The Experts below are selected from a list of 12219 Experts worldwide ranked by ideXlab platform
Andrew R. Marks - One of the best experts on this subject based on the ideXlab platform.
-
high resolution structure of the membrane embedded skeletal Muscle ryanodine receptor
Structure, 2021Co-Authors: Zephan Melville, Kookjoo Kim, Oliver B Clarke, Andrew R. MarksAbstract:Summary The type 1 ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal Muscle Excitation-contraction coupling and is the largest known ion channel, composed of four 565-kDa protomers. Cryogenic electron microscopy (cryo-EM) studies of the RyR have primarily used detergent to solubilize the channel; in the present study, we have used cryo-EM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously. This allowed us to resolve the structure of the channel in the primed and open states at 3.4 and 4.0 A, respectively, with a single dataset. This method offers validation for detergent-based structures of the RyR and offers a starting point for utilizing a chemical gradient mimicking the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol.
-
high resolution structure of the membrane embedded skeletal Muscle ryanodine receptor
Social Science Research Network, 2021Co-Authors: Zephan Melville, Kookjoo Kim, Oliver B Clarke, Andrew R. MarksAbstract:The type 1 ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal Muscle Excitation-contraction coupling and is the largest known ion channel, comprised of four 565 kDa protomers. Cryogenic electron microscopy (cryoEM) studies of the RyR have primarily used detergent to solubilize the channel; in the present study, we have used cryoEM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously, improving the incorporation rate by >20-fold compared to a dialysis-based approach. This allowed us to resolve the structure of the channel in the closed and open states at 3.36 and 3.98 A, respectively. This method offers validation for detergent-based structures of the RyR and offers a method for utilizing an electrochemical gradient mimicking the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol.
-
high resolution structure of the membrane embedded skeletal Muscle ryanodine receptor
bioRxiv, 2021Co-Authors: Zephan Melville, Kookjoo Kim, Oliver B Clarke, Andrew R. MarksAbstract:The type 1 ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal Muscle Excitation-contraction coupling and is the largest known ion channel, comprised of four 565 kDa protomers. Cryogenic electron microscopy (cryoEM) studies of the RyR have primarily used detergent to solubilize the channel, though a recent study resolved the structure with limited resolution in nanodiscs1. In the present study we have used cryoEM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously, a method that improved the incorporation rate by more than 20-fold compared to a dialysis-based approach. In conjunction with new direct-detection cameras, this allowed us to resolve the structure of the channel in the closed and open states at 3.36 and 3.98 [A], respectively. This method offers validation for detergent-based structures of the RyR and lays the groundwork for studies utilizing an electrochemical gradient mimicking the native environment, such as that of the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol of the cell at rest.
-
ca2 calmodulin dependent protein kinase ii phosphorylation regulates the cardiac ryanodine receptor
Circulation Research, 2004Co-Authors: Xander H T Wehrens, Stephan E Lehnart, Steven Reiken, Andrew R. MarksAbstract:The cardiac ryanodine receptor (RyR2)/calcium release channel on the sarcoplasmic reticulum is required for Muscle Excitation-contraction coupling. Using site-directed mutagenesis, we identified th...
-
pka phosphorylation dissociates fkbp12 6 from the calcium release channel ryanodine receptor defective regulation in failing hearts
Cell, 2000Co-Authors: Steven O Marx, Steven Reiken, Yuji Hisamatsu, Thotalla Jayaraman, Daniel Burkhoff, Nora Rosemblit, Andrew R. MarksAbstract:The ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is the major source of calcium (Ca2+) required for cardiac Muscle Excitation-contraction (EC) coupling. The channel is a tetramer comprised of four type 2 RyR polypeptides (RyR2) and four FK506 binding proteins (FKBP12.6). We show that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (Po). Using cosedimentation and coimmunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein, mAKAP. In failing human hearts, RyR2 is PKA hyperphosphorylated, resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.
Manfred Grabner - One of the best experts on this subject based on the ideXlab platform.
