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The molecular basis of the inhibition of CaV1 calcium-dependent inactivation by the distal carboxy tail

Open AccessPublished:March 02, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100502
      Ca2+/calmodulin-dependent inactivation (CDI) of CaV channels is a critical regulatory process that tunes the kinetics of Ca2+ entry for different cell types and physiologic responses. CDI is mediated by calmodulin (CaM), which is bound to the IQ domain of the CaV carboxy tail. This modulatory process is tailored by alternative splicing such that select splice variants of CaV1.3 and CaV1.4 contain a long distal carboxy tail (DCT). The DCT harbors an inhibitor of CDI (ICDI) module that competitively displaces CaM from the IQ domain, thereby diminishing CDI. While this overall mechanism is now well described, the detailed interactions required for ICDI binding to the IQ domain are yet to be elucidated. Here, we perform alanine-scanning mutagenesis of the IQ and ICDI domains and evaluate the contribution of neighboring regions to CDI inhibition. Through FRET binding analysis, we identify functionally relevant residues within the CaV1.3 IQ domain and the CaV1.4 ICDI and nearby A region, which are required for high-affinity IQ/ICDI binding. Importantly, patch-clamp recordings demonstrate that disruption of this interaction commensurately diminishes ICDI function resulting in the re-emergence of CDI in mutant channels. Furthermore, CaV1.2 channels harbor a homologous DCT; however, the ICDI region of this channel does not appear to appreciably modulate CaV1.2 CDI. Yet coexpression of CaV1.2 ICDI with select CaV1.3 splice variants significantly disrupts CDI, implicating a cross-channel modulatory scheme in cells expressing both channel subtypes. In all, these findings provide new insights into a molecular rheostat that fine-tunes Ca2+-entry and supports normal neuronal and cardiac function.

      Keywords

      Abbreviations:

      apoCaM (Ca2+ free calmodulin), Ca2+ (calcium), CaM (calmodulin), CaV (voltage-gated calcium channel), CDI (calcium/CaM-dependent inactivation), CFP (cyan fluorescent protein), DCRD (distal C-terminal regulatory domain), DCT (distal carboxy tail), FR (fret ratio), FRET (fluorescence resonance energy transfer), ICDI (inhibitor of CDI), iTL (individually transformed Langmuir), PCRD (proximal C-terminal regulatory domain), YFP (yellow fluorescent protein)
      L-type voltage-gated calcium channels (CaV1.1–1.4) are an important conduit for extracellular Ca2+ entry into many excitable cells including cardiac myocytes, neurons, smooth muscle, and skeletal muscle (
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      ). Second, CaV1.2 appears to harbor a highly homologous ICDI region (
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      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ), yet CDI for this channel is known to be robust, both when evaluated as full-length channels in heterologous expression system and in primary cells where the carboxy-tail containing ICDI is believed to be cleaved off the channel (
      • Alseikhan B.A.
      • DeMaria C.D.
      • Colecraft H.M.
      • Yue D.T.
      Engineered calmodulins reveal the unexpected eminence of Ca2+ channel inactivation in controlling heart excitation.
      ,
      • Peterson B.Z.
      • DeMaria C.D.
      • Adelman J.P.
      • Yue D.T.
      Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L- type calcium channels.
      ,
      • Zuhlke R.D.
      • Pitt G.S.
      • Deisseroth K.
      • Tsien R.W.
      • Reuter H.
      Calmodulin supports both inactivation and facilitation of L-type calcium channels.
      ,
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ). As such, the function of the ICDI module within CaV1.2 channels remains unclear. Third, the inhibition of CDI by ICDI is the result of competitive binding by apoCaM versus ICDI with the channel IQ domain (
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ,
      • Singh A.
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      • Fritsch R.
      • Sinnegger-Brauns M.J.
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      • Engel J.
      • Romanin C.
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      Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.
      ). In addition to diminishing CDI, the displacement of apoCaM results in a dramatic decrease in baseline channel open probability (
      • Adams P.J.
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      • Dick I.E.
      • Inoue T.
      • Yue D.T.
      Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation.
      ). Fourth, adding to the richness the modulatory role of ICDI, RNA editing, and/or fluctuations in cytosolic CaM concentrations can tune the extent of this competition, enabling different degrees of CDI tailored to specific cell types or physiologic states (
      • Adams P.J.
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      • Dick I.E.
      • Inoue T.
      • Yue D.T.
      Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation.
      ,
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      RNA editing of the IQ domain in Ca(v)1.3 channels modulates their Ca(2)(+)-dependent inactivation.
      ). Importantly, pathologic changes to this system may be linked to altered CaM concentrations in Parkinson’s disease and heart failure (
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      Calcium signaling and neurodegenerative diseases.
      ,
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      Parkinson's disease is associated with altered expression of CaV1 channels and calcium-binding proteins.
      ), and mutations within the ICDI of CaV1.4 channels are known to be causative of congenital stationary night blindness (
      • Singh A.
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      • Koschak A.
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      C-terminal modulator controls Ca2+-dependent gating of CaV1.4 L-type Ca2+ channels.
      ,
      • Hoda J.C.
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      Congenital stationary night blindness type 2 mutations S229P, G369D, L1068P, and W1440X alter channel gating or functional expression of Ca(v)1.4 L-type Ca2+ channels.
      ,
      • Strom T.M.
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      • Apfelstedt-Sylla E.
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      • Weber B.H.
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      • Ruther K.
      • Drescher B.
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      • Meindl A.
      An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness.
      ). Thus, the modulation of CDI by ICDI stands as a critical and robust mechanism for adapting channel regulation to select cell types and conditions. Moreover, as the number of known pathogenic mutations within LTCCs continues to grow, the ability to map these mutations to a locus with known mechanistic impact would enable rapid insight into the pathogenesis of LTCC channelopathies.
      Although the overall competitive nature of ICDI regulation of L-type channels is now well established (
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ,
      • Singh A.
      • Gebhart M.
      • Fritsch R.
      • Sinnegger-Brauns M.J.
      • Poggiani C.
      • Hoda J.C.
      • Engel J.
      • Romanin C.
      • Striessnig J.
      • Koschak A.
      Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.
      ), the precise binding interfaces involved in this regulation are yet to be identified. This gap in understanding is critical as mutations in the carboxy tail of LTCCs result in neurological disease (
      • Singh A.
      • Hamedinger D.
      • Hoda J.C.
      • Gebhart M.
      • Koschak A.
      • Romanin C.
      • Striessnig J.
      C-terminal modulator controls Ca2+-dependent gating of CaV1.4 L-type Ca2+ channels.
      • Hoda J.C.
      • Zaghetto F.
      • Koschak A.
      • Striessnig J.
      Congenital stationary night blindness type 2 mutations S229P, G369D, L1068P, and W1440X alter channel gating or functional expression of Ca(v)1.4 L-type Ca2+ channels.
      ,
      • Strom T.M.
      • Nyakatura G.
      • Apfelstedt-Sylla E.
      • Hellebrand H.
      • Lorenz B.
      • Weber B.H.
      • Wutz K.
      • Gutwillinger N.
      • Ruther K.
      • Drescher B.
      • Sauer C.
      • Zrenner E.
      • Meitinger T.
      • Rosenthal A.
      • Meindl A.
      An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness.
      ). Furthermore, a residue-level analysis may shed light upon structural differences between the ICDI domain of CaV1.3 and the homologous segment of CaV1.2 that engender differential functional regulation. To characterize the landscape of the IQ/ICDI interaction of L-type channels, here we undertook systematic alanine scanning mutagenesis of both IQ and ICDI domains. Through live-cell FRET two-hybrid binding assays and electrophysiological analysis, we identified several novel hotspots on both IQ and ICDI segments that mediate a high-affinity interaction and are functionally relevant for CDI inhibition. Systematic analysis of these mutations revealed a strong inverse correlation between the strength of CDI and the binding affinity of the ICDI domain for the IQ segment, as predicted for a competitive inhibitor (
      • Bazzazi H.
      • Ben Johny M.
      • Adams P.J.
      • Soong T.W.
      • Yue D.T.
      Continuously tunable Ca(2+) regulation of RNA-edited CaV1.3 channels.
      ,
      • Ben Johny M.
      • Yang P.S.
      • Bazzazi H.
      • Yue D.T.
      Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels.
      ,
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ). Thus, we have identified residues that alter binding in a functionally relevant manner. Moreover, similar critical residues were identified in adjacent regions, defining a comprehensive interface map of the IQ/ICDI interaction. Finally, extending our analysis to CaV1.2 channels, we found that the ICDI module binds to the CaV1.2 IQ domain with a reduced affinity and that this binding is insufficient to cause more than a nominal change in the CDI of full-length channels. However, the ICDI from CaV1.2 is capable of binding the CaV1.3 IQ region with high affinity, resulting in a much larger decrease in CDI of these channels. Given the propensity of the carboxy tail of CaV1.2 to exist as a separate peptide within myocytes and neurons (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ,
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ,
      • Gomez-Ospina N.
      • Tsuruta F.
      • Barreto-Chang O.
      • Hu L.
      • Dolmetsch R.
      The C terminus of the L-type voltage-gated calcium channel Ca(V)1.2 encodes a transcription factor.
      ,
      • Schroder E.
      • Byse M.
      • Satin J.
      L-type calcium channel C terminus autoregulates transcription.
      ), these findings raise the prospect of a cross-channel feedback scheme in some cell types. Overall, these results elucidate the detailed binding interface between components of the carboxy tail of L-type Ca2+ channels, lending new insight into normal and pathologic channel regulation.

