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

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8 figures, 1 table and 1 additional file

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Figure 1 with 1 supplement

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Stac specifically abolishes Ca²⁺/CaM-regulation in Ca_V1 channels.

(A) Stac2 diminishes CDI of Ca_V1.2. Left, cartoon schematic shows Ca_V1.2. 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 (r_Ca and r_Ba) and CDI = 1 – r_Ca/r_Ba. (B) Bar graph displays population data of CDI₃₀₀ for Ca_V1.2 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 Ca_V1.3_S, 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 Ca_V1.4_43* assessed in response to +20 mV test pulse. Format as in (A) and (B). (**G–H**) Stac2 spares CDF of Ca_V2.1, as evaluated using a prepulse protocol. An isolated test pulse to 0 mV elicits Ca²⁺ currents with biphasic activation (gray, G). With a + 20 mV prepulse, channels are partially facilitated and the slow component of activation is reduced (red, G). The area between the two current traces (ΔQ), divided by τ_slow, yields facilitation (g). Bar graph plots, CDF = g_Ca – g_BaH). Each bar, mean ±S.E.M from specified number of cells (n). (**I–J**) Stac2 spares CDI of Ca_V2.2 assessed in response to +30 mV test pulse. Here, CDI is evaluated following 800 ms of depolarization to accommodate slow inactivation kinetics, yielding CDI₈₀₀. Format as in (A) and (B). (**K–L**) Stac2 spares CDI of Ca_V2.3. Format as in (A) and (B). (**M–N**) Stac2 spares CDI of Na_V1.4. Both in the absence and presence of stac, Na_V1.4 peak currents decline following a Ca²⁺ step (rose fit) (M). Gray dots, peak currents before uncaging. CDI = 1 – average peak I_Na of last three to four responses after Ca²⁺ uncaging / peak I_Na before uncaging. Bar graph plots maximal CDI observed with Ca²⁺ steps > 5 μM (N). Each bar, mean ±S.E.M.

https://doi.org/10.7554/eLife.35222.002

Figure 1—figure supplement 1

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Extended data highlight selectivity of stac in modulating Ca_V1 versus Ca_V2 and Na_V1 channels.

(A) Stac1-3 isoforms strongly diminish CDI of Ca_V1.2 (schematized in the far left subpanel). In the absence of stac, Ca_V1.2 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 Ca_V1.3. Format as in (A). (C) Stac2 co-expression abolishes CDI of Ca_V1.4_43* alternatively spliced variant. Format as in (A). (D) Stac2 does not alter CDF of Ca_V2.1. 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 0 mV has a markedly blunted slow component (Figure 1G). The area between the two current traces (ΔQ), divided by τ_slow, yields the parameter g, an estimate of facilitation triggered by the prepulse. Here, g plotted as a function of prepulse potentials reveals a U-shaped dependence of Ca²⁺-dependent facilitation. Facilitation is similar in the absence (middle subpanel) and presence of stac2 (right subpanel). (E) Stac2 does not suppress CDI of Ca_V2.2. The steady-state extent of inactivation in Ca²⁺ (red) and Ba²⁺ (black), here, is estimated by the metric r₈₀₀, the ratio of current remaining at 800 ms to peak current during a depolarizing pulse, plotted as a function of voltage. Robust CDI is detected under both control conditions (middle subpanel) and stac2 co-expression (right subpanel). (F) Similarly, CDI of Ca_V2.3 is not greatly affected. Format as in (A). (G) Stac2 spares CDI of Na_V1.4. Mean data for CDI are plotted versus Ca²⁺-step amplitude. CDI = 1 − average peak I_Na of last three to four responses after Ca²⁺uncaging/peak I_Na before uncaging. Symbols, mean ±S.E.M. of ∼four uncaging events compiled from denoted number of cells (n).

https://doi.org/10.7554/eLife.35222.003

Figure 2 with 1 supplement

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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-Ca_V1.3 channels in the presence of auxiliary β_2A and α₂δ subunits. (B) Cartoon shows FRET pairs, CFP-stac3 with YFP-CI, YFP-PCI, and YFP-IQ of Ca_V1.3. (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, K_a,EFF, of stac2 binding to major channel intracellular domains. (**E–F**) Transferring Ca_V1.3_S CI to Ca_V2.3 (Ca_V2.3/1.3 CI) unveils latent stac2-mediated suppression of CDI. Format as in Figure 1A – B.

https://doi.org/10.7554/eLife.35222.004

Figure 2—figure supplement 1

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Systematic FRET 2-hybrid scan of major intracellular loops of Ca_V1.3 with stac.

