Allosteric regulators selectively prevent Ca2+-feedback of CaV and NaV channels

Figures

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  Figure 1.. Stac specifically abolishes Ca2+/CaM-regulation in CaV1 channels. (A) Stac2 diminishes CDI of CaV1.2. Left, cartoon schematic shows CaV1.2. Middle, exemplar current traces evoked in response to +10 mV voltage-step shows robust CDI (rose shaded area) evident as enhanced current decay with Ca2+ (red) versus Ba2+ (black) as the charge carrier. Right, stac2 abolishes CDI. Steady-state levels of inactivation are assessed as the fraction of peak current remaining following 300 ms depolarization (rCa and rBa) and CDI = 1 – rCa/rBa. (B) Bar graph displays population data of CDI300 for CaV1.2 in the absence and in the presence of stac1, stac2, or stac3. Dashed line shows baseline CDI in the absence of stac for comparison. Each bar, mean ±S.E.M. obtained from specified number of cells (n). (C–D) Stac isoforms suppress CDI of CaV1.3S, the canonical short variant, as confirmed by both exemplar traces (C) and population data (D). Format as in (A) and (B). (E–F) Stac2 abolishes CDI of CaV1.443* assessed in response to +20 mV test pulse. Format as in (A) and (B). (G–H) Stac2 spares CDF of CaV2.1, as evaluated using a prepulse protocol. An isolated test pulse to 0 mV elicits Ca2+ currents with…

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  Figure 1—figure supplement 1.. Extended data highlight selectivity of stac in modulating CaV1 versus CaV2 and NaV1 channels. (A) Stac1-3 isoforms strongly diminish CDI of CaV1.2 (schematized in the far left subpanel). In the absence of stac, CaV1.2 exhibits robust CDI (red shaded area, middle left subpanel). Co-expression of stac2 (middle), stac1 (middle right), and stac3 (far right), all suppress CDI. The steady-state extent of inactivation in Ca2+ (red) and Ba2+ (black), here, is estimated by the metric r300, the ratio of current remaining at 300 ms to peak current during a depolarizing pulse, plotted as a function of voltage. Each symbol, mean ±S.E.M. from specified number of cells (n). (B) Stac1-3 isoforms suppress CDI of CaV1.3. Format as in (A). (C) Stac2 co-expression abolishes CDI of CaV1.443* alternatively spliced variant. Format as in (A). (D) Stac2 does not alter CDF of CaV2.1. A prepulse protocol is used to estimate CDF. A step depolarization to 0 mV evokes an inward current containing rapid and slow components caused by a superposition of voltage activation and CDF. With a preceding voltage step to 20 mV, sufficient Ca2+ enters to partially facilitate the channels, such that the subsequent step to…

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  Figure 2.. Stac interacts with the channel carboxy-terminus. (A) Live-cell FRET 2-hybrid assay shows high-affinity interaction between CFP-tagged stac3 with YFP-tethered holo-CaV1.3 channels in the presence of auxiliary β2A and α2δ subunits. (B) Cartoon shows FRET pairs, CFP-stac3 with YFP-CI, YFP-PCI, and YFP-IQ of CaV1.3. (C) FRET-binding curves show robust binding of stac3 to both the CI and PCI segment while binding to IQ is weaker. (D) Bar graph summarizes the relative association constant, Ka,EFF, of stac2 binding to major channel intracellular domains. (E–F) Transferring CaV1.3S CI to CaV2.3 (CaV2.3/1.3 CI) unveils latent stac2-mediated suppression of CDI. Format as in Figure 1A – B.

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  Figure 2—figure supplement 1.. Systematic FRET 2-hybrid scan of major intracellular loops of CaV1.3 with stac. (A) Schematic depicts design of YFP-tagged CaV1.3 intracellular loop constructs (NT, I-II loop, II-III loop, III-IV loop, CI region, PCI region, IQ domain). The exact sequence of the N- and C-termini of each peptide as well as locations on the CaV1.3 α subunit are denoted. (B–E) FRET 2-hybrid binding curves for stac3 interaction with various channel intracellular loops (black symbol and fit). Each symbol denotes FRET measurements from a single cell. Stac3 binds very weakly to the amino terminal (NT) peptide (B) that includes the CaM-binding segment, NSCaTE. Both the I-II (C) and the II-III loop peptides (D) also showed little or no FRET binding. By contrast, the III-IV loop peptide bound appreciably to stac3 (E). (F) Transfer of CI module of CaV1.3 onto CaV2.3 confers stac modulation. Format as in Figure 1—figure supplement 1A. In the absence of stac2, CaV2.3/1.3 CI exhibits robust CDI triggered by local Ca2+ isolated using 10 mM BAPTA (middle subpanel). Stac2 abolishes CDI of CaV2.3/1.3 CI (right subpanel).

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  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).