      Results

      Identification of critical residues within the IQ domain necessary for ICDI binding

      To identify key residues that support a high-affinity IQ/ICDI interaction, we undertook systematic alanine substitution of the IQ domain and evaluated both the relative binding affinity and the strength of ICDI-mediated inhibition of CaM regulation. Importantly, the ICDI domains of both CaV1.3 and CaV1.4 are highly homologous and have been shown to interact with IQ domains in a similar manner evoking similar functional effects (
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ,
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ,
      • Wahl-Schott C.
      • Baumann L.
      • Cuny H.
      • Eckert C.
      • Griessmeier K.
      • Biel M.
      Switching off calcium-dependent inactivation in L-type calcium channels by an autoinhibitory domain.
      ). Even so, the ICDI domain from CaV1.4 (ICDI1.4) has a greater binding affinity for the IQ domains of both CaV1.3 and CaV1.4, with FRET binding assays yielding more robust measurements with enhanced signal-to-noise ratio as compared with ICDI1.3 (
      • Sang L.
      • Dick I.E.
      • Yue D.T.
      Protein kinase A modulation of CaV1.4 calcium channels.
      ). We therefore focus on this canonical ICDI motif for our studies. However, robust expression of the holo-CaV1.4 channel in recombinant systems is notoriously challenging, largely due to their diminutive open probability (
      • Adams P.J.
      • Ben-Johny M.
      • Dick I.E.
      • Inoue T.
      • Yue D.T.
      Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation.
      ,
      • Doering C.J.
      • Hamid J.
      • Simms B.
      • McRory J.E.
      • Zamponi G.W.
      Cav1.4 encodes a calcium channel with low open probability and unitary conductance.
      ). We therefore chose to explore the interaction between the IQ domain of CaV1.3 channels (IQ1.3) and ICDI1.4. To this end, we utilized a chimeric channel in which the DCT of CaV1.4 is spliced onto the backbone of CaV1.3 (CaV1.3Δ/DCT1.4) (Fig. 1A), which has previously proven useful in dissecting the mechanisms underlying ICDI modulation of the channel (
      • Adams P.J.
      • Ben-Johny M.
      • Dick I.E.
      • Inoue T.
      • Yue D.T.
      Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation.
      ,
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ). This chimera furnishes a strong IQ/ICDI interaction coupled with a robust functional readout, enabling quantitative analysis.
      Figure thumbnail gr1
      Figure 1Identifying the hotspots on the IQ region required for ICDI binding and function. A, cartoon depicting the chimeric CaV1.3Δ/DCT1.4 channel. B, exemplar whole-cell recording showing minimal inactivation in response to a 10 mV step depolarization in Ba2+ (black) or Ca2+ (red) for CaV1.3Δ/DCT1.4 demonstrating the function of ICDI1.4. Ba2+ current is scaled to enable comparison of the kinetics of the two traces. Red scale bar refers to the Ca2+ trace and black corresponds to the Ba2+ trace here and throughout. C, exemplar whole-cell recording of the truncated CaV1.3Δ channel, lacking the DCT containing ICDI. Robust CDI is seen as the strong decay of the Ca2+ current (red) as compared to the Ba2+ current. D, FRET two-hybrid assay of IQ/ICDI interaction. FRET binding partners are displayed on the left, with star indicating the locus of alanine mutations. Strong binding was measured for wild-type preIQ3-IQ-A1.3 with ICDI1.4 (black), while the mutation F[−2]A (blue) decreased the binding affinity compared with WT (gray). E, summary of Ka values for mutant Venus-preIQ3-IQ-A1.3 versus Cerulean-ICDI1.4 peptides measured with FRET two-hybrid assay as in panel D. Alanine was systematically substituted into the IQ region of the Venus-preIQ3-IQ-A1.3 peptide, with the identity of the amino acid displayed on the top and bottom of the bar graph such that the canonical “I” of the IQ region is given position 0. The dashed line indicates the WT value and the blue bar corresponds to F[−2]A displayed in panel D. Data are displayed as mean ± SE. F, exemplar patch clamp data corresponding to F[−2]A in CaV1.3Δ/DCT1.4 demonstrating a partial recovery of CDI due to the mutation. G, average CDI values for each mutation, colors correspond to the data in other panels (ANOVA summary analysis indicates a likelihood of significant difference at a level of p < 0.0001. Post-hoc Dunnett’s multiple comparisons versus WT indicated as ∗∗∗∗p < 0.0001; ∗p < 0.05). Data is displayed as mean ± SD. H, CDI and binding data for the mutations is well fit by Equation , validating the competitive mechanism. Colored circles correspond to the colored data in the figure panels.
      To begin, we confirm the functional impact of ICDI in our chimeric channel by evaluating the extent of CDI in HEK293 cells. Indeed, CDI is entirely abolished in CaV1.3Δ/DCT1.4, as seen by the identical Ba2+ and Ca2+ current decay in response to a depolarizing pulse (Fig. 1B). However, removal of DCT1.4 restores robust CDI, as seen by the rapid decay of the Ca2+ current (Fig. 1C, red). In contrast, when Ba2+ (which binds poorly to CaM) is used as the charge carrier, there is minimal inactivation (Fig. 1C, black). We therefore define the extent of CDI as the ratio of Ca2+ current remaining after 300 ms of depolarization versus Ba2+ current at the same time point.
      We next utilized a FRET 2-hybrid binding assay (
      • Erickson M.G.
      • Alseikhan B.A.
      • Peterson B.Z.
      • Yue D.T.
      Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.
      ,
      • Erickson M.G.
      • Liang H.
      • Mori M.X.
      • Yue D.T.
      FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation.
      ) to evaluate the relative strength of interactions between the IQ and ICDI regions. FRET binding pairs were constructed by tagging Cerulean fluorescent protein to ICDI1.4, and Venus fluorescent protein to PreIQ3-IQ-A1.3, a peptide that includes IQ1.3 as well as ∼30 residues upstream (PreIQ3) and ∼150 residues downstream (A-region) of the IQ domain (Fig. 1D, Fig. S1). Both PreIQ3 and A regions were included initially to ensure that all likely interacting residues were included. Strong binding was detected between the Venus-PreIQ3-IQ-A1.3 and Cerulean-ICDI1.4, as can be appreciated by the steep FRET binding curve determined by the FRET Ratio (FR) of each cell plotted as a function of the free donor concentration (Cerulean tagged ICDI1.4) (Fig. 1D, black). After calibration, the FRET binding curve for WT Venus-PreIQ3-IQ-A1.3 versus Cerulean-ICDI1.4 yielded a Ka of 21.4 μM−1. To identify key residues that support a high-affinity IQ/ICDI interaction, we undertook systematic alanine substitution of the IQ domain and evaluated the effect on binding affinity in our FRET assay. Within IQ1.3, we substituted each residue with an alanine or, at loci where the wild-type channel featured an alanine, we replaced the residue with a threonine. For identification of each residue, the canonical isoleucine is assigned position 0. Application of our FRET assay to each mutated peptide identified three residues, Y[−5]A, F[−2]A, and F[+4]A, which severely perturbed the IQ/ICDI interaction (Fig. 1E, Fig. S1). Focusing on F[−2]A, FRET binding produced a shallower curve as compared with WT (Fig. 1D, blue versus gray), resulting in a Ka of 5.8 μM−1 (Fig. 1E, blue). Introducing this mutation into the chimeric channel resulted in a partial rescue of CDI (Fig. 1F), indicating that this interaction site is functionally relevant. However, the IQ domain substitutions Y[−5]A and F[+4]A, which also had a marked effect on Ka, resulted in minimal CDI rescue (Fig. 1, F and G, Fig. S2). Importantly, these residues also serve as anchors for apoCaM binding to the CaV1.3 IQ domain, resulting in weak baseline CDI even in the absence of the ICDI domain (Table S1) (
      • Bazzazi H.
      • Ben Johny M.
      • Adams P.J.
      • Soong T.W.
      • Yue D.T.
      Continuously tunable Ca(2+) regulation of RNA-edited CaV1.3 channels.
      ). Of note, this apoCaM effect may also underly the apparent increase in binding affinity demonstrated by several of the mutations, where a decrease in apoCaM binding would enhance the apparent binding of the IQ/ICDI regions due to altered competition with endogenous CaM.
      In order to confirm the functional relevance of each binding loci identified, we turned to a previously described analysis known as individually transformed Langmuir (iTL) analysis (
      • Ben Johny M.
      • Yang P.S.
      • Bazzazi H.
      • Yue D.T.
      Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels.
      ). This approach allows us to rigorously correlate relative changes in binding with functional changes in CDI. As iTL was initially derived to evaluate the binding interfaces critical to CaM-mediated channel regulation (
      • Bazzazi H.
      • Ben Johny M.
      • Adams P.J.
      • Soong T.W.
      • Yue D.T.
      Continuously tunable Ca(2+) regulation of RNA-edited CaV1.3 channels.
      • Ben Johny M.
      • Yang P.S.
      • Bazzazi H.
      • Yue D.T.
      Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels.
      ), we adjust the model to reflect the competitive binding scheme between CaM and ICDI (
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ). To do so, we first account for the ambiguity caused by binding sites, which are important for both apoCaM and ICDI binding. We therefore incorporate both the IQ domain’s intrinsic affinity for apoCaM (Ka-CaM) and that for the ICDI segment (Ka-ICDI), the competitive inhibitor, by adjusting our measured Ka-ICDI such that:
      KaICDIcorr=KaCaMWTKaCaMmutKaICDI
      (1)


      Where KaCaMWT is the apoCaM binding affinity of WT PreIQ3-IQ-A1.3, and KaCaMmut represents the apoCaM binding affinity of each mutant peptide, values which were previously measured (
      • Bazzazi H.
      • Ben Johny M.
      • Adams P.J.
      • Soong T.W.
      • Yue D.T.
      Continuously tunable Ca(2+) regulation of RNA-edited CaV1.3 channels.
      ) and are listed in Table S1. This compensation remains valid provided that the local concentration of ICDI is much greater than Kd-ICDI ([ICDI]>> 1/Ka-ICDI). With this adjustment made, CDI can be defined by a modified Langmuir function as follows:
      CDICDImax=[apoCaM]KaCaM1+[apoCaM]KaCaM+[ICDI]KaICDIcorr
      (2)


      where CDI is the strength of CDI under endogenous levels of CaM; CDImax is the CDI in saturating concentrations of CaM (Table S1); Ka-CaM is the association constant for apoCaM binding to PreIQ3-IQ-A1.3; [apoCaM] is the free apoCaM concentration in the cell; and [ICDI] is the effective local concentration of ICDI (see Supporting information for full derivation). Equation 2 predicts an inverse correlation between KaICDIcorr and relative CDI, which we can fit to our data (Fig. 1H). For channels containing WT IQ1.3 and ICDI1.4 domains, the strong binding between IQ and ICDI results in minimal CDI, as seen by the black data point on the plot. In contrast, CaV1.3Δ channels, which lack an ICDI domain such that KaICDIcorr=0 by definition, display large CDI values (cyan). The F[−2]A mutation, which produced a partial restoration of CDI, resides in an intermediate position (blue). Overall, our data can be well fit by Equation 2 (Fig. 1H), confirming the functional relevance of the identified residues in a competitive model.