(A) Schematic depicts design of YFP-tagged Ca_V1.3 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 Ca_V1.3 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 Ca_V1.3 onto Ca_V2.3 confers stac modulation. Format as in Figure 1—figure supplement 1A. In the absence of stac2, Ca_V2.3/1.3 CI exhibits robust CDI triggered by local Ca²⁺ isolated using 10 mM BAPTA (middle subpanel). Stac2 abolishes CDI of Ca_V2.3/1.3 CI (right subpanel).

https://doi.org/10.7554/eLife.35222.005

Figure 3 with 1 supplement

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

(**A–B**) Stac2 suppresses CDI of Ca_V1.3_S tethered to CaM. In contrast, fusion of CaM protects Ca_V1.3_S from competitive inhibitors such as CaM₁₂₃₄. Format as in Figure 1A–B. (**C–D**) Stac2 suppresses CDI of Ca_V1.2 tethered to CaM. Format as in Figure 1A–B. (E) FRET 2-hybrid assay shows the high-affinity interaction of YFP-tagged Ca_V1.3 CI to CFP-tagged CaM with relative dissociation constant K_d,EFF ~4000 ± 291 D_free units. (F) Co-expression of untagged CaM₁₂₃₄ with FRET pairs YFP-tagged Ca_V1.3 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 Ca_V1.3 CI with CFP-tagged CaM, yielding an identical E_A-D_free relationship to that in the absence of stac3 (E).

https://doi.org/10.7554/eLife.35222.006

Figure 3—figure supplement 1

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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 Ca_V1.3_S. Left, cartoon schematizes Ca_V1.3_S variant. Right, CDI of Ca_V1.3_S 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 Ca_V1.3_S upon co-expression of CaM₁₂₃₄. Format as in Figure 1—figure supplement 1A. (C) Fusion of CaM_WT protects Ca_V1.3_S from CaM₁₂₃₄, a competitive inhibitor of CDI. Exemplar currents show robust CDI of Ca_V1.3_S-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 Ca_V1.3_S-CaM despite overexpression of CaM₁₂₃₄. Format as in Figure 1—figure supplement 1A. These results demonstrate that CaM-fusion can protect Ca_V1.3 from competitive inhibition of CDI. (**E–F**) Co-expression of stac2 (E) and stac3 (F) abolishes CDI of Ca_V1.3_S-CaM. (G) Stac2 diminishes CDI of Ca_V1.3 despite CaM overexpression. (H) Population data of r₃₀₀ demonstrate that stac2 abolishes CDI of Ca_V1.2 with CaM tethered to the carboxy-terminus (Ca_V1.2-CaM).

https://doi.org/10.7554/eLife.35222.007

Figure 4 with 3 supplements

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Stac enhances the P_O of Ca_V1.3.

(A) A general four-state scheme for stac and CaM modulation. (1) Ca_V1.3_S devoid of CaM and stac possess a low baseline P_O (P_O/E). (2) Without stac, apoCaM binding to Ca_V1.3_S upregulates baseline P_O (P_O/A). Baseline P_O of Ca_V1.3_S bound to stac in the absence (configuration 3, P_O/E*) and the presence of apoCaM (configuration 4, P_O/A*) are unknown. (B) Schematic outlines three mechanistic possibilities for stac binding to Ca_V1 and their functional outcomes. Scenario 1, stac uniformly suppresses P_O of Ca_V1 (P_O/E) and abolishes CDI. Scenario 2, apoCaM tunes baseline P_O of Ca_V1 despite concurrent stac binding. Stac, nonetheless, abrogates CDI. Scenario 3, stac uniformly upregulates the baseline P_O of Ca_V1 and abolishes CDI (P_O/A). (C) Top, cartoon shows the canonical Ca_V1.3_S variant with a high apoCaM binding affinity. Single-channel analysis of recombinant Ca_V1.3_S 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 Ca_V1.3 openings are detected in the absence and presence of stac. (D) Average single-channel P_O-voltage relationship for Ca_V1.3_S obtained from multiple patches in the absence (gray) and presence of stac2 (blue). Error bars indicate ±S.E.M. for specified number of patches and 80–150 stochastic records per patch. (E) Histogram shows distribution of single-trial average P_O ( $\bar{P}$ _O) for the voltage range -30 mV ≤ V ≤ +25 mV under control (top), stac-bound (middle), and CaM-bound (bottom) conditions. $\bar{P}$ _O-distribution is bimodal in the absence of stac corresponding to high P_O (gray) and low P_O (rose) gating modes. With stac, $\bar{P}$ _O-distribution is largely restricted to the high P_O mode. (**F–H**) Single-channel analysis of a recombinant Ca_V1.3_RNA-edited variant reveals a marked upregulation in the baseline P_O in the presence of stac compared with control conditions in which apoCaM preassociation is weak. Absent stac or CaM, single-trial $\bar{P}$ _O-distribution is restricted to the low P_O limits, With either stac or CaM, the high P_O gating mode re-emerges. Format as in (**C–E**). (**I–K**) Stac also upregulates the baseline P_O of Ca_V1.3_L, an alternatively spliced variant, by stabilizing the high P_O gating configuration. Format as in (**C–E**).

