QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 25 April 2006
doi: 10.1242/jcs.02935

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Anderson, A. A. Articles by Zorzato, F. Search for Related Content PubMed PubMed Citation Articles by Anderson, A. A. Articles by Zorzato, F. Social Bookmarking                         What's this?

The junctional SR protein JP-45 affects the functional expression of the voltage-dependent Ca²⁺ channel Ca_v1.1

¹ Departments of Anaesthesia and Research, Basel University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland
² INSERM U607/CEA/UJF, Lab CCFP/DRDC, Rue des Martyrs 17, 38054, Grenoble, Cedex 9 France
³ Department of Physiology and Pharmacology, Gerontology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
⁴ Department of Internal Medicine, Gerontology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
⁵ Department of Experimental and Diagnostic Medicine, General Pathology Section, University of Ferrara, Via Borsari 46, 44100 Ferrara, Italy

View larger version (25K): [in a new window]   Fig. 1. Domains of JP-45 used to identify the Cav1.1 binding sites. (A) Schematic representation. (B) 10% SDS-PAGE and Coomassie Brilliant Blue staining of the GST-JP-45 fusion proteins purified by glutathione-Sepharose. The numbers above each lane indicate the aa residues that were fused in frame in the pGex plasmid to yield the GST-JP-45 fusion protein.

View larger version (35K): [in a new window]   Fig. 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 Materials and Methods. Proteins present in the void (V), last wash (LW) and bound to the beads (B) were separated on a 10% SDS-PAGE, transferred onto nitrocellulose and the presence of the bound Cav1.1 was revealed by western blotting with commercial anti-Cav1.1 Abs. (B) Co-immunoprecipitation experiment with monoclonal anti-JP-45 Ab to pull-down Cav1.1. Experiments were performed as described in the Materials and Methods. Where indicated, recombinant GST-JP-45 domain-2 fusion protein was used to compete-out the interaction between endogenous proteins present in the microsomes forming a supramolecular complex.

View larger version (41K): [in a new window]   Fig. 3. Identification of Cav1.1 domains interacting with JP-45. Cav1.1 domains I-II, C-terminal-distal and C-terminal-proximal His-tag fusion proteins, and subunit ß1a interact with the cytosolic domain of JP-45. (A) Immunoblot of purified His-tagged fusion proteins encompassing the different Cav1.1 domains (10% tricine SDS-PAGE) and ß1a (10% SDS-PAGE). Immunostaining was carried out using commercial anti-poly-His Abs followed by HR-conjugated anti-mouse IgG; bands were visualized by chemiluminescence. (B) Pull-down of His-tagged fusion proteins with domain 2 of JP-45 (aa residues 1-80). GST-JP-45 domain-2 fusion protein was bound to glutathione-Sepharose beads and incubated with the His-tagged recombinant Cav1.1 proteins. Proteins present in the void (V) or bound to the beads (B) were separated on a 10% tricine SDS-PAGE or 10% SDS-PAGE (for the ß1a subunit), blotted onto nitrocellulose and visualized in western blots by 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 SR vescicles isolated from rabbit skeletal muscle in the presence of the indicated concentration of competing His-tagged I-II loop or C-terminal-distal His-tag fusion proteins. (D) Monoclonal anti-JP-45 Abs 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 C-terminal-distal fusion proteins. Proteins bound to the beads were separated in a 10% SDS-PAGE, transferred onto nitrocellulose and probed with anti-Cav1.1 Abs.

View larger version (47K): [in a new window]   Fig. 4. Effect of the ß1a 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 ß1a 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 ß1a subunit. His-tagged I-II loop fusion proteins bound to glutathione-Sepharose beads coated with GST-JP-45 domain-2 fusion protein were separated on a 10% tricine SDS-PAGE and probed with anti-poly-His Ab as described in the legend to Fig. 3. (B) Solubilized rabbit skeletal-muscle light SR vesicles were incubated with glutathione-Sepharose beads coated with GST-JP-45 domain-2 fusion protein in the absence or presence of purified ß1a subunit. Proteins present in the void (V) and bound to the beads (B), were separated on a 10% SDS-PAGE, blotted onto nitrocellulose and probed with anti-1.1 subunit Ab. (C) Solubilized rabbit skeletal-muscle light SR vesicles were incubated with anti-JP-45 Ab followed by incubation with Sepharose-protein G beads in the absence or presence of competing purified ß1a subunit. Proteins present in the void (V) and bound to the beads (B), were separated on a 10% SDS-PAGE, blotted onto nitrocellulose and probed with anti-Cav1.1 Ab.