      Alanine scanning of the ICDI domain reveals complementary hotspots

      Having identified several critical residues within the IQ domain required for ICDI binding, we next probed the ICDI for critical determinates of binding to the IQ. In order to scan a more extensive segment of the channel, we made triple alanine substitutions for every three contiguous residues within the ICDI domain. The ICDI domain has previously been localized to amino acids 1868 to 1956 of the CaV1.3 DCT (
      • Wahl-Schott C.
      • Baumann L.
      • Cuny H.
      • Eckert C.
      • Griessmeier K.
      • Biel M.
      Switching off calcium-dependent inactivation in L-type calcium channels by an autoinhibitory domain.
      ,
      • Singh A.
      • Hamedinger D.
      • Hoda J.C.
      • Gebhart M.
      • Koschak A.
      • Romanin C.
      • Striessnig J.
      C-terminal modulator controls Ca2+-dependent gating of CaV1.4 L-type Ca2+ channels.
      ,
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ). We therefore undertook our alanine scan on this segment of the channel. Importantly, this region includes the distal C-terminal regulatory domain (DCRD), which was previously identified as playing an important role in the ICDI-mediated inhibition of CDI (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ,
      • Singh A.
      • Gebhart M.
      • Fritsch R.
      • Sinnegger-Brauns M.J.
      • Poggiani C.
      • Hoda J.C.
      • Engel J.
      • Romanin C.
      • Striessnig J.
      • Koschak A.
      Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.
      ,
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ,
      • Liu N.
      • Yang Y.
      • Ge L.
      • Liu M.
      • Colecraft H.M.
      • Liu X.
      Cooperative and acute inhibition by multiple C-terminal motifs of L-type Ca(2+) channels.
      ). Finally, we included a selective V[1907]A mutation, as this amino acid change has previously been shown to dramatically alter the function of ICDI (
      • Lieb A.
      • Scharinger A.
      • Sartori S.
      • Sinnegger-Brauns M.J.
      • Striessnig J.
      Structural determinants of CaV1.3 L-type calcium channel gating.
      ). To evaluate the effect of these alanine substitutions on IQ/ICDI binding, we again utilized our FRET two-hybrid binding assay, pairing Venus-PreIQ3-IQ-A1.3 with Cerulean-ICDI1.4 (Fig. 2A, Fig. S3). Indeed, measured Ka values revealed multiple hotspots within the ICDI domain, with a wide range of binding affinities with the IQ containing peptide (Fig. 2B). Interestingly several mutations, largely toward either end of the peptide, appeared to modestly increase in Ka. However, these changes may reflect a structural stabilization of the isolated ICDI peptide by promoting helicity of end regions and may not necessarily yield a corresponding change in intact channels. As such, we did not pursue further analysis of these mutants. The two mutation sites displayed in gray (KQE[1911]AAA and YSD[1941]AAA) were not evaluated as they failed to express. Notably, the effects of the hotspots on the ICDI domain were significantly larger than those observed within the IQ domain (Fig. 2B versus Fig. 1E).
      Figure thumbnail gr2
      Figure 2Identifying the hotspots on ICDI1.4 required for ICDI binding and function. A, FRET binding partners are displayed on the left, with the star indicating the locus of alanine mutations within ICDI1.4. Compared with WT (gray line), IAD[1914]AAA (red) and SLV[1886]AAA (green) significantly decreased binding affinity between the FRET pairs to differing degrees. B, summary of Ka values for Venus-PreIQ3-IQA1.3 versus mutant Cerulean-ICDI1.4 peptides measured with FRET two-hybrid as in panel A. Alanine was systematically substituted three amino acids at a time into Cerulean-ICDI1.4, with the identity of the amino acids displayed on the top of the bar graph. Amino acids labeled in gray (KQE, YSD) were not evaluated due to poor expression. The dashed line indicates the WT value and the colored bars correspond to data in other panels of the figure here and throughout. Data are displayed ±SE. C, summary of CDI values measured for CaV1.3Δ/DCT1.4 harboring mutations corresponding to mutations that produced large effects on binding affinity in B. Gray dashed line represents the robust CDI of the channel without ICDI1.4, while the black dashed line indicates the nominal CDI of WT CaV1.3Δ/DCT1.4. Data are displayed ±SD (ANOVA summary analysis indicates a likelihood of significant difference at a level of p < 0.0001. Post-hoc Dunnett’s multiple comparisons versus WT indicated as ∗∗∗∗p < 0.0001; ∗p < 0.05). D, exemplar whole-cell recordings of CaV1.3Δ/DCT1.4, demonstrating a significant restoration of CDI due to IAD[1914]AAA and near complete CDI due to SLV[1886]AAA. E, robust fit of CDI (C) and FRET binding data (B) to Equation , using identical parameters as in H. Light gray circles indicate data from , while dark gray and colored circles indicate mutations within ICDI corresponding to B and C, with green and red circles indicating exemplars shown in other panels for reference. The PKA phosphorylation site Ser1883 is plotted as the orange circle.
      In order to correlate loss of binding affinity with function, we measured the CDI of those mutations that exhibited a large change in binding affinity (Fig. 2C, Fig. S4). As predicted, mutations that resulted in a significant loss of IQ/ICDI binding also exhibited a corresponding restoration of CDI. Focusing on two examples, IAD[1914]AAA moderately reduced IQ/ICDI binding (Fig. 2, A and B, red), while introduction of the same mutations into our chimeric channel enabled a partial restoration of CDI (Fig. 2, C and D, red). On the other hand, SLV[1886]AAA displayed a drastic reduction in IQ/ICDI binding (Fig. 2, A and B green), and CDI was fully restored to the level seen in CaV1.3Δ (Fig. 2, C and D green). Importantly, all identified loci are well fit by our Langmuir function, such that the same set of Equation 2 parameters describes both the IQ region and ICDI (Fig. 2E). Of note, as mutations in ICDI do not affect the binding of apoCaM, the correction factor for Ka is no longer required, and KaICDIcorr=KaICDI. Having identified critical loci within ICDI, we note that one of these amino acids (S[1866]) has previously been identified as a phosphorylation site, which reduces the binding affinity of ICDI1.4 by about tenfold, while increasing CDI of CaV1.3Δ/DCT1.4 (
      • Sang L.
      • Dick I.E.
      • Yue D.T.
      Protein kinase A modulation of CaV1.4 calcium channels.
      ). We therefore included the results of phosphorylation at this site in our analysis (Fig. 2E, orange). Indeed, phosphorylation of this amino acid results in a change in binding affinity, which correlates with CDI according to the same Langmuir function. Thus, we have identified numerous residues within ICDI1.4, which are critical determinants of a functional competition between ICDI and apoCaM for the IQ region of CaV1.3.