https://doi.org/10.7554/eLife.35222.008

Figure 4—figure supplement 1

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Extended data show that stac2 preferentially biases a high P_O gating mode for Ca_V1.3.

(A) Cartoon schematizes Ca_V1.3_S. (B) Left, 10 sequential single-channel trials of Ca_V1.3_S under endogenous levels of CaM illustrate high P_O gating mode with rare sojourns into a low P_O 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 P_O computed for the voltage-range -30 mV ≤ V ≤ +25 mV ( ${\bar{P}}_{O}$ (-30 ≤ V ≤ 25)). Dashed line discriminates traces to low P_O (red shaded area) or high P_O (gray shaded area) categories as previously established. (C) Average P_O at each voltage calculated for all traces within the high P_O range (gray region in (B)) and the low P_O range (red shaded region in (B)). These relationships estimate the P_O-V relationship for high P_O and low P_O gating modes. (**D–E**) In the presence of stac2, Ca_V1.3_S preferentially adopts the high P_O gating configuration. Format as in (**B–C**).

https://doi.org/10.7554/eLife.35222.009

Figure 4—figure supplement 2

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Extended data show that stac2 and CaM enhance the P_O of Ca_V1.3_RNA-edited variant via discreet transitions into a high P_O gating mode.

(A) Cartoon schematizes Ca_V1.3_RNA-edited. RNA-editing results in a methionine substitution of the central isoleuicine residue, resulting in sharply diminished CaM binding. Functionally, CaM binding enhances the P_O of Ca_V1.3_RNA-edited. (**B–C**) Under endogenous levels of CaM, Ca_V1.3_RNA-edited exhibits a uniformly low P_O as evident from individual trials. The average P_O – V relation is consistent with that for the low P_O gating mode. Format as in Figure 4—figure supplement 1B–C. (**D–E**) CaM overexpression enhances the P_O of Ca_V1.3_RNA-edited variant. Exemplar traces depict channels switching between discrete high and low P_O gating modes. A P_O-V relationship for high P_O and low P_O gating modes is evident. Format as in Figure 4—figure supplement 1B–C. (**F–G**) Similar to CaM, stac2 overexpression also enhances the P_O of Ca_V1.3_RNA-edited variant by stabilizing the high P_O gating mode. Exemplar traces depict channels largely adopting the high gating mode. Format as in Figure 4—figure supplement 1B–C.

https://doi.org/10.7554/eLife.35222.010

Figure 4—figure supplement 3

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Extended data show that both stac2 and CaM enhance the P_O of Ca_V1.3_L variant.

