immunoprecipitation RyR1 was from these samples as were reticulum were added to the and to with from The was of with a was by added to the After of a Ca2+ release channel, the was by of the used for channel were as follows: 250 mm mm mm pH 250 mm HEPES, mm 1 mm 0.5 pH Ca2+ concentration was determined using the G. A.P. Google Scholar). The was to the of an 200 using a and The was at with a similar The single-channel were at 1 with an and at 4 were on a using and a The was used for single-channel and the of open and gating frequency were identified by using at min of The was used for To identify for the binding of FKBP12 to the RyR1 channel we generated four mutant of the RyR1 channel In each case we substitutions for which is in the to to as the to a binding site for FKBP12 on RyR1 (2Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, A.M. Nucifora Jr., F.C. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The of to for Val2461 was by data that the the for peptidyl-prolyl isomerization by FKBP12 of peptide (13Albers M.W. Walsh C.T. Schreiber S.L. J. Org. Chem. 1990; 55: 4984-4986Crossref Scopus (128) Google Scholar). and in the in the whereas and reduced the and the (13Albers M.W. Walsh C.T. Schreiber S.L. J. Org. Chem. 1990; 55: 4984-4986Crossref Scopus (128) Google Scholar). we generated four mutant of RyR1, V2461G, V2461E, V2461H, and V2461I 1 and mutant RyR1 were in HEK293 cells were performed to FKBP12 was with the wild-type and mutant RyR1 as we previously for RyR1 T. A.P. H. Fleischer S. Tempst P. Marks A.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and for RyR1 in cells (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar). FKBP12 with the wild-type RyR1 and with the mutant but not with the V2461G, V2461E, or V2461I mutant of RyR1 1 These data identify the residue Val2461 as critical in the binding of FKBP12 to FKBP12 can be from RyR1 using the immunosuppressant drugs FK506 or at concentrations (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar). were performed in which containing wild-type or mutant RyR1 were from transfected HEK293 and assayed by to FKBP12 is to RyR1 the 1 of the with dissociates FKBP12 that in the with RyR1, sensitivity of the mutant channel was increased FKBP12 from the mutant channel at a concentration of 10 and of FKBP12 was with with for the wild-type channel 1 The V2461E, V2461G, and V2461I mutant RyR1 channels not bind FKBP12 in this not which is in with the To the functional of reduced for FKBP12 in the mutant RyR1 the single-channel properties of proteins were examined in One of the of RyR1 in the absence of FKBP12 is an in the gating frequency with RyR1 or wild-type RyR1 channels with FKBP12 (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar). The wild-type RyR1 channel in HEK293 cells the same single-channel properties A and as reported previously for RyR1 and for wild-type RyR1 with FKBP12 in cells (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar, K. A.M. Scott A. Ehrlich B.E. Marks A.R. 1996; Google Scholar). The mutant RyR1 channel that binds FKBP12 but with reduced normal single-channel properties and However, the mutant RyR1 channel exhibit a significant in the gating frequency and as the mutant RyR1 of the of increased gating frequency in the mutant RyR1 channels with the increased of FKBP12 for with in the with those with Glu, or The for the and V2461I 4 mutant RyR1 channels that these mutant channels exhibit or as reported previously for RyR1 in the absence of FKBP12 (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar). However, given the increased gating frequency of these mutant channels and the of we are not to the reported previously (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google channel properties of wild-type and mutant of to wild-type to wild-type to wild-type to wild-type to wild-type to wild-type to wild-type to wild-type to wild-type are as were using 150 to wild-type to wild-type in a new not FKBP12 restores normal channel function to the V2461I mutant RyR1 of and mutant V2461I V2461I FKBP12 and V2461I FKBP12.6 RyR1 channels is Ca2+ 150 and the addition of (5 are shown were at in Ca2+ 150 Ca2+ mm were generated from 5 × the closed state of the are are as were using 150 The open dwell gating frequency, and open of the wild-type RyR1 and mutant channels were similar In the open dwell times were and the gating frequency was for the and mutant channels with the wild-type channel These data that the and mutant channels are in the open or closed as suggested previously for RyR1 channels in the absence of FKBP12 (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar). One for the observed in single-channel function in the RyR1 that not bind FKBP12 is that the structural in the channel that result in altered function of FKBP12 To this specifically we a mutant RyR1 that does not bind FKBP12 but does bind FKBP12.6 and be by the addition of FKBP12.6. The to normal function to the mutant RyR1 channel the that the substitutions the observed in channel function of FKBP12 we showed that the V2461I mutant RyR1 in HEK293 cells that FKBP12 but not binds FKBP12.6 but not FKBP12 the V2461I mutant channel was examined in the same increased gating frequency as the mutant RyR1 4 However, FKBP12.6 was added to the RyR1 V2461I normal channel function with that of the wild-type RyR1 was restored 4 In the addition of FKBP12 to the V2461I mutant RyR1 channel to normal channel function 4 The FKBP12 isomerization of peptidyl-prolyl is to bind to and the of the twisted-amide transition state intermediate A. M. Schreiber S.L. Science. 