      The role of the A region in the IQ/ICDI interaction

      While our analysis identified several functionally relevant IQ domain loci, the impact of these mutations was far less than those identified within the ICDI (Fig. 1 versus Fig. 2). This suggests that additional regions outside the IQ domain may contribute to ICDI binding. In order to identify such regions, we generated truncated variations of our Venus-PreIQ3-IQ-A1.3 construct and paired them with Cerulean-ICDI1.4 in the FRET two-hybrid binding assays (Fig. 3A, Fig. S5). We began by removing the PreIQ3, and found no change in FRET binding, indicating that all relevant interaction loci are contained within the IQ and A regions (Fig. 3B, blue). However, removal of the IQ domain, leaving only the A region intact, resulted in a complete loss of FRET binding (Fig. 3B, open circles). Likewise, the IQ region alone displayed no binding with ICDI (Fig. 3C), suggesting that both the IQ and the A region are necessary for interaction with ICDI. In order to further localize the critical interaction sites, we undertook successive truncation of the vernus-IQ-A1.3 peptide (Fig. 3D). FRET measurements demonstrated minimal effect of truncations up to 34 amino acids from the end of the A region, as demonstrated by the strong binding of Venus-IQ-A1.3Δ34 with ICDI1.4 (Fig. 3E, green). However, our next truncation, Venus-IQ-A1.3Δ28, exhibited a marked decrease in FRET binding (Fig. 3, E and F, red) Thus, the 34 residues immediately downstream of the IQ domain critically augment ICDI binding. Of note, this region includes the previously identified proximal C-terminal regulatory domain (PCRD), which is reported to play an important role in the ICDI interaction (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      • Singh A.
      • Gebhart M.
      • Fritsch R.
      • Sinnegger-Brauns M.J.
      • Poggiani C.
      • Hoda J.C.
      • Engel J.
      • Romanin C.
      • Striessnig J.
      • Koschak A.
      Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.
      ,
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ,
      • Liu N.
      • Yang Y.
      • Ge L.
      • Liu M.
      • Colecraft H.M.
      • Liu X.
      Cooperative and acute inhibition by multiple C-terminal motifs of L-type Ca(2+) channels.
      ). Having identified a subset of the A region, which is vital to ICDI binding, we again undertook systematic alanine substitutions, replacing each of the three contiguous residues with three alanine residues and undertook our FRET-2-hybrid binding assay (Fig. 3G). Disruptions in binding were identified as the result of a number of mutations, spanning both the previously identified PCRD region and a previously unidentified region upstream of this motif (Fig. 3, H and I, Fig. S6). Thus, both the IQ and the distal A region of the channel are required for high-affinity interaction with ICDI.
      Figure thumbnail gr3
      Figure 3Elucidating the role of the A region for ICDI interaction. A, cartoon depicting FRET interacting pairs for panels B and C. Portions of the PreIQ3-IQ-A1.3 were paired with ICDI1.4 to identify critical regions. B, IQ-A1.3 is sufficient to support robust FRET (blue), indicating that the pre-IQ3 region is not involved in the interaction. However, the A region alone is not sufficient to support binding (open circles). C, Ka values for each channel fragment indicate that neither IQ1.3 nor A1.3 is sufficient for robust binding with ICDI1.4; however, both regions are necessary as indicated by the robust binding of IQ-A1.3 with ICDI1.4. Data are displayed as mean ± SE here and throughout. D, cartoon depicting FRET interacting pairs for panels E and F. Cerulean-ICDI1.4 was paired with various truncations of Venus-PreIQ3-IQ-A1.3. The blue box shows the sequence of the A region and illustrates the truncation strategy. E and F, truncating up to 34 amino acids distal to the IQ region (Δ34) had nominal effects on binding (green); however, removal of six additional amino acids (Δ28) dramatically reduced binding affinity (red). G, cartoon depicting FRET interacting pairs for panels H and I. Alanine substitutions were made within the A region, as indicated by the yellow star. H, mutation of KPY[1626]AAA within the A region had no effect on binding (cyan), while ALQ[1635]AAA (purple) diminished the binding affinity between IQ-A1.3 and ICDI1.4. I, summary of Ka values for alanine mutations in the A region indicate multiple critical amino acids.

      The functional relevance of ICDI in CaV1.2 channels

      Similar to CaV1.3 and CaV1.4, CaV1.2 channels also feature a highly homologous ICDI segment, argued to function as a channel inhibitor (
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ,
      • Fuller M.D.
      • Emrick M.A.
      • Sadilek M.
      • Scheuer T.
      • Catterall W.A.
      Molecular mechanism of calcium channel regulation in the fight-or-flight response.
      ) or as a transcriptional factor (
      • Gomez-Ospina N.
      • Tsuruta F.
      • Barreto-Chang O.
      • Hu L.
      • Dolmetsch R.
      The C terminus of the L-type voltage-gated calcium channel Ca(V)1.2 encodes a transcription factor.
      ,
      • Schroder E.
      • Byse M.
      • Satin J.
      L-type calcium channel C terminus autoregulates transcription.
      ). We therefore considered the impact of ICDI1.2 on both CaV1.2 and CaV1.3 channels. We interrogated the binding of Cerulean-ICDI1.2 with Venus-PreIQ3-IQ-A1.2 via FRET two-hybrid (Fig. 4, A and B) and found that the interaction is significantly weaker than the prototypic Venus-PreIQ3-IQ-A1.2 and Cerulean-ICDI1.4 interaction (Fig. 4B versus Fig. 1D). However, when paired with Venus-PreIQ3-IQ-A1.3, binding with ICDI1.2 is significantly larger, and only about half that of the strong binding of ICDI1.4 (Fig. 4C). Thus, it appears that ICDI1.2 is poised to have a larger effect in the context of CaV1.3 channels as compared with its native channel backbone. Nonetheless, the limited binding observed between Cerulean-ICDI1.2 and Venus-PreIQ3-IQ-A1.2 prompted us to evaluate the possibility of a functional role for ICDI1.2 within CaV1.2 channels. Interestingly, truncation of CaV1.2 at the known carboxy-tail cleavage site (
