The junctional SR protein JP-45 affects the functional expression of the voltage-dependent Ca2+ channel Cav1.1

Abstract

JP-45, an integral protein of the junctional face membrane of the skeletal muscle sarcoplasmic reticulum (SR), colocalizes with its Ca2+ -release channel (the ryanodine receptor), and interacts with calsequestrin and the skeletal-muscle dihydropyridine receptor Cav1. We have identified the domains of JP-45 and the Cav1.1 involved in this interaction, and investigated the functional effect of JP-45. The cytoplasmic domain of JP-45, comprising residues 1-80, interacts with Cav1.1. JP-45 interacts with two distinct and functionally relevant domains of Cav1.1, the I-II loop and the C-terminal region. Interaction between JP-45 and the I-II loop occurs through the alpha-interacting domain in the I-II loop. beta1a, a Cav1 subunit, also interacts with the cytosolic domain of JP-45, and its presence drastically reduces the interaction between JP-45 and the I-II loop. The functional effect of JP-45 on Cav1.1 activity was assessed by investigating charge movement in differentiated C2C12 myotubes after overexpression or depletion of JP-45. Overexpression of JP-45 decreased peak charge-movement and shifted VQ1/2 to a more negative potential (-10 mV). JP-45 depletion decreased both the content of Cav1.1 and peak charge-movements. Our data demonstrate that JP-45 is an important protein for functional expression of voltage-dependent Ca2+ channels.

Figure 1. Domains of JP-45 used to identify the Cav1.1 binding sites

A. Schematic representation. B. 10% SDS PAG and Coomassie Brilliant Blue staining of the GST- JP-45- fusion proteins purified by glutathione-Sepharose. The numbers above each lane indicate the amino acid residues which were fused in frame in the pGex plasmid to yield the GST-JP-45 fusion protein.

Figure 2. Identification of the JP-45 domain interacting with Cav1.1

A. GST-JP-45 fusion proteins encompassing different domains of JP-45 were bound to glutathione-Sepharose beads and incubated with solubilized rabbit skeletal muscle microsomal vescicles as described in the Methods section. The proteins present in the void (V), last wash (LW) and those bound (B) to the beads were separated on a 10% SDS PAG, transferred onto nitrocellulose and the presence of the bound Cav1.1 was revealed by Western blotting using commercial anti- α1.1 antibodies. B. Co-immunoprecipitation experiment using monoclonal anti-JP-45 Ab to pull down the Cav1.1. Experiments were performed as described in the Methods section: where indicated recombinant GST-JP-45 domain 2 was used to compete out the interaction between endogenous proteins present in the microsomes forming a supramolecular complex.

Figure 3. Identification of Cav1.1 domains interacting with JP-45

α1.1 domains I–II, COOH-distal, COOH-proximal and β1a interact with the cytosolic domain of JP-45. A. Immunoblot of purified His-tagged fusion proteins encompassing the different α1.1 domains (10% Tricine SDS PAG) and β1a (10% SDS PAG). Immunostaining was carried out using commercial anti-poly-His antibodies followed by peroxidase conjugated anti-mouse IgG; bands were visualized by chemiluminescence. B. Pull-down of His-tagged fusion proteins with domain 2 of JP-45 (aa residue 1-80). GST-JP-45 domain 2 fusion protein was bound to glutathione-Sepharose beads and incubated with the His-tagged recombinant α1.1 proteins. Proteins present in the void (V) or bound to the beads (B) were separated on a 10% tricine SDS PAG or 10% SDS PAG (for the β1a subunit), blotted onto nitrocellulose and visualized by Western blot using anti-poly-His Ab as described above. C. GST-JP-45 domain 2 fusion protein was bound to glutathione-Sepharose beads and incubated with solubilized light sarcoplasmic reticulum vescicles isolated from rabbit skeletal muscle in the presence of the indicated concentration of competing His-tagged I–II loop or COOH-distal fusion proteins. D. Monoclonal anti-JP-45 antibodies were used to co-immunoprecipitate the complex from solubilized light microsomal vescicles isolated from rabbit skeletal muscle in the presence of the indicated concentration of competing His-tagged I–II loop or COOH-distal fusion proteins. Proteins bound to the beads were separated in a 10% SDS PAG, transferred onto nitrocellulose and probed with antiα1.1 antibodies.

Figure 4. Effect of the β1a subunit on the interaction between JP-45 and Cav1.1

A. Interaction between GST-JP-45 and His I–II loop in the presence of competing purified β1a subunit. For the fusion- protein protein interaction, 0.57 μM of GST-JP-45 domain 2 and 1.4 μM I–II loop fusion protein were incubated in the presence of the indicated concentration of His-tagged β1a subunit. His tagged I–II loop fusion proteins bound to the GST-JP-45 domain 2 coated glutathione-Sepharose beads were separated on a 10% tricine SDS PAG and probed with anti-poly-His Ab as described in the legend to figure 3. B. Solubilized rabbit skeletal muscle light sarcoplasmic reticulum vesicles were incubated with GST-JP-45 domain 2-coated glutathione-Sepharose beads in the absence or presence of purified β1a subunit. Proteins present in the void (V) and bound to the beads (B), were separated on a 10% SDS PAG, blotted onto nitrocellulose and probed with antiα1.1 subunit Ab. C. Solubilized rabbit skeletal muscle light sarcoplasmic reticulum vesicles were incubated with anti-JP-45 Ab followed by incubation with Sepharose-protein G beads in the absence or presence of competing purified β1a subunit. Proteins present in the void (V) and bound (B) to the beads, were separated on a 10% SDS PAG, blotted onto nitrocellulose and probed with anti-α1.1 subunit Ab.