1990; PubMed Scopus Google Scholar). FKBP12 bind to the twisted-amide transition state intermediate of a peptidyl-prolyl bond on of an bond be of the binding of FKBP12 the RyR1 channel to a that optimal gating (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar) as as coupled gating between neighboring channels S.O. Ondrias K. Marks A.R. Science. 1998; 281: 818-821Crossref PubMed Scopus (354) Google Scholar). this be a role for an immunophilin and that the isomerization of the peptidyl-prolyl bond to or is because the of FKBP12 for the or of the peptidyl-prolyl bond be whereas that in FKBP12 is to the interaction between FKBP12 and RyR1 the site of the in binding to the substrate not be consistent with FKBP12 as a The for the binding of FKBP12 to V2461H, V2461E, and mutant RyR1 channels with the increased of FKBP12 for with a hydrophobic residue at the (13Albers M.W. Walsh C.T. Schreiber S.L. J. Org. Chem. 1990; 55: 4984-4986Crossref Scopus (128) Google the lack of FKBP12 binding to the V2461I mutant RyR1 from this the of for peptide in which the residue at the is is for with at the with with Glu, and with (13Albers M.W. Walsh C.T. Schreiber S.L. J. Org. Chem. 1990; 55: 4984-4986Crossref Scopus (128) Google Scholar). of of the isomerization of peptide has used to the that of the twisted-amide intermediate is the of amide by FKBP12 A. M. Schreiber S.L. Science. 1990; PubMed Scopus Google Scholar). the of the of FKBP12 with the binding of FKBP12 to the wild-type and of the four mutant RyR1 channels FKBP12 the by which FKBP12 modulates RyR1 channel gating binding to and of a twisted-amide transition state intermediate of a peptidyl-prolyl bond in However, the that FKBP12 does not bind to the V2461I mutant RyR1 to some on this with with at the the of was the residue was a and the residue was an Ile (13Albers M.W. Walsh C.T. Schreiber S.L. J. Org. Chem. 1990; 55: 4984-4986Crossref Scopus (128) Google Scholar). the other channels and to bind FKBP12, a 1,4,5-triphosphate 1 and and at the and one has an Ile at the The that the V2461I mutant RyR1 channel does not bind FKBP12 was not on the of the of FKBP12 because the a peptide with Ile in the one with (13Albers M.W. Walsh C.T. Schreiber S.L. J. Org. Chem. 1990; 55: 4984-4986Crossref Scopus (128) Google Scholar). data show that FKBP12.6 binds with to the V2461I mutant RyR1 as binds to the wild-type RyR2 channel that has an Ile in the corresponding 1 The binding of FKBP12.6 but not FKBP12 to the V2461I RyR1 mutant be the by hydrophobic were in that FKBP12.6 the of an whereas FKBP12 does be that is not the site of FKBP12/12.6 that binds to the residue at in RyR1, open the that the site is with peptidyl-prolyl bond in the channel. The of FKBP12 to the 1 receptor that the residue and the in the FKBP12 binding site not with FKBP12. However, this that are other between FKBP12 and receptor M. J. J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The data in the present study that the FKBP12 binding site on RyR1 have a from that on the receptor because mutations of the residue binding of FKBP12 but does not the that other in RyR1 by in the residue in FKBP12/12.6 binding to the the present study the critical role of Val2461 in FKBP12 data defects in mutant RyR1 channels that not bind FKBP12 are consistent with data from RyR1 channels in cells that lack FKBP12 (3Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (705) Google Scholar) and RyR1 channels from the FKBP12 mouse that similar single-channel defects B. S. S.L. 1998; PubMed Scopus Google Scholar). In addition to the on single-channel properties a role for FKBP12 in coupled gating between neighboring RyR1 channels has proposed S.O. Ondrias K. Marks A.R. Science. 1998; 281: 818-821Crossref PubMed Scopus (354) Google Scholar). One for FKBP12 can gating of channels is that each of the of RyR1 a the sarcoplasmic reticulum and FKBP12 the channel in a that the four of the channel to A.R. 1996; PubMed Scopus Google Scholar). To FKBP12 can coupled gating between the have to of a state that between neighboring channels A.R. 1996; PubMed Scopus Google Scholar). The present study the that FKBP12 binding to RyR1 the channel in a this is a for regulating channel gating. The substitution of for Val2461 at the be to increased the peptidyl-prolyl bond at this because of the of the bond the channel to the in dwell times and the corresponding in gating frequency by the mutant RyR1 The that FKBP12.6 can normal function to the V2461I mutant channel the that the observed defects in single-channel function are caused by the substitution of FKBP12 is to the channel. data that Val2461 is a critical of FKBP12 binding to RyR1, which is required for normal channel function. However, these data not the that are other between FKBP12 and RyR1 and other in RyR1 that are required for binding of FKBP12 to the channel. We for