      • Hulme J.T.
      • Konoki K.
      • Lin T.W.
      • Gritsenko M.A.
      • Camp D.G.
      • Bigelow D.J.
      • Catterall W.A.
      Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle.
      ) for this channel (CaV1.2Δ1800) resulted in a minimal, yet statistically significant (p ≤ 0.05), increase in CDI (Fig. 4, D and F, Fig. S7). Next, to test the effect of ICDI1.2 on CaV1.3 channels, we replaced the native ICDI1.3 of CaV1.3long channels with ICDI1.2. Indeed, the loss of CDI surpassed that of CaV1.2 channels (Fig. 4E), as predicted based on the stronger PreIQ3-IQ-A1.3/ICDI1.2 interaction (Fig. 4C). In fact, the CDI exhibited by CaV1.3-ICDI1.2 channels was not statistically different than the CDI measured in the native CaV1.3long splice variant (Fig. 4F).
      Figure thumbnail gr4
      Figure 4Residual functionality of ICDI1.2 in modulating CaV channels. A, cartoon depicting FRET interacting pairs for panels B and C. Venus-PreIQ3-IQ-A1.2 or Venus-PreIQ3-IQ-A1.3 was paired with Cerulean-ICDI1.2 in order to evaluate the role of the ICDI contained within CaV1.2 channels. B, Venus-PreIQ3-IQ-A1.2 displays moderate binding with Cerulean-ICDI1.2 (blue) as compared with the robust binding between Venus-PreIQ3-IQ-A1.3 and Cerulean-ICDI1.4 which is reproduced as the gray line for comparison. C, Venus-PreIQ3-IQ-A1.3 is able to bind strongly with Cerulean-ICDI1.2. D, Left, channel cartoon with cleavage site indicated by the yellow arrowhead. Right, exemplar data demonstrating that truncation of CaV1.2 at the cleavage site has a minor effect on CDI. E, Left, channel cartoon indicating introduction of ICDI1.2 into the CaV1.3 channel backbone. Right, introduction of ICDI1.2 into CaV1.3 channels causes a large decrease in CDI. F, average CDI data demonstrating a modest but statistically significant effect of ICDI1.2 on CaV1.2 channels (blue), and a large effect on CaV1.3, which is comparable to the native effect of ICDI1.3 contained within CaV1.3long (gray). Data are plotted as mean ± SD. (CaV1.2: ∗p < 0.05 based on a Student’s t-test; CaV1.3: ANOVA summary analysis indicates a likelihood of significant difference at a level of p < 0.0001. Post-hoc Dunnett’s multiple comparisons versus CaV1.3short indicated as ∗∗∗∗p < 0.0001.) G, Left, cartoon of human CaV1.343S, with the sequence of the end of the channel displayed below. This splice variant contains a portion of the A region (blue highlight), which contains the identified hotspots required for ICDI binding (bold), as well as a short sequence of unique amino acids prior to truncation of the channel (pink). Right, exemplar whole-cell patch clamp data demonstrates robust CDI in WT CaV1.343S, which is reduced when the predicted cleavage fragment of the human CaV1.2 channel is coexpressed, or when ICDI1.4 is expressed as a peptide. H, average CDI effects for the channels described in panel G, DCT1.2 and ICDI1.4 both have significant effects on the CDI of CaV1.343S (ANOVA summary analysis indicates a likelihood of significant difference at a level of p < 0.0001. Post-hoc Dunnett’s multiple comparisons versus CaV1.343s indicated as ∗∗∗∗p < 0.0001; data are displayed ±SD). I, cartoon proposing a cross-channel effect of ICDI1.2, such that cleaved or independently transcribed DCT1.2 may modulate CaV1.343S channels.
      Multiple studies have shown that the DCT of CaV1.2, containing ICDI1.2, exists as a peptide within neurons and cardiomyocytes, either due to proteolysis (
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ,
      • Hulme J.T.
      • Konoki K.
      • Lin T.W.
      • Gritsenko M.A.
      • Camp D.G.
      • Bigelow D.J.
      • Catterall W.A.
      Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle.
      ) or as a result of alternative transcriptional initiation sites (
      • Gomez-Ospina N.
      • Tsuruta F.
      • Barreto-Chang O.
      • Hu L.
      • Dolmetsch R.
      The C terminus of the L-type voltage-gated calcium channel Ca(V)1.2 encodes a transcription factor.
      ). Moreover, it has previously been demonstrated that ICDI domains can exert their effects on L-type channels when expressed as separate peptides (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ). We therefore sought to recreate the potential interaction of select CaV1.3 channel variants with the DCT of CaV1.2. To begin, we choose the human CaV1.343S splice variant of CaV1.3, as these channels terminate just past the A region and thus lack an inherent ICDI module (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ). In addition, the inclusion of the A region within CaV1.343S has been demonstrated to be important for ICDI binding, both in functional experiments done by others (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ) and in our alanine scan of the A region (Fig. 3). We therefore generated the proteolytic product of human CaV1.2 channels (DCT1.2) and evaluated the effect of this peptide on the CDI of CaV1.343S. Indeed, consistent with previous studies (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ), coexpression of DCT1.2 significantly reduced the CDI of CaV1.343S (Fig. 4G, Fig. S7). For comparison, we also coexpressed these channels with ICDI1.4 expressed as a peptide, which we have shown has a Ka about double that of ICDI1.2 (Fig. 4C versus Fig. 1D). Indeed, ICDI1.4 results in an even larger CDI deficit when expressed with CaV1.343S (Fig. 4, G and H). Thus, the proteolytically cleaved DCT1.2 is well poised to exert a significant modulation of select CaV1.3 channel variants, such that the ambient concentrations of CaM and DCT1.2 are able to tune the CDI of CaV1.343S channels in a competitive manner (Fig. 4I).