(A) Cartoon schematizes Ca_V1.3_L. 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, Ca_V1.3_L exhibits a uniformly low P_O as evident from individual trials. The average P_O – V relation is consistent with that for the low P_O gating mode. Format as in Figure 4—figure supplement 1B–C. (**D–E**) CaM overexpression enhances the P_O of Ca_V1.3_L variant. Exemplar traces depict channels switching between discrete high and low P_O gating modes. Format as in Figure 4—figure supplement 1B–C. (**F–G**) Similar to CaM, stac2 overexpression also enhances the P_O of Ca_V1.3_L variant by stabilizing the high P_O gating mode. Exemplar traces depict channels largely adopting the high gating mode. Format as in Figure 4—figure supplement 1B–C.

https://doi.org/10.7554/eLife.35222.011

Figure 5 with 2 supplements

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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.

https://doi.org/10.7554/eLife.35222.012

Figure 5—figure supplement 1

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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).

https://doi.org/10.7554/eLife.35222.013

Figure 5—figure supplement 2

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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).

https://doi.org/10.7554/eLife.35222.014

Figure 6 with 2 supplements

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Synthetic U-domain peptide is sufficient for physiological perturbations.

(A) Schematic illustrates pipette dialysis of custom synthesized U-domain peptide in Ca_V1.2 heterologously expressed in HEK293 cells, a strategy that emulates acute elevation of cytosolic stac2 levels. (**B–C**) Exemplar traces and population data confirm that pipette dialysis of U-domain suppresses CDI of recombinant Ca_V1.2 in HEK293 cells. Format as in Figure 1A–B. Control relation in (C) is duplicated from Figure 1B. (**D–F**) Pipette dialysis of U-domain abolishes CDI of endogenous L-type current in freshly dissociated ventricular myocytes from adult guinea pigs as evident from exemplar traces and bar graph summary of population data. To eliminate T-type current, the cells were depolarized to −40 mV for a period of 100 ms. Format as in (**A-–C**). (G) Exemplar action potential traces of aGPVMs paced at 0.5 Hz with (blue) and without (black) 0.5 μM U-domain in the internal solution. In the presence of U-domain, the action potentials are markedly prolonged (blue shaded area) consistent with a loss of CDI of native L-type current. (H) Complement of cumulative distribution (P(*APD*₈₀ >t) of action potential durations (*APD*₈₀) obtained in the presence (blue) and absence (black) of U-domain in the internal solution.

https://doi.org/10.7554/eLife.35222.015

Figure 6—figure supplement 1

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Baseline expression of stac in cardiac myocytes.

(A) HEK293 transfected with stac1-3 (top) and immunostained with DAPI (cyan) and stac1 (red, top) or stac2 (red, bottom) antibodies confirm selectivity of stac antibodies. (B) Population analysis shows the complement of the cumulative distribution of fluorescence intensities ( $P (F > \hat{F})$ ) from 80 to 100 individual HEK293 cells stained with either stac1 (top) or stac2 (bottom) antibodies. Black line, untransfected cells. Data from cells transfected with recombinant stac1, stac2, or stac3 are shown in green, red, and blue, respectively. When probed with stac1 antibody, only cells transfected with exogenous stac1 show appreciable fluorescence above background. Similarly, when probed with stac2 antibody, only cells transfected with exogenous stac2 show fluorescence intensities above background level. (C) Acutely dissociated aGPVM immunostained with α-actinin (green) and stac1 (red, left) and stac2 (red, right). Images representative of three fields of views for stac1 and seven fields of view for stac2. (D) Stac2 immunostaining reveals weak t-tubular localization in aGPVM. Image reproduced from panel C. Yellow line, line-scan used in (E). (E) Fluorescence intensity of stac2 (red, top) and α-actinin (green, bottom) across the line-scan shown in (D) from left to right. Vertical lines correspond to location of the peak fluorescence intensity for α-actinin. (F) Spatial Fourier transform of line scans subpanel shows prominent spatial frequency of 0.52 µm⁻¹ with both α-actinin (green) and stac2 (red). (G) Immunoblotting with stac2 antibody shows stac2 expression,~50 kDa band, in HEK293 cells transfected with recombinant stac2, but not in untransfected HEK293 cells. Analysis of freshly dissociated aGPVM confirms basal stac2 expression. Image representative of five independent biological replicates.

https://doi.org/10.7554/eLife.35222.016

Figure 6—figure supplement 2

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Pipette dialysis of U-motif as a peptide abolishes CDI of both recombinant and native L-type currents in ventricular myocytes.