-
Pore mutation N617D in the skeletal Muscle DHPR blocks Ca2+ influx due to atypical high-affinity Ca2+ binding
'eLife Sciences Publications Ltd', 2021Co-Authors: Anamika Dayal, Monica L Fernández-quintero, Klaus R Liedl, Manfred GrabnerAbstract:Skeletal Muscle Excitation-contraction (EC) coupling roots in Ca2+-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other Muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here, we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region
-
domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal Muscle
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Anamika Dayal, Clara Franziniarmstrong, Vinayakumar Bhat, Manfred GrabnerAbstract:The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement—the three prerequisites of skeletal Muscle Excitation–contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca2+ release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal Muscle Excitation–contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3–C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal Muscle.
-
intramolecular cav1 1 chimeras reveal the molecular mechanism determining the characteristic gating behaviour of the skeletal Muscle calcium channel
Biophysical Journal, 2011Co-Authors: Petronel Tuluc, Manfred Grabner, Bernhard E FlucherAbstract:The Ca2+ channel CaV1.1 is the voltage sensor of skeletal Muscle Excitation-contraction coupling. The classical skeletal Muscle CaV1.1 isoform has poor voltage sensitivity and conducts a small, slowly activating Ca2+ current. In contrast, a splice variant lacking exon 29 (α1S-ΔE29) (Tuluc et al.,2009) has an 8-fold higher current amplitude, fast activation-kinetics, and a 30mV left-shifted voltage-dependence of activation. Therefore, the extracellular loop in repeat IV (IVS3-IVS4) mainly coded by exon 29 is a critical determinant of the characteristic gating properties of CaV1.1. Here we used intramolecular chimeras between repeats I and IV to characterize the structural basis of the gating properties of CaV1.1.Inserting exon 29 (alone or in combination with IVS3) into the corresponding region of repeat I was ineffective. However, in combination with the voltage sensor (IVS4) it fully restored α1S-like amplitude and voltage-sensitivity to α1S-ΔE29. Interestingly, all three chimeras exhibit faster activation kinetics. Secondary structure predictions showed that the long IVS3-IVS4 loop contains a beta-sheet while the short loop forms a coil. Point mutations in exon 29 which abolish the beta-sheet fully mimic the effects of deleting exon 29 regarding the kinetic properties and increase the current amplitude by 3-fold and left-shift the voltage dependence by −15mV. Together with previous findings (Nakai et al., 1994) our data suggest that the S3-S4 loop of the first repeat determines activation kinetics, while the corresponding loop plus voltage sensor in the fourth repeat with its unique secondary structure dictate the voltage-dependence, amplitude, and kinetics of skeletal Muscle Ca2+ currents.Grants: PT (MFI-2007-417), BEF (FWF-P20059-B05).
-
skeletal Muscle Excitation contraction coupling is independent of a conserved heptad repeat motif in the c terminus of the dhprβ1a subunit
Cell Calcium, 2010Co-Authors: Anamika Dayal, Johann Schredelseker, Clara Franziniarmstrong, Manfred GrabnerAbstract:In skeletal Muscle Excitation–contraction (EC) coupling the sarcolemmal L-type Ca2+ channel or 1,4-dihydropyridine receptor (DHPR) transduces the membrane depolarization signal to the sarcoplasmic Ca2+ release channel RyR1 via protein–protein interaction. While it is evident that the pore-forming and voltage-sensing DHPRα1S subunit is essential for this process, the intracellular DHPRβ1a subunit was also shown to be indispensable. We previously found that the β1a subunit is essential to target the DHPR into groups of four (tetrads) opposite the RyR1 homotetramers, a prerequisite for skeletal Muscle EC coupling. Earlier, a unique hydrophobic heptad repeat motif (L⋯V⋯V) in the C-terminus of β1a was postulated by others to be essential for skeletal Muscle EC coupling, as substitution of these residues with alanines resulted in 80% reduction of RyR1 Ca2+ release. Therefore, we wanted to address the question if the proposed β1a heptad repeat motif could be an active element of the DHPR–RyR1 signal transduction mechanism or already contributes at the ultrastructural level i.e. DHPR tetrad arrangement. Surprisingly, our experiments revealed full tetrad formation and an almost complete restoration of EC coupling in β1-null zebrafish relaxed larvae and isolated myotubes upon expression of a β1a-specific heptad repeat mutant (LVV to AAA) and thus contradict the earlier results.
