Neuronal junctophilins recruit specific CaV and RyR isoforms to ER-PM junctions and functionally alter CaV2.1 and CaV2.2
- Department of Physiology and Biophysics, Anschutz Medical Campus, University of Colorado, United States
Figures
Figure 1
Junctions between the endoplasmic reticulum and plasma membrane (ER-PM junctions) are induced in tsA201 cells by expression of JPH3 or JPH4 N-terminally tagged with fluorescent proteins.
(A) Schematic representation of JPH3 and JPH4, indicating the ‘MORN’ motifs that bind to the plasma membrane and C-terminal segment (‘TM’) that traverses the ER membrane. Except for the ‘Divergent’ …
Figure 2
Three neuronal, high-voltage-activated calcium channels (CaV1.2, CaV2.1, and CaV2.2), but not a low-voltage-activated one (CaV3.1), localize at junctions induced between the endoplasmic reticulum and plasma membrane by JPH3 or JPH4.
Mid-level or bottom-surface optical sections are shown for tsA201 cells transfected with the designated CaV constructs (represented in green) either in the absence of junctophilins (A) or together …
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Figure 2—source data 1
Numerical data and statistical analyses to support graphs in Figure 2.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig2-data1-v2.docx
Figure 3 with 1 supplement
JPH3 and JPH4 slow the inactivation of Ca2+ currents via CaV2.1 and CaV2.2 but not those via CaV1.2.
Ca2+ currents were measured in tsA201 cells transfected with CaV1.2 (A), CaV2.1 (B), or CaV2.2 (C) either without junctophilins (black) or together with either JPH3 (red) or JPH4 (green). Constructs …
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Figure 3—source data 1
Numerical data to support graphs in Figure 3.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig3-data1-v2.docx
Figure 3—figure supplement 1
Similar to its effects on Ca2+ currents, JPH4 slows inactivation of Ba2+ currents via CaV2.1 and CaV2.2, with less effect on inactivation of Ba2+ currents via CaV1.2.
Ba2+ currents were measured in tsA201 cells transfected with CaV1.2 (A), CaV2.1 (B), or CaV2.2 (C) either without junctophilins (open symbols) or together with JPH4 (closed symbols). Constructs for …
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Figure 3—figure supplement 1—source data 1
Numerical data to support graphs in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig3-figsupp1-data1-v2.docx
Figure 4
The ability of JPH3 and JPH4 to slow inactivation of CaV2.1 and CaV2.2 does not depend upon the formation of junctions induced between the endoplasmic reticulum and plasma membrane.
Cells were transfected with the calcium channels together with JPH3(1 – 707) or JPH4(1 – 576), which lack the ER-spanning membrane segment and thus associate with the cell surface without inducing …
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Figure 4—source data 1
Numerical data to support graphs in Figure 4.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig4-data1-v2.docx
Figure 5 with 1 supplement
JPH3 recruits all three RyR isoforms to junctions between the endoplasmic reticulum and plasma membranes, whereas JPH4 only recruits RyR3.
Representative, red/green merged images of the mid-level or bottom surface of tsA201 cells expressing GFP-RyR1, YFP-RyR2, or YFP-RyR3 (left to right, represented in green) together with either …
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Figure 5—source data 1
Numerical data and statistical analyses to support graphs in Figure 5.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig5-data1-v2.docx
Figure 5—figure supplement 1
Levels of RyRs endogenously expressed in tsA201 cells are very low.
The change in fluorescence (ΔF/F0) of Fluo8-AM-loaded cells is plotted as a function of time. The blue and red traces represent the responses of RyR1-stable cells and non-transfected tsA201 cells, …
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Figure 5—figure supplement 1—source data 1
Numerical data to support graph in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig5-figsupp1-data1-v2.docx
Figure 6
The additional expression of CaV1.2 does not affect colocalization between JPH3 and RyR2 or between JPH4 and RyR3.
Pearson’s coefficients, calculated from bottom-surface scans, for mCherry-JPH3 versus YFP-RyR2 (left), and for mCherry-JPH4 versus YFP-RyR3 (right) expressed either alone or together with CFP-CaV1.2 …
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Figure 6—source data 1
Numerical data and statistical analyses to support graphs in Figure 6.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig6-data1-v2.docx
Figure 7
CaV2.1 and CaV2.2 colocalize with RyR1 in the presence of JPH3 but not of JPH4.