      Discussion

      CaM regulation of CaV channels is vital to normal physiology and thus has been the subject of intense study (
      • Pitt G.S.
      • Zuhlke R.D.
      • Hudmon A.
      • Schulman H.
      • Reuter H.
      • Tsien R.W.
      Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels.
      ,
      • Peterson B.Z.
      • DeMaria C.D.
      • Adelman J.P.
      • Yue D.T.
      Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L- type calcium channels.
      ,
      • Zuhlke R.D.
      • Pitt G.S.
      • Deisseroth K.
      • Tsien R.W.
      • Reuter H.
      Calmodulin supports both inactivation and facilitation of L-type calcium channels.
      ,
      • Griessmeier K.
      • Cuny H.
      • Roetzer K.
      • Griesbeck O.
      • Harz H.
      • Biel M.
      • Wahl-Schott C.
      Calmodulin is a functional regulator of CaV1.4 l-type Ca2+ channels.
      ,
      • Chaudhuri D.
      • Issa J.B.
      • Yue D.T.
      Elementary mechanisms producing facilitation of Cav2.1 (P/Q-type) channels.
      ,
      • Kim J.
      • Ghosh S.
      • Nunziato D.A.
      • Pitt G.S.
      Identification of the components controlling inactivation of voltage-gated Ca2+ channels.
      ,
      • Lee A.
      • Zhou H.
      • Scheuer T.
      • Catterall W.A.
      Molecular determinants of Ca(2+)/calmodulin-dependent regulation of Ca(v)2.1 channels.
      ). The competitive mechanism of ICDI within select CaV1 channels forms a basis with which CaM regulation can be tuned (
      • Adams P.J.
      • Ben-Johny M.
      • Dick I.E.
      • Inoue T.
      • Yue D.T.
      Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation.
      ,
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ,
      • Singh A.
      • Hamedinger D.
      • Hoda J.C.
      • Gebhart M.
      • Koschak A.
      • Romanin C.
      • Striessnig J.
      C-terminal modulator controls Ca2+-dependent gating of CaV1.4 L-type Ca2+ channels.
      ). Numerous processes designed to modulate this regulation include splice variation, RNA editing, variations in ambient CaM concentration, and phosphorylation (
      • Adams P.J.
      • Ben-Johny M.
      • Dick I.E.
      • Inoue T.
      • Yue D.T.
      Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation.
      ,
      • Bazzazi H.
      • Ben Johny M.
      • Adams P.J.
      • Soong T.W.
      • Yue D.T.
      Continuously tunable Ca(2+) regulation of RNA-edited CaV1.3 channels.
      ,
      • Haeseleer F.
      • Williams B.
      • Lee A.
      Characterization of C-terminal splice variants of Cav1.4 Ca2+ channels in human retina.
      ,
      • Huang H.
      • Tan B.Z.
      • Shen Y.
      • Tao J.
      • Jiang F.
      • Sung Y.Y.
      • Ng C.K.
      • Raida M.
      • Kohr G.
      • Higuchi M.
      • Fatemi-Shariatpanahi H.
      • Harden B.
      • Yue D.T.
      • Soong T.W.
      RNA editing of the IQ domain in Ca(v)1.3 channels modulates their Ca(2)(+)-dependent inactivation.
      ,
      • Sang L.
      • Dick I.E.
      • Yue D.T.
      Protein kinase A modulation of CaV1.4 calcium channels.
      ). Identification of critical loci involved in this regulation is therefore key to understanding how CaM regulation may vary in different physiological and pathological states. As such, in-depth residue-level analysis not only reveals interfaces utilized by cells to tune channel regulation, but may offer targets in the search for novel regulators of the channel, which may have therapeutic benefit. In particular, the dramatically different efficacy of ICDI across channel subtypes may offer the possibility of subtype selective drug targeting, which remains challenging for CaV1 channels.
      Given the importance of understanding these interactions within the carboxy-tail of CaV1 channels, we quantified the structure–function relationship of these interactions using a variant of previously described iTL analysis (
      • Ben Johny M.
      • Yang P.S.
      • Bazzazi H.
      • Yue D.T.
      Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels.
      ). This provided a major advantage in that the quantitative agreement of our results with Equation 2 demonstrates that each identified locus is functionally relevant. This overcomes a common limitation of binding assays between channel fragments, which may identify sites that are inaccessible or inconsequential in the context of the holochannel. Moreover, by fitting to a specific Langmuir curve, we can distinguish mutations that may alter channel function through ancillary mechanisms such as transduction or altered folding of the channel. Thus, in addition to identifying critical loci, our results confirm the competitive mechanism described for ICDI modulation of CDI.
      A number of previous studies have identified regions within the carboxy tail of CaV1 channels, which are critical to the competitive mechanism of ICDI inhibition. Among these are the PCRD and DCRD regions, which were identified within CaV1.2 channels as potential interaction sites such that the DCRD region of the proteolytically cleaved CaV1.2 DCT may interact with the PCRD on the channel via electrostatic interaction with the negatively charged amino acids (
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ). Homologous regions within CaV1.3 and CaV1.4 were later shown to be important for ICDI regulation and for the interaction with modular peptides (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ,
      • Lieb A.
      • Scharinger A.
      • Sartori S.
      • Sinnegger-Brauns M.J.
      • Striessnig J.
      Structural determinants of CaV1.3 L-type calcium channel gating.
      ,
      • Singh A.
      • Gebhart M.
      • Fritsch R.
      • Sinnegger-Brauns M.J.
      • Poggiani C.
      • Hoda J.C.
      • Engel J.
      • Romanin C.
      • Striessnig J.
      • Koschak A.
      Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.
      ,
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ,
      • Liu N.
      • Yang Y.
      • Ge L.
      • Liu M.
      • Colecraft H.M.
      • Liu X.
      Cooperative and acute inhibition by multiple C-terminal motifs of L-type Ca(2+) channels.
      ). In this study, the PCRD resides within the A region and overlaps with the identified locus of critical amino acids required for high-affinity binding between the IQ-A and ICDI. Interestingly, these critical amino acids were identified both within the PCRD and upstream of the motif, arguing for a larger interacting region, which is highly conserved across CaV channels (Fig. S8). However, our binding assay also demonstrated that the A region, in itself, is insufficient for high-affinity binding, but also requires the upstream IQ region (Fig. 3A). This fits with previous findings in which neutralization of the PCRD arginines was not sufficient to prevent the ICDI inhibition of CDI (