(A) Cartoon schematic shows pipette dialysis of U-motif into HEK293 cells expressing recombinant Ca_V1.2. (B) Pipette dialysis of minimal effector stac2 U-motif suppresses Ca²⁺/CaM regulation. Format as in Figure 1—figure supplement 1A. (C) Cartoon schematic shows pipette dialysis of U-motif into freshly dissociated adult guinea pig ventricular myocytes that feature endogenous L-type currents. (D) Population data show that endogenous L-type Ca²⁺-current in ventricular myocytes elicits strong CDI. Format as in Figure 1—figure supplement 1A. Each symbol, mean ±S.E.M. from six cells. (E) In comparison to control conditions, pipette dialysis of minimal effector stac2 U-motif strongly diminishes Ca²⁺/CaM regulation of endogenous Ca²⁺ current in ventricular myocytes. Format as in Figure 1—figure supplement 1A.

https://doi.org/10.7554/eLife.35222.017

Figure 7 with 1 supplement

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Fhf uses an allosteric mechanism to abrogate Ca²⁺-feedback of Na_V1.4.

(A) Structural comparison of Na_V1.5 CI (green) in the presence of CaM alone (cyan, left) or both CaM (cyan) and fhf1b (purple). Fhf binding changes baseline conformation of CaM on Na_V1 CI. (**B–C**) Co-expression of fhf1b abolishes CDI in Na_V1.4 evoked via Ca²⁺ photo-uncaging. Format as in Figure 1M–N. Control data are reproduced from Figure 1M–N for comparison. (**D–E**) In sharp contrast, strong overexpression of fhf1b does not alter CDI of Ca_V1.3_S. Format as in Figure 1A–B. Control data reproduced from Figure 1D for comparison. (**F–G**) Fhf1 suppresses CDI of Na_V1.4 tethered to CaM. Fusion of CaM protects Na_V1.4 from competitive inhibitors such as CaM₁₂₃₄ (G). Format as in Figure 1M–N. (H) Structure of Ca_V1.1 upstream CI elements (blue) composed of dual vestigial EF hands and preIQ segments isolated from cryo-EM structure of Ca_V1.1 (PDBID, 5GJV). This domain is the primary interface for stac interaction in the Ca_V1 CI. (I) Structural overlay of upstream CI elements of Ca_V1.1 (PDBID, 5GJV) and Na_V1.5 (PDBID, 4DCK) shows highly conserved dual vestigial EF hand segments while the fhf binding site is structurally divergent. (J) The divergence in the fhf binding interface in Ca_V1.1 in comparison to Na_V1.5 would introduce a steric clash that prohibits fhf binding to Ca_V channels.

https://doi.org/10.7554/eLife.35222.018

Figure 7—figure supplement 1

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Extended data confirm the selectivity of fhf in modulating Na_V versus Ca_V channels.

(A) Population data show that Fhf1 abolishes CDI of Na_V1.4 channels. Mean data for CDI are plotted versus Ca²⁺-step amplitude. Symbols, mean ±S.E.M. of around four uncaging events compiled from denoted number of cells. (B) Fhf does not alter CDI of Ca_V1.3_S demonstrating selectivity of fhf1 in modulating Na_V channels. Strong CDI of Ca_V1.3_S persists in the presence of fhf as confirmed by population data of r₃₀₀ values. (C) Na_V1.4 fused to CaM (Na_V1.4-CaM) exhibits strong CDI identical to untethered channels as illustrated by identical fit reproduced from (A). (D) Fusion of CaM_WT protects Na_V1.4 from CaM₁₂₃₄, a competitive inhibitor of CDI. Exemplar currents shows robust CDI of Na_V1.4-CaM in the presence of CaM₁₂₃₄. Format as in Figure 1M and (A). (E) Fhf1b suppresses CDI of Na_V1.4-CaM confirming the dominance of fhf over CaM in modulation of Na_V1.4. Format as in (A).

https://doi.org/10.7554/eLife.35222.019

Figure 8 with 1 supplement

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Engineering synthetic modulation of Ca_V1 channels.

(A) Left, schematic shows the atomic structure of Mona SH3 domain in complex with RxxK motif. Right, sequence alignment outlines strategy for insertion of RxxK motif into Ca_V1.3, yielding Ca_V1.3_RxxK to confer synthetic suppression of Ca_V1.3 CDI by Mona SH3. (**B–C**) Ca_V1.3_S expressed with and without Mona SH3 shows full CDI, confirming that wildtype Ca_V1.3 CDI is insensitive to Mona SH3. Format as in Figure 1A–B. Control data are reproduced from Figure 1D for comparison. (**D–E**) Mona SH3 strongly diminishes CDI of Ca_V1.3_RxxK. Format as in Figure 1A–B. (F) Cartoon summarizes selective modulation of Ca²⁺/CaM signaling to Ca_V1, and Na_V1 channels with CaM, stac, and fhf.

https://doi.org/10.7554/eLife.35222.020

Figure 8—figure supplement 1

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Extended data demonstrate feasibility of engineering synthetic modulators of CaM signaling to Ca_V1.3.

