Duplex signaling by CaM and Stac3 enhances CaV1.1 function and provides insights into congenital myopathy

Figures

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  Figure 1.. Robust expression of CaV1.1 is dependent on its CT. (a) CaV1.3 with β2a and α2δ auxiliary subunits exhibits robust currents in HEK293 cells. Top: Cartoon depicts the CaV1.3 pore-forming α1 subunit with auxiliary subunits cotransfected. Middle: Exemplar current traces in response to a voltage-step protocol from −80 mV to +50 mV. Gray dashed line, baseline of 0 pA. Bottom: Population data for current density–voltage relationship from −30 to +80 mV in 10-mV increments for indicated number of cells (n). Error bars show mean ± SEM. Gray solid line, baseline of 0 pA. (b) CaV1.1 with auxiliary subunits exhibits minimal ionic currents. Format as in a. (c) Chimeric CaV1.1 with the CaV1.3 CT partially rescues functional expression. Format as in a. (d) Coexpression of stac3 also elicits robust Ca2+ currents through CaV1.1. Format as in a. (e and f) Restoration of CaM to CaV1.1 through overexpression of CaM or direct linkage to the auxiliary β2A subunit produces robust functional expression. Format as in a.

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  Figure 2.. Quantifying CaV1.1 surface–membrane trafficking using a dual-labeling approach. (a) Top: To label surface–membrane CaV1.1 channels, we inserted BBS into the domain II S5–S6 linker and a GFP to the amino terminus yielding CaV1.1BBS. Middle: CaV1.1BBS yields robust Ca2+ currents when coexpressed with β2A-CaM. Exemplar trace shows Ca2+ current elicited with a +50-mV voltage-step. Right: Jpeak (mean ± SEM) computed from indicated number of cells (n). Blue relation shows Jpeak relationship for WT CaV1.1. (b) CaV1.1 in the presence of α2δ and β2A auxiliary subunits traffics poorly and exhibits weak extracellular labeling. Left: Schematic shows external-epitope labeling of GFP-tagged CaV1.1BBS in the presence of α2δ and β2A auxiliary subunits. In this figure, the external epitope is α-bungarotoxin conjugated to QD. Right: Transmitted-light, intracellular GFP (SG), extracellular QD (SR), and merged images for transfected and labeled cells were collected by confocal microscopy. Bars, 5 μm. (c) Flow-cytometric analysis confirms weak surface–membrane expression for CaV1.1BBS with α2δ and β2A auxiliary subunits cotransfected. QD fluorescence, SR, is plotted as a function of GFP fluorescence,…

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  Figure 3.. CaM and Stac3 enhance surface–membrane trafficking of CaV1.1. (a and b) Confocal imaging and flow cytometry show that cotransfection of β2a-CaM augments CaV1.1 surface–membrane labeling. α2δ was cotransfected. Format as in Fig. 2 (b and c). Blue circles and fit in d correspond with binned data in the presence of β2a-CaM, and black fit is the relation with β2A reproduced from Fig. 2 c to facilitate comparison with baseline CaV1.1 trafficking. (c and d) Confocal imaging and flow cytometry of CaV1.1BBS cotransfected with stac3, β2A, and α2δ subunits confirms enhanced surface–membrane trafficking of CaV1.1. Format as in Fig. 2 (b and c). Bars, 5 µm. (e) Bar graph summary shows marked enhancement in saturating surface–membrane trafficking limit ϕmax, with coexpression of β2A-CaM but not β2A tethered to RYR1 P2 domain (β2A-RYR1[P2]) in comparison with β2A alone. Similarly, stac3 coexpression also enhances ϕmax. (f) Bar graph summary illustrates enhancement in surface–membrane trafficking of CaV1.1 bound to β1A subunit after coexpression of CaM or stac3 alone. *, P < 10−5 with Welch’s t test. Error bars show mean ± SEM.

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  Figure 4.. CaV1.1 CI harbors CaM and stac3 and is critical for plasmalemmal trafficking. (a) Cartoon schematic shows the potential interaction of CaV1.1 CI with CaM and stac3. (b) Left: Schematic shows FRET binding pairs, Cer-CaMWT and Ven-CaV1.3 CI. The CI module consists of the dual vestigial EF hand and pre-IQ and IQ segments of the channel CT. Middle: The CI region of CaV1.3 binds with a high affinity to apoCaM (Kd,EFF = 3,000 Dfree units ∼98 nM). FRET efficiency (EA) is plotted as a function of donor fluorophore–tagged molecule (Dfree) concentration (right). Gray dashed line indicates baseline for no binding. Right: CaV1.3 binds well to Ca2+/CaM (Kd,EFF = 8,000 Dfree units ∼260 nM). (c) In contrast, the CI region of CaV1.1 binds weakly to both apoCaM and Ca2+/CaM (Kd,EFF = 70,000 Dfree units ∼2.3 µM). Format as in b. (d) Stac3 binds with a high affinity to the CI region of CaV1.1 (Kd,EFF ∼12,000 Dfree units ∼400 nM). (e) Bar graph summarizes binding affinities from FRET curves (mean ± SEM). (f) Deletion of the entire CaV1.1 CT (CaV1.1[ΔCT]BBS) enhances baseline plasmalemmal trafficking. However, coexpression of β2A-CaM or stac3 fails to enhance surface–membrane trafficking, suggesting…

