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. 2018 Sep 10;7:e35222. doi: 10.7554/eLife.35222

Figure 3. Allosteric regulation of stac by interaction with the channel carboxy-terminus.

(A–B) Stac2 suppresses CDI of CaV1.3S tethered to CaM. In contrast, fusion of CaM protects CaV1.3S from competitive inhibitors such as CaM1234. Format as in Figure 1A–B. (C–D) Stac2 suppresses CDI of CaV1.2 tethered to CaM. Format as in Figure 1A–B. (E) FRET 2-hybrid assay shows the high-affinity interaction of YFP-tagged CaV1.3 CI to CFP-tagged CaM with relative dissociation constant Kd,EFF ~4000 ± 291 Dfree units. (F) Co-expression of untagged CaM1234 with FRET pairs YFP-tagged CaV1.3 CI and CFP-tagged CaM results in a marked reduction in FRET efficiency. (G) Co-expression of untagged stac3 spares the binding of YFP-tagged CaV1.3 CI with CFP-tagged CaM, yielding an identical EA-Dfree relationship to that in the absence of stac3 (E).

Figure 3.

Figure 3—figure supplement 1. Extended data show that stac acts concurrently with CaM.

Figure 3—figure supplement 1.

(A) Dominant-negative CaM1234 lacking Ca2+-binding to all four EF hands abolishes strong CDI of CaV1.3S. Left, cartoon schematizes CaV1.3S variant. Right, CDI of CaV1.3S is absent in the presence of CaM1234. Here, CaM1234 competitively replaces prebound wildtype CaM to suppress CDI. (B) Population data of r300 confirm the abolition of CDI for CaV1.3S upon co-expression of CaM1234. Format as in Figure 1—figure supplement 1A. (C) Fusion of CaMWT protects CaV1.3S from CaM1234, a competitive inhibitor of CDI. Exemplar currents show robust CDI of CaV1.3S-CaM under both control conditions and in the presence of CaM1234. Control data here reproduced from main text Figure 3A for symmetry. (D) Population data of r300 confirm the presence of CDI for CaV1.3S-CaM despite overexpression of CaM1234. Format as in Figure 1—figure supplement 1A. These results demonstrate that CaM-fusion can protect CaV1.3 from competitive inhibition of CDI. (E–F) Co-expression of stac2 (E) and stac3 (F) abolishes CDI of CaV1.3S-CaM. (G) Stac2 diminishes CDI of CaV1.3 despite CaM overexpression. (H) Population data of r300 demonstrate that stac2 abolishes CDI of CaV1.2 with CaM tethered to the carboxy-terminus (CaV1.2-CaM).