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

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  Figure 4.. Stac enhances the PO of CaV1.3. (A) A general four-state scheme for stac and CaM modulation. (1) CaV1.3S devoid of CaM and stac possess a low baseline PO (PO/E). (2) Without stac, apoCaM binding to CaV1.3S upregulates baseline PO (PO/A). Baseline PO of CaV1.3S bound to stac in the absence (configuration 3, PO/E*) and the presence of apoCaM (configuration 4, PO/A*) are unknown. (B) Schematic outlines three mechanistic possibilities for stac binding to CaV1 and their functional outcomes. Scenario 1, stac uniformly suppresses PO of CaV1 (PO/E) and abolishes CDI. Scenario 2, apoCaM tunes baseline PO of CaV1 despite concurrent stac binding. Stac, nonetheless, abrogates CDI. Scenario 3, stac uniformly upregulates the baseline PO of CaV1 and abolishes CDI (PO/A). (C) Top, cartoon shows the canonical CaV1.3S variant with a high apoCaM binding affinity. Single-channel analysis of recombinant CaV1.3S in the absence (middle) and presence of stac (bottom). In both panels, the unitary Ba2+ currents during voltage-ramp are shown between −50 mV and +40 mV (slanted gray lines, GHK fit). Robust CaV1.3 openings are detected in the absence and presence of stac. (D) Average single-channel PO-voltage…

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  Figure 4—figure supplement 1.. Extended data show that stac2 preferentially biases a high PO gating mode for CaV1.3. (A) Cartoon schematizes CaV1.3S. (B) Left, 10 sequential single-channel trials of CaV1.3S under endogenous levels of CaM illustrate high PO gating mode with rare sojourns into a low PO gating mode. Here, traces show Ba2+ currents elicited every 12 s in response to a voltage ramp shown between -40 mV and +40 mV. Right, diary plot displays single trial average PO computed for the voltage-range -30 mV ≤ V ≤ +25 mV (P-O (-30 ≤ V ≤ 25)). Dashed line discriminates traces to low PO (red shaded area) or high PO (gray shaded area) categories as previously established. (C) Average PO at each voltage calculated for all traces within the high PO range (gray region in (B)) and the low PO range (red shaded region in (B)). These relationships estimate the PO-V relationship for high PO and low PO gating modes. (D–E) In the presence of stac2, CaV1.3S preferentially adopts the high PO gating configuration. Format as in (B–C).

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  Figure 4—figure supplement 2.. Extended data show that stac2 and CaM enhance the PO of CaV1.3RNA-edited variant via discreet transitions into a high PO gating mode. (A) Cartoon schematizes CaV1.3RNA-edited. RNA-editing results in a methionine substitution of the central isoleuicine residue, resulting in sharply diminished CaM binding. Functionally, CaM binding enhances the PO of CaV1.3RNA-edited. (B–C) Under endogenous levels of CaM, CaV1.3RNA-edited exhibits a uniformly low PO as evident from individual trials. The average PO – V relation is consistent with that for the low PO gating mode. Format as in Figure 4—figure supplement 1B–C. (D–E) CaM overexpression enhances the PO of CaV1.3RNA-edited variant. Exemplar traces depict channels switching between discrete high and low PO gating modes. A PO-V relationship for high PO and low PO gating modes is evident. Format as in Figure 4—figure supplement 1B–C. (F–G) Similar to CaM, stac2 overexpression also enhances the PO of CaV1.3RNA-edited variant by stabilizing the high PO gating mode. Exemplar traces depict channels largely adopting the high gating mode. Format as in Figure 4—figure supplement 1B–C.

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  Figure 4—figure supplement 3.. Extended data show that both stac2 and CaM enhance the PO of CaV1.3L variant. (A) Cartoon schematizes CaV1.3L. The long-splice variant includes a distal carboxy-tail (DCT) that interacts with the IQ domain and competitively displaces CaM. (B–C) Under endogenous levels of CaM, CaV1.3L exhibits a uniformly low PO as evident from individual trials. The average PO – V relation is consistent with that for the low PO gating mode. Format as in Figure 4—figure supplement 1B–C. (D–E) CaM overexpression enhances the PO of CaV1.3L variant. Exemplar traces depict channels switching between discrete high and low PO gating modes. Format as in Figure 4—figure supplement 1B–C. (F–G) Similar to CaM, stac2 overexpression also enhances the PO of CaV1.3L variant by stabilizing the high PO gating mode. Exemplar traces depict channels largely adopting the high gating mode. Format as in Figure 4—figure supplement 1B–C.

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  Figure 5.. Stac U-domain is a minimal effect domain for suppression of CaV1 CDI. (A–C) To localize an effector motif for stac2, CDI of CaV1.2-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 CaV1.2-CaM in the presence of C1 (A), and SH3-SH3 (C) domains. Co-expression of the linker-region is sufficient to suppress CDI of CaV1.2-CaM (B). Format as in Figure 1A. (D) Bar graph summarizes population data for CaV1.2-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 CaV1.2-CaM (F) and CaV1.3-CaM (G). Format as in Figure 1A. (H) Bar graph displays population data for CDI of CaV1.2-CaM and CaV1.3-CaM in the presence of U-domain. Each bar,…

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  Figure 5—figure supplement 1.. Extended data demonstrate that the U-motif is a minimal domain for suppressing CDI of CaV1.2 and CaV1.3. (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 CaV1.2-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 CaV1.2-CaM, fully recapitulating the effect of stac on CaV1 channels (D). The well-conserved minimal U-motif from stac2 is also sufficient to fully suppress CDI of CaV1.2-CaM (E). Format as in Figure 1—figure supplement 1A. Each symbol, mean ±S.E.M. of r300 values. (F) Cartoon shows CaV1.3S-CaM. (G) Stac2 U-domain also diminished CDI of CaV1.3S-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 CaV1.3S-CaM (Figure 3—figure supplement 1E).