Figure 5. Stac U-domain is a minimal effect domain for suppression of Ca_V1 CDI.

(A–C) To localize an effector motif for stac2, CDI of Ca_V1.2-CaM was quantified in the presence of three stac subdomains: (1) C1, (2) linker region, and (3) SH3-SH3. Exemplar traces in response to a +10 mV voltage-step depolarization show robust CDI of Ca_V1.2-CaM in the presence of C1 (A), and SH3-SH3 (C) domains. Co-expression of the linker-region is sufficient to suppress CDI of Ca_V1.2-CaM (B). Format as in Figure 1A. (D) Bar graph summarizes population data for Ca_V1.2-CaM CDI in the presence of the three stac subdomains. Each bar, mean ±S.E.M of CDI₃₀₀ at +10 mV from specified number of cells. CDI levels in the presence (solid blue line) and absence (dashed gray line) of full-length stac2 is reproduced for comparison. (E) Bar graph shows degree of conservation for the linker region across 770 orthologs of stac2. A well conserved subsegment termed U-domain is shaded blue. (F–G) Co-expression of U-domain is sufficient to abolish CDI of Ca_V1.2-CaM (F) and Ca_V1.3-CaM (G). Format as in Figure 1A. (H) Bar graph displays population data for CDI of Ca_V1.2-CaM and Ca_V1.3-CaM in the presence of U-domain. Each bar, mean ±S.E.M of CDI₃₀₀ at +10 mV from specified number of cells. Dashed line, baseline CDI for both channels in the absence of stac2. Blue line, CDI of both channels in the presence of full-length stac2. (I) Systematic alanine scanning mutagenesis of the U-domain reveals critical determinants for stac-mediated suppression of Ca_V1.2 CDI. For comparison, Ca_V1.2 CDI in the presence (blue line) and absence (black dashed line) of stac2 are shown. Stac2 mutants ₂₀₀KVD/AAA, ₂₀₃PVY/AAA, ₂₀₆ETL/AAA fully abolish stac2-mediated CDI suppression. (J) Exemplar currents show that stac2 mutant ₂₀₆ETL/AAA eliminates stac’s ability to suppress Ca_V1.2 CDI. Format as in Figure 1A. (K) Stac2 ₂₀₆ETL/AAA also fails to inhibit CDI of Ca_V1.3_S. Format as in Figure 1A.

Figure 5—figure supplement 1. Extended data demonstrate that the U-motif is a minimal domain for suppressing CDI of Ca_V1.2 and Ca_V1.3.

(A–C) Population data confirm that co-expression of either the cysteine-rich domain (C1; B) or dual SH3 domains (SH3-SH3; C) of stac2 in isolation spares CDI of Ca_V1.2-CaM (cartoon in A). Format as in Figure 1—figure supplement 1A. Each symbol, mean ±S.E.M. of r₃₀₀ values obtained from the specified number of cells (n). Here, tethered CaM was used to protect from potential competitive displacement of CaM by stac2 subdomains. (D–E) Population data confirm that the U-linker that connects the C1 and dual SH3 domains is sufficient to suppress CDI of Ca_V1.2-CaM, fully recapitulating the effect of stac on Ca_V1 channels (D). The well-conserved minimal U-motif from stac2 is also sufficient to fully suppress CDI of Ca_V1.2-CaM (E). Format as in Figure 1—figure supplement 1A. Each symbol, mean ±S.E.M. of r₃₀₀ values. (F) Cartoon shows Ca_V1.3_S-CaM. (G) Stac2 U-domain also diminished CDI of Ca_V1.3_S-CaM as evident from population data of r₃₀₀ values. Residual CDI here (red shaded area) is reminiscent of that observed with full-length stac2 and Ca_V1.3_S-CaM (Figure 3—figure supplement 1E).

Figure 5—figure supplement 2. Systematic alanine scanning mutagenesis of minimal U-motif reveals structural determinants for stac modulation of Ca_V1.

(A) Triple alanine substitution of residues ₂₀₀KVD₂₀₂ abolishes stac modulation of Ca_V1.2. In comparison to stac2 that fully suppresses CDI of Ca_V1.2, co-expression of stac2 ₂₀₀KVD/AAA spares strong CDI of Ca_V1.2 suggesting that residues ₂₀₀KVD₂₀₂are critical for stac modulation. Left, cartoon schematizes the location of ₂₀₀KVD/AAA mutation on full-length stac2. Middle, exemplar current traces show robust CDI of Ca_V1.2 in the presence of stac2 ₂₀₀KVD/AAA. Right, population data showing r₃₀₀ values as a function of voltage for Ca²⁺ (red) and Ba²⁺ currents (black). Each symbol, mean ±S.E.M. from five cells. (B–C) Triple alanine substitution of stac2 residues ₂₀₃PVY₂₀₅ and ₂₀₆ETL₂₀₈ abolishes stac modulation of Ca_V1.2 as evident from strong CDI present despite overexpression of mutant stac. Format as in (A). (D–G) Ca_V1.2 CDI is suppressed by stac2 despite alanine substitution of residues, ₂₀₉RYG₂₁₁D), ₂₁₂TSL₂₁₄ (E), ₂₁₈NRS₂₂₀(F), and ₂₂₁S (G), suggesting that these residues are not necessary for stac modulation of Ca_V1 channels. Format as in (A).