      • Singh A.
      • Gebhart M.
      • Fritsch R.
      • Sinnegger-Brauns M.J.
      • Poggiani C.
      • Hoda J.C.
      • Engel J.
      • Romanin C.
      • Striessnig J.
      • Koschak A.
      Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.
      ), pointing to the existence of additional interaction loci. In a similar manner, our scan of the ICDI region validated the importance of the DCRD, while also identifying numerous critical interacting loci upstream of the motif (Fig. S8). Thus, this study has expanded our knowledge of the important interactions required for ICDI inhibition, providing a comprehensive map of the critical loci within the carboxy tail.
      The impact of ICDI in CaV1.3 and CaV1.4 channels has been well recognized; however, its role in CaV1.2 has been uncertain. It has been demonstrated that the truncation of CaV1.2 results in increased current density, altered voltage dependence of channel activation, and disrupted targeting of the channel to the membrane (
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ,
      • Gerhardstein B.L.
      • Gao T.
      • Bunemann M.
      • Puri T.S.
      • Adair A.
      • Ma H.
      • Hosey M.M.
      Proteolytic processing of the C terminus of the alpha(1C) subunit of L-type calcium channels and the role of a proline-rich domain in membrane tethering of proteolytic fragments.
      ,
      • Gao T.
      • Bunemann M.
      • Gerhardstein B.L.
      • Ma H.
      • Hosey M.M.
      Role of the C terminus of the alpha 1C (CaV1.2) subunit in membrane targeting of cardiac L-type calcium channels.
      ); however, no impact on CDI has been reported. Here, we find that the impact of ICDI1.2 within CaV1.2 channels is minimal, allowing cleavage of this DCT region without significant disruption of CDI. Yet ICDI1.2 is capable of causing significant disruption of CDI in the context of CaV1.3 (Fig. 4, EI) (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ). This selective effect of ICDI1.2 on CaV1.3 channels is intriguing as it represents a difficult to achieve discrimination between CaV1.2 and CaV1.3 (
      • Striessnig J.
      • Ortner N.J.
      • Pinggera A.
      Pharmacology of L-type calcium channels: Novel drugs for old targets?.
      ,
      • Huang H.
      • Ng C.Y.
      • Yu D.
      • Zhai J.
      • Lam Y.
      • Soong T.W.
      Modest CaV1.342-selective inhibition by compound 8 is beta-subunit dependent.
      ). The strong homology between these two channels has resulted in challenges to dissecting the contribution of each channel to the function of cells, which express both channel subtypes, and hinders therapeutic options for neuropsychiatric disorders, which may benefit from blockade of CaV1.3 (
      • Striessnig J.
      • Ortner N.J.
      • Pinggera A.
      Pharmacology of L-type calcium channels: Novel drugs for old targets?.
      ). As such, the interface defined in this study between the IQ-A region and ICDI may represent a promising interface with which to selectively target CaV1.3.
      The DCT of CaV1.2 is routinely cleaved in neurons and myocytes, with the cleavage product able to either remain associated with the channel (
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ) or translocate to the nucleus (
      • Gomez-Ospina N.
      • Tsuruta F.
      • Barreto-Chang O.
      • Hu L.
      • Dolmetsch R.
      The C terminus of the L-type voltage-gated calcium channel Ca(V)1.2 encodes a transcription factor.
      ). Our FRET binding data would argue that this association between CaV1.2 and the DCT may be relatively weak, leaving DCT1.2 available to other binding partners. Moreover, it has been shown that an alternate start site exists within the carboxy tail of CaV1.2, such that alternative transcription of CaV1.2 will produce a DCT peptide containing a calcium channel-associated transcription regulator (CCAT) (
      • Gomez-Ospina N.
      • Tsuruta F.
      • Barreto-Chang O.
      • Hu L.
      • Dolmetsch R.
      The C terminus of the L-type voltage-gated calcium channel Ca(V)1.2 encodes a transcription factor.
      ). Importantly, ICDI would be intact within this peptide, providing an additional source of DCT1.2 within cells. In addition, CCAT has been shown to localize to the cytosol in a Ca2+-dependent manner, providing a source of ICDI in proximity to the membrane, which can be tuned by activity (
      • Gomez-Ospina N.
      • Tsuruta F.
      • Barreto-Chang O.
      • Hu L.
      • Dolmetsch R.
      The C terminus of the L-type voltage-gated calcium channel Ca(V)1.2 encodes a transcription factor.
      ). Our FRET binding analysis (Fig. 4A) suggests that the CaV1.2 DCT is capable of binding upstream calmodulatory elements in CaV1.2, albeit weakly. Functional analysis, however, suggests only minimal effects of this segment on CaV1.2 CDI. By comparison, the CaV1.343S channel variant contains all the elements required for high-affinity binding with DCT1.2 and exhibits functional inhibition of CDI (Fig. 4G), consistent with previous studies (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ). Moreover, CaV1.343S is widely expressed in the brain, accounting for up to 39% of transcripts, and is often expressed within the same neuron type as CaV1.2 (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ). Thus, it seems likely that DCT1.2 may interact with this channel, altering the normally robust CDI. It is interesting to note, however, that while DCT1.2 is poised to modulate some CaV1.3 channels, the same cannot be said of DCT1.3. Not only is the IQ-A region of CaV1.2 suboptimal for binding to ICDI, but there is little evidence that CaV1.3 is cleaved in neurons (
      • Singh A.
      • Gebhart M.
      • Fritsch R.
      • Sinnegger-Brauns M.J.
      • Poggiani C.
      • Hoda J.C.
      • Engel J.
      • Romanin C.
      • Striessnig J.
      • Koschak A.
      Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.
      ). Thus, this cross-channel modulation may be unidirectional. Finally, since CaV1.2 and CaV1.3 often exist within the same neuron, this mode of cross-channel modulation may represent an important method for tuning CDI in different regions of the brain.