Bernhard E Flucher - One of the best experts on this subject based on the ideXlab platform.
-
multiple sequence variants in stac3 affect interactions with cav1 1 and Excitation contraction coupling
Structure, 2020Co-Authors: Britany Rufenach, Marta Campiglio, Bernhard E Flucher, Darren Christy, Jennifer M Bui, Jorg Gsponer, Filip Van PetegemAbstract:STAC3 is a soluble protein essential for skeletal Muscle Excitation-contraction (EC) coupling. Through its tandem SH3 domains, it interacts with the cytosolic II-III loop of the skeletal Muscle voltage-gated calcium channel. STAC3 is the target for a mutation (W284S) that causes Native American myopathy, but multiple other sequence variants have been reported. Here, we report a crystal structure of the human STAC3 tandem SH3 domains. We analyzed the effect of five disease-associated variants, spread over both SH3 domains, on their ability to bind to the CaV1.1 II-III loop and on Muscle EC coupling. In addition to W284S, we find the F295L and K329N variants to affect both binding and EC coupling. The ability of the K329N variant, located in the second SH3 domain, to affect the interaction highlights the importance of both SH3 domains in association with CaV1.1. Our results suggest that multiple STAC3 variants may cause myopathy.
-
correcting the r165k substitution in the first voltage sensor of cav1 1 right shifts the voltage dependence of skeletal Muscle calcium channel activation
Channels, 2019Co-Authors: Yousra El Ghaleb, Marta Campiglio, Bernhard E FlucherAbstract:The voltage-gated calcium channel CaV1.1a primarily functions as voltage-sensor in skeletal Muscle Excitation-contraction (EC) coupling. In embryonic Muscle the splice variant CaV1.1e, which lacks ...
-
intramolecular cav1 1 chimeras reveal the molecular mechanism determining the characteristic gating behaviour of the skeletal Muscle calcium channel
Biophysical Journal, 2011Co-Authors: Petronel Tuluc, Manfred Grabner, Bernhard E FlucherAbstract:The Ca2+ channel CaV1.1 is the voltage sensor of skeletal Muscle Excitation-contraction coupling. The classical skeletal Muscle CaV1.1 isoform has poor voltage sensitivity and conducts a small, slowly activating Ca2+ current. In contrast, a splice variant lacking exon 29 (α1S-ΔE29) (Tuluc et al.,2009) has an 8-fold higher current amplitude, fast activation-kinetics, and a 30mV left-shifted voltage-dependence of activation. Therefore, the extracellular loop in repeat IV (IVS3-IVS4) mainly coded by exon 29 is a critical determinant of the characteristic gating properties of CaV1.1. Here we used intramolecular chimeras between repeats I and IV to characterize the structural basis of the gating properties of CaV1.1.Inserting exon 29 (alone or in combination with IVS3) into the corresponding region of repeat I was ineffective. However, in combination with the voltage sensor (IVS4) it fully restored α1S-like amplitude and voltage-sensitivity to α1S-ΔE29. Interestingly, all three chimeras exhibit faster activation kinetics. Secondary structure predictions showed that the long IVS3-IVS4 loop contains a beta-sheet while the short loop forms a coil. Point mutations in exon 29 which abolish the beta-sheet fully mimic the effects of deleting exon 29 regarding the kinetic properties and increase the current amplitude by 3-fold and left-shift the voltage dependence by −15mV. Together with previous findings (Nakai et al., 1994) our data suggest that the S3-S4 loop of the first repeat determines activation kinetics, while the corresponding loop plus voltage sensor in the fourth repeat with its unique secondary structure dictate the voltage-dependence, amplitude, and kinetics of skeletal Muscle Ca2+ currents.Grants: PT (MFI-2007-417), BEF (FWF-P20059-B05).