View larger version (27K): [in a new window]   Fig. 5. JP-45 interacts with the AID domain on the I-II loop of Cav1.1. (A) GST-I-II loop fusion protein or the GST-AID containing domain encompassed within Cav1.1 residues 336-384 were incubated with His-tagged JP-45 domain-2 fusion protein. Pull-down was performed as described in Fig. 3; proteins in the void (V), last wash (LW) or bound to the glutathione resin (B) were separated on a 12.5% SDS-PAGE, transferred onto nitrocellulose and probed with affinity-purified anti-JP-45 Ab. (B) Synthetic biotinylated peptides 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-PAGE, 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 Materials and Methods. Although the fusion protein migrated slower in SDS-PAGE, its identity was verified by direct sequencing (not shown) and by immunoblotting with anti-His Ab. Note that treatment of the fusion protein with DTT+DEPC eliminated its immunoreactivity.

View larger version (31K): [in a new window]   Fig. 6. Overexpression of JP-45 in C2C12 cells does not affect the expression levels of the Cav1.1 subunit. C2C12 cells were transfected either with the pFP-N3-DsRed2-JP-45 vector or with pFP-N3-DsRed2 alone as control. (A) Microsomes were prepared from transfected differentiated C2C12; 5 µg protein were separated on a 10% SDS-PAGE, blotted onto nitrocellulose and probed with anti-JP-45 polyclonal Ab, followed by HR-coupled protein G. The immunoreactive band was visualized by chemiluminescence. Note that control cells show the endogenous JP-45 immunoreactive band alone, whereas cells transfected with pFP-N3-DsRed2-JP-45 show an additional band of approximately 70 kDa, representing the DsRed-JP-45 fusion protein. (B) Immunoprecipitation and western blot analysis of Cav1.1 subunit expression in C2C12 cells transfected with pFP-N3-DsRed2 or pFP-N3-DsRed2-JP-45. Results are representative of three different transfection experiments.

View larger version (24K): [in a new window]   Fig. 7. Effect of JP-45 overexpression on Cav1.1 charge movement. (A,B) Maximum charge-movement versus fluorescence 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. (C,D) Charge-movement versus membrane-voltage (Vm) for pFP-N3-DsRed2-JP-45 (C) or pFP-N3-DsRed2 plasmid (D) transfected cells fitted to a Boltzmann equation (see text). The best fitting parameters are included in Table 1. (E) Charge-movement versus Vm for the five highest and lowest Qmax values (E) from JP-45 transfected cells (C).

View larger version (48K): [in a new window]   Fig. 8. JP-45 gene silencing in differentiated C2C12 myotubes. (A) Total RNA was extracted from transfected and differentiated C2C12 cells and converted into cDNA. The cDNA encoding JP-45 and ß-actin was amplified by PCR. Amplified DNA obtained from 50 ng or 100 ng RNA was separated on a 7.5% acrylamide gel (JP-45, top panel) or a 1% agarose gel (ß-actin, bottom panel). (B) Microsomal proteins from transfected and differentaiated C2C12 cells were prepared, separated on a 10% SDS-PAGE, blotted onto nitrocellulose and probed with anti-JP-45 Abs (central panel) or commercial anti-ß-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 in three different transfection experiments.

View larger version (23K): [in a new window]   Fig. 9. JP-45 gene silencing modifies Cav1.1 charge movement. Charge-movement versus membrane-voltage (Vm) recorded in C2C12 cells transfected with JP-45 siRNA (n=18). Control cells were transfected with pSHAG vector (n=17). (A) Data points, expressed as mean ± s.e.m., were fitted to a Boltzmann equation (see text). Best fitting parameters are shown in Table 2. (B,C) Charge-movement records in the -30 mV to +30 mV range. Numbers on the left indicate the membrane potential. Dotted lines represent the baseline. (D,E) Immunoprecipitation and western blot analysis of Cav1.1 expression in C2C12 cells transfected with siRNA JP-45 and control pSHAG vector. Two assays from a total of four. The Cav1.1 expression in D and E decreased by 69% and 28%, respectively. The location of the molecular mass standards in kDa are depicted on the right.

View larger version (49K): [in a new window]   Fig. 10. Model depicting the potential functional role of JP-45.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter