tsa201 cells  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: JPH3 and JPH4 interact with the Ca V 2.1 II-III loop. Mid-level confocal scans of tsA201 cells transfected with mCherry-tagged JPH3 or JPH4 together with the isolated, GFP-tagged Ca V 2.1 cytoplasmic domains: N-terminus ( A ), I-II loop ( B ), II-III loop ( C ), III-IV loop ( D ), and C-terminus ( E ). For each combination of constructs, the three panels arrayed vertically illustrate the distribution of the junctophilin ( red , topmost), cytoplasmic domain ( green , center), and the overlay of these two images ( bottom -most). At the cell periphery, the Ca V 2.1 II-III loop colocalized with both JPH3 and JPH4, as revealed in the overlaid images by the presence of yellow puncta, some of which are indicated by arrowheads ( C ). There appeared to be no colocalization of any other Ca V 2.1 cytoplasmic domains with either JPH3 or JPH4. Bars represent 5 μm. F , Pearson's colocalization coefficients calculated from confocal sections at the bottom surface of the cell. Data are reported as values from individual cells ( circles ), with the mean indicated by the height of the superimposed rectangles and ± SD by the horizontal black lines. For each construct combination, the numbers indicate total number of analyzed cells/number of separate transfected dishes. The colocalization between the II-III loop and JPH4 appeared to be somewhat greater than that between the loop and JPH3 (∗∗ p = 0.0019, t test with Welch's correction).

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Transfection, Isolation, Construct

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: JPH3 and JPH4 lacking the ER transmembrane segment retain the ability to interact with the Ca V 2.1 II-III loop, and this interaction appears to rescue junction formation. A , schematic representation of JPH-ΔTM constructs, JPH3 1-707 and JPH4 1-576 , both of which lack the TM domain that anchors them to the ER but retain the N-terminal regions responsible for association with the plasma membrane. B , bottom surface optical sections of tsA201 cells transfected with mCherry-JPH3 1-707 or mCherry-JPH4 1-576 alone ( a ), full-length mCherry-JPHs alone ( b ), and mCherry-JPH3 1-707 or mCherry-JPH4 1-576 in combination with the GFP-Ca V 2.1 II-III loop ( c ). In the absence of the II-III loop, the truncated junctophilins were distributed evenly on the plasma membrane ( a ). The interaction between the II-III loop, which is associated with the ER, and the JPH-ΔTMs, which are associated with the plasma membrane, was strong enough to rescue ER-PM junction formation, which caused redistribution of the truncated junctophilins into a punctate pattern ( c ) resembling the one observed for full-length junctophilins ( b ). C , mid-level confocal scans of tsA201 cells transfected with mCherry-JPH3 1-707 or mCherry-JPH4 1-576 in combination with the GFP-Ca V 2.1 II-III loop. Bars represent 5 μm in ( B ) and (C). D , Pearson's colocalization coefficients for the specified combinations of II-III loop and junctophilin constructs were calculated from optical sections of the bottom of the cell. Data are reported as values from individual cells ( circles ), with the mean indicated by the height of the superimposed rectangles, and ± SD by the horizontal black lines ( p = 0.0613, t test with Welch's correction). Numbers in parentheses represent total number of analyzed cells/separate transfected dishes.

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Construct, Clinical Proteomics, Membrane, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: JPH3-ΔTM and JPH4-ΔTM both interact with the synprint-containing, N-terminal half of the Ca V 2.1 II-III loop. Only JPH4-ΔTM interacts with the C-terminal half of the II-III loop. A , top : schematic representation of Ca V 2.1 II-III 715-1084 with the synprint domain indicated in red . Bottom : midlevel image of a tsA201 cell transfected only with GFP-tagged II-III 715-1084 . B , midlevel optical sections of tsA201 cells expressing mCherry-JPH3 1-707 or mCherry-JPH4 1-576 together with GFP-Ca V 2.1 II-III 715-1084 . Co-expression with either JPH3 1-707 or JPH4 1-576 caused Ca V 2.1 II-III 715-1084 to become concentrated at the cell periphery, where it colocalized with the JPH-ΔTM constructs. C , Pearson's coefficients calculated from midlevel optical scans. (n.s. p = 0.478, t test with Welch's correction). D , top : schematic representation of Ca V 2.1 II-III 1037-1253 . Bottom : midlevel image of a tsA201 cell transfected only with GFP-tagged II-III 1037-1253 . E , midlevel optical sections of tsA201 cells expressing mCherry-JPH3 1-707 or mCherry-JPH4 1-576 together with GFP-II-III 1037-1253 . Ca V 2.1 II-III 715-1084 did not colocalize with JPH3 1-707 but did colocalize with JPH4 1-576 . F , Pearson's coefficients calculated from bottom surface scans (∗∗∗∗ p < 0.0001, t test with Welch's correction). In ( C ) and ( F ), circles indicate values for individual cells, with the mean ± SD indicated by the superimposed rectangle and horizontal black lines, respectively; numbers in parentheses indicate the total number of analyzed cells/number of separate transfected dishes. Bars represents 5 μm in ( A ), ( B ), ( D ), and ( E ).

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Transfection, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: Recruitment to ER-PM junctions and slowing of inactivation of the Ca V 2.1 Δ2-like variant, which lacks most of the synprint domain, are mediated by JPH4 but not by JPH3. A , schematic representation of rabbit Ca V 2.1 showing the synprint domain ( red ), with its first and last residues indicated. The cyan bracket and numbers indicate the residues deleted to obtain the Δ2-like rabbit variant, based on the rat Δ2 splice variant and the alignment illustrated in Fig. S3 . B , midlevel optical sections of tsA201 cells transfected with GFP-Ca V 2.1 Δ2-like variant, β1b, α2δ1 in combination with either mCherry-JPH3 ( left column) or mCherry-JPH4 ( right column). Accumulation of the channel at JPH4-induced junction is visible ( white arrowheads). C , Pearson's colocalization coefficients for the specified combinations of Ca V 2.1 and junctophilins, calculated from optical sections of the bottom of the cell. Pearson’s coefficients for Ca V 2.1 with the intact synprint domain are part of a data set previously described in Perni and Beam, 2021 . All data are reported as values from individual cells ( circles ), with the mean indicated by the height of the superimposed rectangles, ± SD [2-way ANOVA: “no JPH v.s. JPH3”: p = 0.93, F (1,110) = 0.008. 2-way ANOVA “no JPH” v.s. “JPH4”, p < 0.0001, F (1, 111) = 172.9; post hoc Sidak's test: ∗∗ p = 0.0035, n.s. p = 0.135]. D , percentage of peak Ca 2+ current remaining 700 ms after the peak (I 700 /I peak ) is plotted (mean ± SD) as a function of test potential in tsA201 cells transfected with GFP-tagged Ca V 2.1 Δ2-like variant, β1b and α2δ1 together with either no junctophilins ( black ), JPH3 ( blue ), or JPH4 ( gold ), which were tagged with mCherry. The inset illustrates representative Ca 2+ currents (scaled to match in amplitude) elicited by an 800 ms depolarization to +50 mV, with the vertical dotted line indicating the current 700 ms after the peak. On average, the inactivation of the Ca V 2.1 Δ2-like variant was unaffected by JPH3 but significantly reduced by JPH4 (2-way ANOVA, post hoc Sidak's test: ∗ p = 0.032; ∗∗ p = 0.002; ∗∗∗∗ p < 0.0001). Numbers in parentheses indicate total number of analyzed cells/number of separate transfected dishes. Bars represent 2 μm.

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Variant Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: JPH3 and JPH4 constructs lacking the divergent and TM domains interact with the Ca V 2.1 II-III loop, but this interaction is lost after the additional removal of all or most of the α-helical domain. A , schematic representation of C-terminally truncated JPH constructs JPH3 1-423 and JPH4 1-417 , in which the divergent and transmembrane domains have been removed and midlevel optical sections of tsA201 cells expressing either of these two mCherry-tagged constructs in combination with GFP-tagged Ca V 2.1 II-III loop. The II-III loop colocalized with both JPH3 1-707 and JPH4 1-576, as indicated by the yellow segments at the cell periphery in the overlaid images ( e.g. , yellow arrowheads). Pearson's coefficients for these construct combinations, calculated from bottom -surface scans, are plotted in ( B ). C , schematic representation of JPH3 1-336 and JPH4 1-364 in which C-terminal truncation removed all, or part, respectively, of the α-helical domain and midlevel optical sections of tsA201 cells expressing mCherry-JPH3 1-336 or mCherry-JPH4 1-364 together with the GFP-tagged Ca V 2.1 II-III loop. There was no apparent colocalization of the II-III loop with either of these constructs. Pearson's coefficients, calculated from bottom-surface scans, are plotted in ( D ). In ( B ) and ( D ), circles indicate values for individual cells, with the mean ± SD indicated by the superimposed rectangle and horizontal black lines, respectively. Numbers in parentheses indicate total number of analyzed cells/number of separate transfected dishes. ∗∗∗∗ p < 0.0001; n.s. p = 0.84 ( t test with Welch's correction). Bars represent 5 μm in ( A ) and ( C ).

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Construct, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: The α-helical domain is responsible for the ability of JPH4 to interact with the distal half of the Ca V 2.1 II-III loop. A , schematic representation of constructs JPH3 1-333 -JPH4 α-Hlx , which contains the MORN and joining domains of JPH3 fused with the α-helical domain of JPH4, and the complementary construct JPH4 1-327 -JPH3 α-Hlx , containing the MORN and joining domains of JPH4 and the α-helical domain of JPH3. The first and last residues of the swapped α-helical domains are indicated in gold (JPH4 α-helical domain) and blue (JPH3 α-helical domain). B , midlevel optical sections of tsA201 cells expressing mCherry-tagged JPH3 1-333 -JPH4 α-Hlx or JPH4 1-327 -JPH3 α-Hlx together with the full-length Ca V 2.1 II-III loop tagged with GFP. The full-length II-III loop colocalized at the surface with both these junctophilin chimeras ( yellow segments in overlays, some indicated by arrowheads). Pearson's coefficients for these construct combinations, calculated from bottom-surface scans, are plotted in ( C ). D , midlevel optical sections of tsA201 cells expressing mCherry-tagged JPH3 1-333 -JPH4 α-Hlx or JPH4 1-327 -JPH3 α-Hlx together with the distal half of the Ca V 2.1 II-III loop, II-III 1037-1253 , tagged with GFP. Unlike the full-length II-III loop, which showed clear colocalization with both junctophilin chimeras, the only apparent colocalization of the distal half of the II-III loop was with JPH3 1-333 -JPH4 α-Hlx . Pearson's coefficients for these combinations of constructs, calculated from bottom-surface scans, are plotted in ( E ). In ( C ) and ( E ), circles indicate values calculated for individual cells, with the mean ± SD indicated by the superimposed rectangle and horizontal black lines, respectively. Numbers in parentheses indicate total number of analyzed cells/number of separate transfected dishes. In ( C ) and ( E ), n.s p = 0.548; ∗∗∗∗ p < 0.0001 ( t test with Welch's correction). Bars represent 5 μm in ( B ) and ( D ).

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Construct, Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Interaction between Ca V 2.1 and Junctophilin3/4 depends on the II-III loop of Ca V 2.1 and on the α-helical region of Junctophilin3/4

doi: 10.1016/j.jbc.2025.108424

Figure Lengend Snippet: The greater slowing of Ca V 2.1 inactivation by JPH4 than by JPH3 is not attributable to the α-helical domain. A , representative Ca 2+ currents (scaled to match in amplitude) elicited by an 800 ms depolarization to +40 mV in tsA201 cells transfected with YFP-Ca V 2.1, β1b, and α2δ1 together with either no junctophilins ( black ) or with mCherry-tagged JPH3 ( blue ), JPH4 ( gold ), JPH3 1-333 -JPH4 α-Hlx ( orange ), or JPH4 1-327 -JPH3 α-Hlx ( green ) designated in the figure as JPH3-JPH4 α-Hlx and JPH4-JPH3 α-Hlx , respectively. The vertical dotted line indicates the current 700 ms after the peak. B , percentage of peak current remaining 700 ms after the peak (I 700 /I peak ) is plotted (mean ± SD) as a function of test potential for Ca 2+ currents recorded from tsA201 cells transfected with Ca V 2.1, β1b, and α2δ1 together with either no junctophilins ( black ), JPH3 1-333 -JPH4 α-Hlx ( orange ), or JPH4 1-327 -JPH3 α-Hlx ( green ). In ( B ), numbers in parentheses indicate total number of analyzed cells/number of separate transfected dishes. The currents for JPH3 and JPH4, and the current and I 700 /I peak values for no junctophilins, are part of a data set previously described in Perni and Beam, 2021 [2-way ANOVA: “JPH4 1-327 -JPH3α-Hlx v.s. JPH3 1-333 -JPH4α-Hlx”: p < 0.0001, F (1,168) = 197.9; post hoc Sidak's test: ∗∗∗ p = 0.0004; ∗∗∗∗ p < 0.0001].

Article Snippet: tsA201 cells (ECACC 96121229) were cultured in high-glucose Dulbecco's Modified Eagle Medium (Mediatech), supplemented with 10% (vol/vol) fetal bovine serum and 2 mM glutamine in a humidified incubator with 5% (vol/vol) CO 2 .

Techniques: Transfection