(A) Ca_V1.3_S is insensitive to the SH3 domain of Mona/gads protein. Population data of inactivation r₃₀₀ values show strong CDI of Ca_V1.3_S in the presence of Mona SH3. Format as in Figure 1—figure supplement 1A. (B) In sharp contrast, the introduction of RxxK motif to Ca_V1.3 upstream CI elements (left subpanel) confers synthetic modulation of CaM signaling by Mona SH3. Middle, population r₃₀₀ values show CDI of Ca_V1.3_RxxK under endogenous levels of CaM. Right, co-expression of Mona SH3 abolishes CDI of Ca_V1.3_RxxK, illustrating the feasibility of engineering synthetic regulatory proteins that abolish Ca²⁺/CaM signaling to selected targets. Format as in Figure 1—figure supplement 1A.

https://doi.org/10.7554/eLife.35222.021

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (rat)	β_2A	PMID: 1370480	GenBank: M80545
Gene (rat)	α₂δ	PMID: 8107966	NCBI: NM_012919
Gene (Oryctolagus cuniculus)	Ca_V1.2	PMID: 1718988	NCBI: NM_001136522
Gene (rat)	Ca_V1.3_S	PMID: 20139964	GenBank: AF370009.1
Gene (rat)	Ca_V1.3_MQDY	PMID: 24120865, 22284185
Gene (human)	Ca_V1.4_43*	PMID: 22069316		Laboratory of Dr. Soong Tuck Wah (National University of Singapore)
Gene (human)	Ca_V2.1 splice variant 37a(EFa) with 43⁺/44^-/47^-	PMID: 12451115
Gene (human)	Ca_V2.2	PMID: 1321501, 10233069	GenBank: M94172.1
Gene (rat)	Ca_V2.3	PMID: 8388125, 18400181	NCBI: NM_019294.2
Gene (rat)	Na_V1.4	PMID: 2175278
Gene (human)	stac1	Origene	NCBI: NP_003140.1
Gene (mouse)	stac2	Origene	NCBI: NP_666140.1
Gene (human)	stac3	Origene	NCBI: NP_659501.1
Gene (human)	fhf	PMID: 8790420		Laboratory of Dr. Jeremy Nathans (Johns Hopkins University).
Gene (human)	Mona SH3	PMID: 12773374		Synthesized by Genscript based on sequence in publication
Peptide (mouse)	U-peptide	this paper		Peptide sequence KVDPVYETLRYGTSLALM NRSS synthesized by Genscript
Competent cells (E. coli)	DH5α	Invitrogen: 18265017
Cell line (human)	HEK293	other	RRID: CVCL_0045
Biological sample (guinea pig)	aGPVM	PMID: 24076394		Generated from Hartley strain guinea pigs
Antibody	anti-stac1	Abcam: ab181157		1:100
Antibody	anti-stac2	Abcam: ab156080		IHC – 1:100 WB – 1:250
Antibody	anti-α-actinin	Sigma Aldrich: A7811	RRID: AB_476766	1:300
Antibody	goat anti- rabbit IgG Alexa Fluor 594	Abcam: ab150080	RRID: AB_2650602	1:1000
Antibody	goat anti-mouse IgG1 Alexa Fluor 488	Thermo Fischer: A21121	RRID: AB_2535764	1:1000
Antibody	Goat Anti-Rabbit IgG (H + L)	Jackson ImmunoResearch: 111-035-144	RRID: AB_2307391	1:10,000
Recombinant DNA reagent	Ca_V1.3_L	PMID: 20139964		Engineered from Ca_V1.3_S and human long distal carboxyl tail (NCBI: NM_000718)
Recombinant DNA reagent	Ca_V2.3/1.3 CI	PMID: 24441587
Recombinant DNA reagent	Ca_V1.3- CaM_WT	PMID: 24441587
Recombinant DNA reagent	Ca_V1.2- CaM_WT	PMID: 15087548
Recombinant DNA reagent	Na_V1.4-CaM	this paper		Engineered by fusing CaM_WT carboxy-tail of Na_V1.4
Recombinant DNA reagent	Ca_V1.3_RxxK	this paper		Engineered from Ca_V1.3_S
Recombinant DNA reagent	CFP-stac3	this paper		stac3 was cloned into CFP vector with NotI and XbaI
Recombinant DNA reagent	YFP-Ca_V1.3 CI	PMID: 23591884
Recombinant DNA reagent	YFP-Ca_V1.3 PCI	PMID: 23591884
Recombinant DNA reagent	YFP-Ca_V1.3 IQ	PMID: 23591884
Recombinant DNA reagent	Ven-C1	this paper		stac2 C1 was cloned into Venus vector (PMID: 26997269) with NotI and XbaI