-
a cav1 1 ca2 channel splice variant with high conductance and voltage sensitivity alters ec coupling in developing skeletal Muscle
Biophysical Journal, 2009Co-Authors: Petronel Tuluc, Bernhard E Flucher, Natalia Molenda, Bettina Schlick, Gerald J Obermair, Karin JurkatrottAbstract:The Ca2+ channel α1S subunit (CaV1.1) is the voltage sensor in skeletal Muscle Excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca2+ release from internal stores and conducts a slowly activating Ca2+ current. However, this Ca2+ current is not essential for skeletal Muscle EC coupling. Here, we identified a CaV1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse Muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged α1S subunit and expressed it in dysgenic (α1S-null) myotubes. GFP-α1SΔ29 was correctly targeted into triads and supported skeletal Muscle EC coupling. However, the Ca2+ currents through GFP-α1SΔ29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca2+ influx contributed substantially to the depolarization-induced Ca2+ transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary α2δ-1 subunit. Thus, characterizing the CaV1.1Δ29 splice variant revealed the structural bases underlying the specific gating properties of skeletal Muscle Ca2+ channels, and it suggests the existence of a distinct mode of EC coupling in developing Muscle.
Francesco Zorzato - One of the best experts on this subject based on the ideXlab platform.
-
the novel skeletal Muscle sarcoplasmic reticulum jp 45 protein molecular cloning tissue distribution developmental expression and interaction with α1 1 subunit of the voltage gated calcium channel
Journal of Biological Chemistry, 2003Co-Authors: Ayuk A Anderson, Susan Treves, Donatella Biral, Romeo Betto, Doriana Sandona, Michel Ronjat, Francesco ZorzatoAbstract:JP-45 is a novel integral protein constituent of the skeletal Muscle sarcoplasmic reticulum junctional face membrane. We identified its primary structure from a cDNA clone isolated from a mouse skeletal Muscle cDNA library. Mouse skeletal Muscle JP-45 displays over 86 and 50% identity with two hypothetical NCBI data base protein sequences from mouse tongue and human Muscle, respectively. JP-45 is predicted to have a cytoplasmic domain, a single transmembrane segment followed by an intralumenal domain enriched in positively charged amino acids. Northern and Western blot analyses reveal that the protein is mainly expressed in skeletal Muscle. The mRNA encoding JP-45 appears in 17-day-old mouse embryos; expression of the protein peaks during the second month of postnatal development and then decreases approximately 3-fold during aging. Double immunofluorescence of adult skeletal Muscle fibers demonstrates that JP-45 co-localizes with the sarcoplasmic reticulum calcium release channel. Co-immunoprecipitation experiments with a monoclonal antibody against JP-45 show that JP-45 interacts with the alpha1.1 subunit voltage-gated calcium channel and calsequestrin. These results are consistent with the localization of JP-45 in the junctional sarcoplasmic reticulum and with its involvement in the molecular mechanism underlying skeletal Muscle Excitation-contraction coupling.
-
the novel skeletal Muscle sarcoplasmic reticulum jp 45 protein molecular cloning tissue distribution developmental expression and interaction with α1 1 subunit of the voltage gated calcium channel
Journal of Biological Chemistry, 2003Co-Authors: Ayuk A Anderson, Susan Treves, Donatella Biral, Romeo Betto, Doriana Sandona, Michel Ronjat, Francesco ZorzatoAbstract:JP-45 is a novel integral protein constituent of the skeletal Muscle sarcoplasmic reticulum junctional face membrane. We identified its primary structure from a cDNA clone isolated from a mouse skeletal Muscle cDNA library. Mouse skeletal Muscle JP-45 displays over 86 and 50% identity with two hypothetical NCBI data base protein sequences from mouse tongue and human Muscle, respectively. JP-45 is predicted to have a cytoplasmic domain, a single transmembrane segment followed by an intralumenal domain enriched in positively charged amino acids. Northern and Western blot analyses reveal that the protein is mainly expressed in skeletal Muscle. The mRNA encoding JP-45 appears in 17-day-old mouse embryos; expression of the protein peaks during the second month of postnatal development and then decreases ∼3-fold during aging. Double immunofluorescence of adult skeletal Muscle fibers demonstrates that JP-45 co-localizes with the sarcoplasmic reticulum calcium release channel. Co-immunoprecipitation experiments with a monoclonal antibody against JP-45 show that JP-45 interacts with the α1.1 subunit voltage-gated calcium channel and calsequestrin. These results are consistent with the localization of JP-45 in the junctional sarcoplasmic reticulum and with its involvement in the molecular mechanism underlying skeletal Muscle Excitation-contraction coupling.