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  Figure 5.. CaM and stac3 bind to distinct sites on the CT and exert independent trafficking effects. (a) FRET binding of Ca2+/CaM with EF hands (left), pre-IQ domain (middle), and IQ domain (right) shows weak binding for EF hands and strong binding for pre-IQ and IQ domains. (b) Bar graph summary of binding affinities from a. (c and d) Stac3 binds preferentially to EF hands. Format as in a and b. (e and f) Coexpression of CaM and stac3 results in distinct extracellular labeling and supralinear increase of channels on the membrane. Format as in Fig. 2 (b and c). Bar, 5 µm. (g) Bar graph summarizing ϕmax for CaM and stac3 separately as well as together. Expressing CaM and β2A-CaM does not appreciably change ϕmax. In comparison, coexpression of β2A-CaM with stac3 results in a sixfold increase in ϕmax. *, P < 10−5. Error bars show mean ± SEM.

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  Figure 6.. POof CaV1.1 is increased with CaM and stac3. (a) CaV1.1 with only its auxiliary β2A and α2δ subunits elicits small tail current (Itail) despite large gating charge movement, indicating a low PO for these channels at baseline. Top: Cartoon depicts CaV1.1 α1 subunit bound to auxiliary β2A and α2δ subunits in HEK293 cells. Bottom: Exemplar currents elicited in response to voltage step depolarization from −80 mV to +80 mV show large gating-charge movement at the reversal potential (qrev) and Itail evoked upon repolarization to 0 mV. (b) Exemplar current trace for gating charge movement after pore block with Cd2+/La3+ with voltage step depolarization from −80 mV to +80 mV and back to −80 mV. Trace shows equivalent gating charge movement into the open conformation (QON) and gating charge movement into the closed conformation (QOFF). (c) Population data reveal low PO. In this figure, Itail evoked in response to a voltage-step family with varying prepulse potentials is normalized by the gating charge movement at the reversal potential, qrev. This ratio (Itail/qrev) is proportional to PO of the channel. (d) Population data of normalized QOFF to QON confirms equivalent charge movement and…

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  Figure 7.. Myopathic stac3 mutations reduce binding to the CT and surface membrane trafficking. (a) Amino acid sequence for stac3 mutations in both the C1 and first SH3 domain (SH31). (b) Schematic shows fluorophore-tagged FRET pairs, Ven-CI of CaV1.1 with Cer-tagged stac3 variants. NAM, Native American myopathy. (c) Myopathy-associated stac3 variants weaken binding to CaV1.1 CI. Left: Intron insert results in a frameshift, and truncation of SH3 domains (L[111]Δ) dramatically reduces stac3 binding to CaV1.1 CI. Middle: A point mutation in the first SH3 domain (W[284]S) strongly reduces stac3 binding. Right: Disease-associated stac3 variant with a nonsense mutation in the second SH3 domain (K[288]*) moderately weakens binding to CaV1.1. (d) Stac3 strongly enhances CaV1.1 surface–membrane trafficking in the presence of β1A and α2δ subunits as demonstrated by detectible BTX labeling in exemplar confocal images. Format as in Fig. 2 b. (e) Flow cytometric analysis confirms high expression of CaV1.1 when bound to stac3. Format as in Fig. 2 c. (f and g) Coexpression of myopathy-associated stac3 mutant (W[284]S) results in only modest enhancement in CaV1.1 surface–membrane trafficking as…

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  Figure 8.. Small-molecule modulators partially rescue pathological deficits in CaV1.1 trafficking. (a) Cartoon shows potential enhancement of CaV1.1 plasmalemmal trafficking in the presence of potential pharmacological agents. Left: CaV1.1, when bound to myopathy-associated mutant stac3 (W[284]S) and α2δ and β1A subunits, traffics poorly to the plasma membrane. Right: Addition of a small-molecule trafficking modulator may enhance the fraction of surface–membrane channels. (b) Bar graph summarizes changes in the saturating surface–membrane trafficking limit (ϕmax) of CaV1.1 trafficking after addition of various small-molecule modulators at low and high concentrations. Bottom dashed line corresponds with baseline trafficking with myopathic stac3 (W[284]S; gray); top dashed line corresponds with CaV1.1 trafficking with WT stac3. Both diltiazem and verapamil markedly enhanced ϕmax, whereas dihydropyridines and Na channel modulators ranolazine and mexiletine did not substantially alter CaV1.1 trafficking. Without stac3, 1 µM verapamil increased CaV1.1 trafficking by approximately twofold (*, P < 10−5). Error bars show mean ± SEM.

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  Figure 9.. Molecular determinants for CaV1.1 functional expression. (a–e) Schematic illustrates a simplified model for the effect of various regulatory proteins on CaV1.1 functional expression. For all panels, top and bottom rows identify the regulatory input and functional outcomes, respectively. Middle row schematizes underlying molecular mechanism. (a) Devoid of a β subunit, CaV1.1 fails to traffic to the surface membrane, presumably because of ER retention motifs. (b) Binding of the β subunit enables low basal CaV1.1 surface–membrane trafficking. Channels at the plasma membrane feature a low PO. (c) CaM enhances CaV1.1 surface–membrane trafficking. The same CaM also enhances baseline PO. (d) Stac3 binding also enhances CaV1.1 surface–membrane trafficking and up-regulates baseline PO. (e) Binding of CaM and stac allows for high CaV1.1 trafficking. The channels are presumed to retain a high baseline PO.