Recombinant DNA reagent	Ven-linker region	this paper		stac2 linker region was cloned into Venus vector (PMID: 26997269) with NotI and XbaI
Recombinant DNA reagent	Ven-SH3-SH3	this paper		stac2 SH3-SH3 was cloned into Venus vector (PMID: 26997269) with NotI and XbaI
Recombinant DNA reagent	Ven-U-motif	this paper		stac2 U-motif was cloned into Venus vector (PMID: 26997269) with NotI and XbaI
Recombinant DNA reagent	stac2 (KVD/AAA)	this paper		Quickchange PCR with stac2
Recombinant DNA reagent	stac2 (PVY/AAA)	this paper		Quickchange PCR with stac2
Recombinant DNA reagent	stac2 (ETL/AAA)	this paper		Quickchange PCR with stac2
Recombinant DNA reagent	stac2 (RYG/AAA)	this paper		Quickchange PCR with stac2
Recombinant DNA reagent	stac2 (TSL/AAA)	this paper		Quickchange PCR with stac2
Recombinant DNA reagent	stac2 (NRS/AAA)	this paper		Quickchange PCR with stac2
Recombinant DNA reagent	stac2 (S/A)	this paper		Quickchange PCR with stac2
Sequence-based reagent	Ven-C1 forward primer	this paper		cttctcgcggccgc tatgaccgaa atga gcgagaa
Sequence-based reagent	Ven-C1 reverse primer	this paper		tcagaattctagattat tgctggt gggagatctc
Sequence-based reagent	Ven-linker region forward primer	this paper		cttctcgcggccgcta catctttt cgacgcaact
Sequence-based reagent	Ven-linker region reverse primer	this paper		tcagaattctagatta gtacatg ggccccacg
Sequence-based reagent	Ven-SH3-SH3 forward primer	this paper		cttctcgcggccgc ttcctacgt cgccctct
Sequence-based reagent	Ven-SH3-SH3 reverse primer	this paper		tcagaattctagattat cagatctct gccaaggag
Sequence-based reagent	Ven-U-motif forward primer	this paper		cttctcgcggccgctaagg tggac ccagtttatga
Sequence-based reagent	Ven-U-motif reverse primer	this paper		tcagaattctagattag ctggaa cggttcatcag
Sequence-based reagent	stac2 (KVD/AAA) sense	this paper		ctactgggaccagcgg ggcggcgg ccccagt ttatgagacgc
Sequence-based reagent	stac2 (KVD/AAA) antisense	this paper		gcgtctcataaactggg gccgccgc cccgctgg tcccagtag
Sequence-based reagent	stac2 (PVY/AAA) sense	this paper		ccagcgggaaggtggac gcagc tgctgagacgct gcgctatg
Sequence-based reagent	stac2 (PVY/AAA) antisense	this paper		catagcgcagcgtctc agcagct gcgtccacc ttcccgctgg
Sequence-based reagent	stac2 (ETL/AAA) sense	this paper		ggtggacccagttt atgcggcgg cgcgct atggcacctcc
Sequence-based reagent	stac2 (ETL/AAA) antisense	this paper		ggaggtgccatagcgc gccgcc gcataaact gggtccacc
Sequence-based reagent	stac2 (RYG/AAA) sense	this paper		cccagtttatgagacgc tggccgc tgccacctcc ctggcactgatg
Sequence-based reagent	stac2 (RYG/AAA) antisense	this paper		catcagtgccaggg aggtggca gcggccagc gtctcataaactggg
Sequence-based reagent	stac2 (TSL/AAA) sense	this paper		acgctgcgctatgg cgccgccgc ggcactga tgaaccg
Sequence-based reagent	stac2 (TSL/AAA) antisense	this paper		cggttcatcagtgc cgcggcggc gccatag cgcagcgt
Sequence-based reagent	stac2 (NRS/AAA) sense	this paper		gatgtgctgctga agctggcagcg gccatc agtgccagggaggtg
Sequence-based reagent	stac2 (NRS/AAA) antisense	this paper		cacctccctggc actgatggccgc tgcc agcttcagcagcacatc
Sequence-based reagent	stac2 (S/A) sense	this paper		cactgatgaacc gttccgccttc agcag cacatctg
Sequence-based reagent	stac2 (S/A) antisense	this paper		cagatgtgctgctga aggcgg aacggttca tcagtg
Software, algorithm	PyMOL	http://www.pymol.org/	RRID: SCR_000305