Ca2+ release from the endoplasmic reticulum (ER) is detectable after depolarization of cells expressing RyR1, JPH3, and either CaV2.1 or CaV2.2. (A) Representative images of cells expressing YFP-CaV2…
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Figure 7—source data 1
Numerical data and statistical analyses to support graphs in Figure 7B,D.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig7-data1-v2.docx
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Figure 7—source data 2
Numerical data to support graphs in Figure 7F,G,I,J.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig7-data2-v2.docx
Figure 8 with 1 supplement
The JPH3 divergent domain is important for the junctional recruitment of all RyRs.
(A) Schematic representation of the chimera ‘JPH3-with-JPH4-divergent’ in which the divergent domain of JPH3 has been replaced by that from JPH4. (B) Red only (top row) and red/green merged images …
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Figure 8—source data 1
Numerical data and statistical analyses to support graphs in Figure 8.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig8-data1-v2.docx
Figure 8—figure supplement 1
The JPH3 divergent domain is not important for interactions with voltage-gated calcium channels.
(A) Pearson's coefficients for tsA201 cells co-transfected with YFP-CaV1.2 and mCherry-tagged JPH3, JPH3-with-JPH4-divergent (see Figure 6A), or JPH4. Constructs for β1a and α2δ1were also present. (B…
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Figure 8—figure supplement 1—source data 1
Numerical data and statistical analyses to support graph in Figure 8—figure supplement 1A .
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig8-figsupp1-data1-v2.docx
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Figure 8—figure supplement 1—source data 2
Numerical data to support graph in Figure 8—figure supplement 1B.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig8-figsupp1-data2-v2.docx
Figure 9
The cytoplasmic domains of RyR1 and RyR3, which have been untethered from the endoplasmic reticulum (ER), accumulate at JPH3-induced junctions between the endoplasmic reticulum and plasma membrane, but the untethered cytoplasmic domain of RyR2 does not; none of the untethered RyR cytoplasmic domains accumulate at junctions induced by JPH4.
Mid-level and bottom-surface optical sections of tsA201 cells expressing YFP-RyR11:4300, YFP-RyR21:4226, or YFP-RyR31:4032 (represented in green) together with either mCherry-JPH3 (A) or …
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Figure 9—source data 1
Numerical data and statistical analyses to support graphs in Figure 9.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig9-data1-v2.docx
Figure 10 with 1 supplement
The cytoplasmic domain of RyR1 interacts with a distal segment of the JPH3 divergent region.
(A–E) Confocal sections of tsA201 cells transfected with YFP-RyR11:4300 (represented in green) and the indicated mCherry-tagged constructs. For all the constructs, the leftmost image displays only …
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Figure 10—source data 1
Numerical data and statistical analyses to support graph in Figure 10.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig10-data1-v2.docx
Figure 10—figure supplement 1
Deletion of JPH3 divergent domain residues 681 – 725 does not affect junctional recruitment of RyR1.
(A) Mid-level confocal sections of a tsA201 cell co-expressing mCherry-JPH3Δ(681-725) (red, left panel), GFP-RyR1 (green, center panel), and the red/green merged image (right panel). Scale bar = 5 …
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Figure 10—figure supplement 1—source data 1
Numerical data and statistical analyses to support graph in Figure 10—figure supplement 1.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig10-figsupp1-data1-v2.docx
Figure 11
Absence of colocalization between YFP-RyR31:4032 and mCherry-JPH3(1 – 707) expressed in tsA201 cells.
(A) Mid-level optical sections acquired from a transfected cell before and after photobleaching of YFP. mCherry-JPH3(1 – 707) was associated with the cell surface (leftmost image) but there was only …
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Figure 11—source data 1
Numerical data and statistical analyses to support graph in Figure 11.
- https://cdn.elifesciences.org/articles/64249/elife-64249-fig11-data1-v2.docx
Figure 12
Species alignment of a segment of the JPH3 divergent domain that houses a likely site of interaction with the cytoplasmic domain of RyR1.