Figure 5. JP-45 interacts with the AID domain on the I–II loop of Cav1.1

A. A GST-I–II loop fusion protein or the GST-AID containing domain encompassed within α1.1 residues 336-384 were incubated with His-tagged JP-45 domain 2. Pull down was performed as described in figure 3; proteins in the void (V), last wash (LW) or bound (B) to the glutathione resin were separated on a 12.5% SDS PAG, transferred onto nitrocellulose and probed with affinity-purified anti-JP-45 Ab. B. A synthetic biotinylated peptide corresponding to the AID sequence or an unrelated biotinylated peptide were used to coat Neutroavidine beads, which were subsequently incubated with His-JP-45 domain 2. Proteins present in the void, last wash or bound to the beads were separated on a 12.5% SDS PAG, transferred onto nitrocellulose and the immunopositive band was visualized using anti-His-tag commercial Abs. C. A his-tagged fusion protein encompassing domain 2 JP-45 was prepared as described in the Methods section. Though the fusion protein migrated slower in SDS-PAG, its identity was verified by direct sequencing (not shown), by immunoblotting using anti-His Ab. Note that treatment of the fusion protein with DTT+DEPC eliminated its immunoreactivity.

Figure 6. Overexpression of JP-45 in C2C12 cells does not affect the expression level of α1.1 subunit

C2C12 cells were transfected either with the pFP-N3/DsRed2/JP-45 vector or with pFP-N3/DsRed2 alone as control. Panel A- Microsomes were prepared from transfected differentiated C2C12; 5 μg protein were separated on a 10%SDS PAG, blotted onto nitrocellulose and probed with anti-JP-45 polyclonal Ab, followed by protein G peroxidase. The immunoreactive band was visualized by chemiluminescence. Note that control cells show the endogenous JP-45 immunoreactive band alone, while cells transfected with the pFP-N3/DsRed2/JP-45 vector show an additional band of approximately 70 kDa, representing the DsRed/JP-45 fusion protein. Panel B- immunoprecipitation and Western blot analysis of α1.1 subunit expression in C2C12 cells transfected with pFP-N3/DsRed2 or pFP-N3/DsRed2/JP-45. Results are representative of transfection experiments performed three different times.

Figure 7. Effect of JP-45 Overexpression on Cav1.1 charge movement

Maximum charge movement – fluorescence relationship for C2C12 cells transfected with either pFP-N3/DsRed2 JP-45 (A) or pFP-N3/DsRed2 alone as control (B). The lines in A and B represent the linear regression including all data points. Charge movement – Vm relationship for pFP-N3/DsRed2 JP-45 (C) or pFP-N3/DsRed2 plasmid (D) transfected cells fitted to a Boltzmann equation 1 (see text). The best fitting parameters are included in Table 1. Charge movement-Vm relationship for the five top and bottom Q_max values (E) from JP-45 transfected cells (C).

Figure 8. JP-45 gene silencing in differentiated C2C12 myotubes

A. Total RNA was extracted from transfected and differentiated C2C12 cells, converted into cDNA and the cDNA encoding JP-45 and β-actin was amplified by PCR. Amplified DNA obtained from 50 or 100 ng RNA was separated on a 7.5% acrylamide gel (JP-45, Top Panel) or a 1% agarose gel (β-actin, Bottom Panel). B. Microsomal proteins from transfected and differentaiated C2C12 cells were prepared, separated on a 10% SDS PAG, blotted onto nitrocellulose and probed with anti-JP-45 Abs (central panel) or commercial anti-β-actin Abs, followed by peroxidase-labelled secondary Abs. Immunoreactive bands were visualized by chemiluminescence. Panel on the right shows blotted proteins stained with Ponceau Red. Results are representative of experiments carried out on three different transfection experiments.

Figure 9. JP-45 gene silencing modifies Cav1.1 charge movement

Charge movement – membrane voltage (Vm) relationship recorded in C2C12 cells transfected with JP-45 siRNA (triangles) (n = 18). Control cells were transfected with pSHAG vector (circles) (n = 17). A illustrate data points, expressed as means ± S.E.M., were fitted to a Boltzmann equation (equation 1). Best fitting parameters are shown in Table 2. B and C illustrate charge movement records in the −30 to +30mV range. Numbers on the left indicate the membrane potential. Dotted lines represent the baseline. D and E show immunoprecipitation and western blot analysis of Cav1.1 α1 subunit expression in C2C12 cells transfected with siRNA JP-45 and control pSHAG vector. D and E represent two from a total of 4 assays. The Cav1.1 α1 subunit expression in D and E decreased by 69 and 28%, respectively. The location of the molecular weight standards and their M_r values are depicted on the right.

Figure 10

Model depicting potential functional role of JP-45