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Jacqueline Niu
Ivy E Dick
Wanjun Yang
Moradeke A Bamgboye
David T Yue
Gordon Tomaselli
Takanari Inoue
Manu Ben-Johny

(2018)

Allosteric regulators selectively prevent Ca²⁺-feedback of Ca_V and Na_V channels

eLife 7:e35222.

https://doi.org/10.7554/eLife.35222

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Stac specifically abolishes Ca2+/CaM-regulation in CaV1 channels.

Extended data highlight selectivity of stac in modulating CaV1 versus CaV2 and NaV1 channels.

Stac interacts with the channel carboxy-terminus.

Systematic FRET 2-hybrid scan of major intracellular loops of CaV1.3 with stac.

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

Extended data show that stac acts concurrently with CaM.

Stac enhances the PO of CaV1.3.

Extended data show that stac2 preferentially biases a high PO gating mode for CaV1.3.

Extended data show that stac2 and CaM enhance the PO of CaV1.3RNA-edited variant via discreet transitions into a high PO gating mode.

Extended data show that both stac2 and CaM enhance the PO of CaV1.3L variant.

Stac U-domain is a minimal effect domain for suppression of CaV1 CDI.

Extended data demonstrate that the U-motif is a minimal domain for suppressing CDI of CaV1.2 and CaV1.3.

Systematic alanine scanning mutagenesis of minimal U-motif reveals structural determinants for stac modulation of CaV1.

Synthetic U-domain peptide is sufficient for physiological perturbations.

Baseline expression of stac in cardiac myocytes.

Pipette dialysis of U-motif as a peptide abolishes CDI of both recombinant and native L-type currents in ventricular myocytes.

Fhf uses an allosteric mechanism to abrogate Ca2+-feedback of NaV1.4.

Extended data confirm the selectivity of fhf in modulating NaV versus CaV channels.

Engineering synthetic modulation of CaV1 channels.

Extended data demonstrate feasibility of engineering synthetic modulators of CaM signaling to CaV1.3.

Transparent reporting form

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Stac specifically abolishes Ca²⁺/CaM-regulation in Ca_V1 channels.

Extended data highlight selectivity of stac in modulating Ca_V1 versus Ca_V2 and Na_V1 channels.

Systematic FRET 2-hybrid scan of major intracellular loops of Ca_V1.3 with stac.

Stac enhances the P_O of Ca_V1.3.

Extended data show that stac2 preferentially biases a high P_O gating mode for Ca_V1.3.

Extended data show that stac2 and CaM enhance the P_O of Ca_V1.3_RNA-edited variant via discreet transitions into a high P_O gating mode.

Extended data show that both stac2 and CaM enhance the P_O of Ca_V1.3_L variant.

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

Extended data demonstrate that the U-motif is a minimal domain for suppressing CDI of Ca_V1.2 and Ca_V1.3.

Systematic alanine scanning mutagenesis of minimal U-motif reveals structural determinants for stac modulation of Ca_V1.

Fhf uses an allosteric mechanism to abrogate Ca²⁺-feedback of Na_V1.4.

Extended data confirm the selectivity of fhf in modulating Na_V versus Ca_V channels.

Engineering synthetic modulation of Ca_V1 channels.

Extended data demonstrate feasibility of engineering synthetic modulators of CaM signaling to Ca_V1.3.