Identical residues are shaded pink, and the numbers designate the N- and C-terminal residues, respectively. NCBI Sequence References are AAH36533.1 (Homo sapiens), NP_001100907.1 (Rattus norvegicus),…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | tsA201 | tsA201 | ECACC 96121229 RRID:CVCL_0063 | 100 % STR profile match to ATCC # CRL-3216 |
| Cell line (Homo sapiens) | Spiking-HEK293 | PMID:24391999 | HEK293 cells stably expressing NaV1.3 and KIR2.1 | 86 % STR profile match to ATCC # CRL-1573.3 |
| Cell line (Homo sapiens) | RyR1-stable cells | This paper (Materials and methods) | Spiking-HEK293 stably expressing RyR1 | |
| Transfected construct (Homo sapiens) | JPH3 | GenScript | C96900 | In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS |
| Transfected construct (Homo sapiens) | JPH4 | GenScript | C97908 | In vector: pcDNA3.1-DYK with addition of mCherry or ECFP CDS |
| Transfected construct (Homo sapiens) | JPH3-with-JPH4-divergent | This paper (Materials and methods) | In vector: same as JPH3 | |
| Transfected construct (Homo sapiens) | JPH3(1 – 707) | This paper (Materials and methods) | In vector: pmCHerry-C1 | |
| Transfected construct (Homo sapiens) | JPH4(1 – 576) | This paper (Materials and methods) | In vector: pmCHerry-C1 | |
| Transfected construct (Homo sapiens) | JPH3Δ681-725 | This paper (Materials and methods) | In vector: same as JPH3 | |
| Transfected construct (Homo sapiens) | JPH4(1 – 414)-JPH3(418 – 707) | This paper (Materials and methods) | In vector: same as JPH3 | |
| Transfected construct (Homo sapiens) | JPH3(418 – 748) | This paper (Materials and methods) | In vector: pmCHerry-C1 | |
| Transfected construct (Homo sapiens) | JPH3(653 – 748) | This paper (Materials and methods) | In vector: pmCHerry-C1 | |
| Transfected construct (Oryctolagus cuniculus) | CaV1.2 | PMID:2474130 | NM_001136522.1 | In vector: pEYFP-C1 or pECFP |
| Transfected construct (Oryctolagus cuniculus) | CaV2.2 | PMID:8386525 | GenBank: D14157.1 | In vector: modified pSP72 (see ref 28) |
| Transfected construct (Rattus norvegicus) | CaV3.1 | PMID:9495342 | GenBank: AF027984.1 | |
| Transfected construct (Oryctolagus cuniculus) | CaV2.1 | PMID:1849233 | NM_001101693.1 | In vector: pEYFP-C1 |
| Transfected construct (Oryctolagus cuniculus) | α2δ1 | PMID:28495885 | NM_001082276.1 | |
| Transfected construct (Rattus norvegicus) | β1b | PMID:19996312 | GenBank: X61394.1 | |
| Transfected construct (Oryctolagus cuniculus) | RyR1 | PMID:2725677 | NM_001101718.1 | In vector: pEYFP-C1 or pECFP-C1 or pCEP4 (with oriP removed) |
| Transfected construct (Mus musculus) | RyR2 | PMID:10473538 | NM_023868.2 | In vector: pcDNA3 plus EYFP CDS |
| Transfected construct (Oryctolagus cuniculus) | RyR3 | PMID:12471029 | NM_001082762.1 | In vector: pcDNA3 plus EYFP CDS |
| Transfected construct (Oryctolagus cuniculus) | RyR11:4300 | PMID:29284662 | In vector: pEYFP-C1 | |
| Transfected construct (Mus musculus) | RyR21:4226 | This paper (Materials and methods) | In vector: pcDNA3 plus EYFP CDS | |
| Transfected construct (Mus musculus) | RyR21:3991 | This paper (Materials and methods) | In vector: pcDNA3 plus EYFP CDS | |
| Transfected construct (Oryctolagus cuniculus) | RyR31:4032 | This paper (Materials and methods) | In vector: pcDNA3 plus EYFP CDS | |
| Transfected construct (Oryctolagus cuniculus) | pCMV R-CEPIA1er | Addgene | Cat # 58216 RRID:Addgene_58216 | |
| Recombinant DNA reagent | pmCherry-C1 | TaKaRa/Clontech | Cat # PT3975-5 | |
| Recombinant DNA reagent | mCherry-ER | Addgene | Cat # 55041 RRID:Addgene_55041 | |