Susan Treves - One of the best experts on this subject based on the ideXlab platform.
-
raptor ablation in skeletal Muscle decreases cav1 1 expression and affects the function of the Excitation contraction coupling supramolecular complex
Biochemical Journal, 2015Co-Authors: Ruben Lopez, Susan Treves, Barbara Mosca, Marcin Maj, Leda Bergamelli, Juan C Calderon, Florian C Bentzinger, Klaas RomaninoAbstract:The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal Muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in Muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal Muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal Muscle Excitation-contraction (E-C) coupling is affected by mTORC1 signalling.
-
the novel skeletal Muscle sarcoplasmic reticulum jp 45 protein molecular cloning tissue distribution developmental expression and interaction with α1 1 subunit of the voltage gated calcium channel
Journal of Biological Chemistry, 2003Co-Authors: Ayuk A Anderson, Susan Treves, Donatella Biral, Romeo Betto, Doriana Sandona, Michel Ronjat, Francesco ZorzatoAbstract:JP-45 is a novel integral protein constituent of the skeletal Muscle sarcoplasmic reticulum junctional face membrane. We identified its primary structure from a cDNA clone isolated from a mouse skeletal Muscle cDNA library. Mouse skeletal Muscle JP-45 displays over 86 and 50% identity with two hypothetical NCBI data base protein sequences from mouse tongue and human Muscle, respectively. JP-45 is predicted to have a cytoplasmic domain, a single transmembrane segment followed by an intralumenal domain enriched in positively charged amino acids. Northern and Western blot analyses reveal that the protein is mainly expressed in skeletal Muscle. The mRNA encoding JP-45 appears in 17-day-old mouse embryos; expression of the protein peaks during the second month of postnatal development and then decreases approximately 3-fold during aging. Double immunofluorescence of adult skeletal Muscle fibers demonstrates that JP-45 co-localizes with the sarcoplasmic reticulum calcium release channel. Co-immunoprecipitation experiments with a monoclonal antibody against JP-45 show that JP-45 interacts with the alpha1.1 subunit voltage-gated calcium channel and calsequestrin. These results are consistent with the localization of JP-45 in the junctional sarcoplasmic reticulum and with its involvement in the molecular mechanism underlying skeletal Muscle Excitation-contraction coupling.
-
the novel skeletal Muscle sarcoplasmic reticulum jp 45 protein molecular cloning tissue distribution developmental expression and interaction with α1 1 subunit of the voltage gated calcium channel
Journal of Biological Chemistry, 2003Co-Authors: Ayuk A Anderson, Susan Treves, Donatella Biral, Romeo Betto, Doriana Sandona, Michel Ronjat, Francesco ZorzatoAbstract:JP-45 is a novel integral protein constituent of the skeletal Muscle sarcoplasmic reticulum junctional face membrane. We identified its primary structure from a cDNA clone isolated from a mouse skeletal Muscle cDNA library. Mouse skeletal Muscle JP-45 displays over 86 and 50% identity with two hypothetical NCBI data base protein sequences from mouse tongue and human Muscle, respectively. JP-45 is predicted to have a cytoplasmic domain, a single transmembrane segment followed by an intralumenal domain enriched in positively charged amino acids. Northern and Western blot analyses reveal that the protein is mainly expressed in skeletal Muscle. The mRNA encoding JP-45 appears in 17-day-old mouse embryos; expression of the protein peaks during the second month of postnatal development and then decreases ∼3-fold during aging. Double immunofluorescence of adult skeletal Muscle fibers demonstrates that JP-45 co-localizes with the sarcoplasmic reticulum calcium release channel. Co-immunoprecipitation experiments with a monoclonal antibody against JP-45 show that JP-45 interacts with the α1.1 subunit voltage-gated calcium channel and calsequestrin. These results are consistent with the localization of JP-45 in the junctional sarcoplasmic reticulum and with its involvement in the molecular mechanism underlying skeletal Muscle Excitation-contraction coupling.