| Recombinant DNA reagent | pEYFP-C1 | TaKaRa/Clontech | Cat # 6006- 1 | |
| Recombinant DNA reagent | pECFP-C1 | TaKaRa/Clontech | Cat # 6076 -1 | |
| Recombinant DNA reagent | pCEP4 | Invitrogen | Cat # V044-50 | |
| Sequence-based reagent | #1 | This paper (Materials and methods) | PCR primer | CGGGAGCTGCCAAC CCCCTGCTGGTGGT CATGGTGATCTTGC |
| Sequence-based reagent | #2 | This paper (Materials and methods) | PCR primer | TCTAGCATGGGCTG CAGGTCTTTGGCAG TGATCCTGGCGAT |
| Sequence-based reagent | #3 | This paper (Materials and methods) | PCR primer | TCGCCAGGATCACT GCCAAAGACCTGCA GCCCATGCTAGAGG |
| Sequence-based reagent | #4 | This paper (Materials and methods) | PCR primer | AAGATCACCATGA CCACCAGCAGGG GGTTGGC |
| Sequence-based reagent | #5 | This paper (Materials and methods) | PCR primer | GCTCGCCAGTTTC TGCACG |
| Sequence-based reagent | #6 | This paper (Materials and methods) | PCR primer | CCTATCCTGGTGG TCATGGTG |
| Sequence-based reagent | #7 | This paper (Materials and methods) | PCR primer | GTACGGGCTCAGC GCCTATCGTGGTG GGAGCCGTGG |
| Sequence-based reagent | #8 | This paper (Materials and methods) | PCR primer | TGGAAGGAAGGGG AGAACTCCTGGGC TATCAGTTTGGCCA |
| Sequence-based reagent | #9 | This paper (Materials and methods) | PCR primer | TGGCCAAACTGATAG CCCAGGAGTTCTCCC CTTCCTTCCAGCACC |
| Sequence-based reagent | #10 | This paper (Materials and methods) | PCR primer | AGGGCCACGGCTCC CACCACGATAGGCG CTGAGCCCG |
| Sequence-based reagent | #11 | This paper (Materials and methods) | PCR primer | CCAGGATCACGAAT TCAGAGTTCTCCCC |
| Sequence-based reagent | #12 | This paper (Materials and methods) | PCR primer | AGTGGTACCTTCC AGGGTCAAGG |
| Sequence-based reagent | #13 | This paper (Materials and methods) | PCR primer | GAGATGAATTCCT TGCTGAGGATGG |
| Sequence-based reagent | #14 | This paper (Materials and methods) | PCR primer | ACGATAAGAGCA AGGGCGAGGAGG |
| Sequence-based reagent | #15 | This paper (Materials and methods) | PCR primer | CTCAGCAACACCAT GGTGGCGACC |
| Sequence-based reagent | #16 | This paper (Materials and methods) | PCR primer | CCATGGTGTTGCTG AGGATGGAGACGCAT |
| Sequence-based reagent | #17 | This paper (Materials and methods) | PCR primer | GCCCTTGCTCTTAT CGTCGTCATCCTTG TAATCGATGAA |
| Sequence-based reagent | #18 | This paper (Materials and methods) | PCR primer | GGGCTAGCGCCAC CATGCAGAGACTG CGGTCC |
| Sequence-based reagent | #19 | This paper (Materials and methods) | PCR primer | GTTCAGGGGGA GGTGTGG |
| Sequence-based reagent | #20 | This paper (Materials and methods) | PCR primer | CGTCAGATCCGCT AGCGCTACCG |
| Sequence-based reagent | #21 | This paper (Materials and methods) | PCR primer | GATCCCGGGCTA GCGGTACCGTCG |
| Sequence-based reagent | #22 | This paper (Materials and methods) | PCR primer | CCGGGCTAGCGGT ACCCCGTCGACTGC |
| Sequence-based reagent | #23 | This paper (Materials and methods) | PCR primer | CTGATCCGATACG TGGATGAGGCGC |
| Sequence-based reagent | #24 | This paper (Materials and methods) | PCR primer | CCATCTGTTTGCCT ATGCGGCCGCTCA CCACATTACC |
| Sequence-based reagent | #25 | This paper (Materials and methods) | PCR primer | GCTCCTGCGGCCG CTCCTTCTCACTCTC |
| Commercial assay or kit | jetPRIME transfection reagent | Polyplus | VWR Cat#:89129- 922 | |
| Chemical compound, drug | Caffeine | Sigma-Aldrich | Cat# C-0750 | |
| Chemical compound, drug | Fluo8-AM | Aat Bioquest | Cat # 21082 | |
| Software, algorithm | GraphPad Prism | GraphPad Prism | RRID:SCR_002798 | Graphs and statistics |
| Software, algorithm | Fiji | ImageJ | doi: 10.1038/nmeth.2019 RRID:SCR_002285 | Image analysis |