      Experimental procedures

      Molecular biology

      The rat brain CaV1.3 α1 subunit (in pcDNA6) corresponds to AF370009.1 (
      • Xu W.
      • Lipscombe D.
      Neuronal Ca(V)1.3alpha(1) L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines.
      ) and was incorporated to the mammalian expression plasmid pCDNA6 (Invitrogen) (
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ). This plasmid features a unique BglII restriction site at a locus corresponding to ∼450 amino acids upstream of the IQ domain and a unique XbaI site after the stop codon, which were used for generation of mutant and chimeric plasmids as descried below. The CaV1.2 α1 subunit (in pGW) is identical to rabbit NM001136522 (
      • Wei X.Y.
      • Perez-Reyes E.
      • Lacerda A.E.
      • Schuster G.
      • Brown A.M.
      • Birnbaumer L.
      Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels.
      ), and the CaV1.4 channel (in pcDNA3) is the human clone corresponding to NP005174.2. CaV1.3Δ/DCT1.4 was made by fusing with the DCT of the CaV1.4 α1 subunit to the CaV1.3 α1 subunit (truncated after the IQ domain), as previously described (
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ). CaV1.343S was made by PCR amplification of the channel segment between the BglII site and IQ domain with the appendage of amino acids unique to this splice variant (
      • Bock G.
      • Gebhart M.
      • Scharinger A.
      • Jangsangthong W.
      • Busquet P.
      • Poggiani C.
      • Sartori S.
      • Mangoni M.E.
      • Sinnegger-Brauns M.J.
      • Herzig S.
      • Striessnig J.
      • Koschak A.
      Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.
      ). The PCR product was then inserted into the channel via the BglII/XbaI sites.
      FRET constructs were fluorescent-tagged (either Venus or Cerulean) using similar strategies as previously described (
      • Erickson M.G.
      • Alseikhan B.A.
      • Peterson B.Z.
      • Yue D.T.
      Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.
      ). Briefly, Venus and Cerulean fluorophores (a kind gift from Dr Steven Vogel, NIH) were subcloned into the pcDNA3 vector via unique KpnI and NotI sites. The PCR-amplified channel peptides, as described in Liu et al., (
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ) were then cloned via unique NotI and XbaI sites. Mutations were introduced into the channel or FRET construct via PCR amplification or overlap extension PCR.

      Transfection of HEK293 cells

      For electrophysiology experiments, HEK293 cells were cultured on 10-cm plates, and channels were transiently transfected by a calcium phosphate protocol (
      • Peterson B.Z.
      • DeMaria C.D.
      • Adelman J.P.
      • Yue D.T.
      Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L- type calcium channels.
      ). We applied 8 μg of plasmid DNA encoding the desired pore forming α1 subunit, as well as 8 μg of β2A (M80545) and 8 μg of rat α (NM012919.2) subunits along with 3 μg of SV40 T antigen. For microscope-based FRET assays, HEK293 cells cultured on 3.5-cm culture dishes with integral No. 0 glass coverslip bottoms (In Vitro Scientific) were transiently transfected using polyethylenimine (PEI) reagent (Polysciences).

      Whole-cell patch clamp recordings

      Whole-cell recordings were obtained using an Axopatch 200A amplifier (Axon Instruments). Electrodes were pulled from borosilicate glass capillaries (World Precision Instruments), with 1 to 3 MΩ resistances, which were in turn compensated for series resistance by >60%. Currents were low-pass filtered at 2 kHz before digital acquisition at five times the frequency. A P/8 leak subtraction protocol was used. The internal solution contained (in mM): CsMeSO3, 114; CsCl, 5; MgATP, 4; HEPES (pH 7.4), 10; and BAPTA (1,2-bis(o-aminophenoxy)ethane- N,N,N’,N’-tetraacetic acid), 10; at 295 mOsm adjusted with CsMeSO3. The bath solution contained (in mM): TEA-MeSO3, 102; HEPES (pH 7.4), 10; CaCl2 or BaCl2, 40; at 305 mOsm adjusted with TEA-MeSO3. Data was analyzed using custom Matlab scripts. Inactivation was quantified as the ratio of current remaining after 300 ms (current amplitude measured at 300 ms divided by peak current amplitude) in either Ca2+ or Ba2+ (r300). CDI was then quantified as the r300 in Ca2+ subtracted from the r300 in Ba2+, measured at 10 mV for CaV1.3 channels, and 30 mV for CaV1.2.

      FRET optical imaging

      FRET two-hybrid experiments were performed on an inverted microscope as described (
      • Erickson M.G.
      • Alseikhan B.A.
      • Peterson B.Z.
      • Yue D.T.
      Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.
      ,
      • Erickson M.G.
      • Liang H.
      • Mori M.X.
      • Yue D.T.
      FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation.
      ). The bath solution was a Tyrode’s solution composed of (in mM): NaCl, 138; KCl, 4; MgCl2, 1; HEPES (pH 7.4), 10; CaCl2, 2; at 305 mOsm adjusted with glucose. Background fluorescent signals were measured from cells without expression of the fluorophores and subtracted from cells expressing the fluorophores. Concentration-dependent spurious FRET was subtracted from the raw data prior to binding-curve analysis (
      • Erickson M.G.
      • Alseikhan B.A.
      • Peterson B.Z.
      • Yue D.T.
      Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.
      ,
      • Erickson M.G.
      • Liang H.
      • Mori M.X.
      • Yue D.T.
      FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation.
      ). Cerulean (
      • Rizzo M.A.
      • Springer G.H.
      • Granada B.
      • Piston D.W.
      An improved cyan fluorescent protein variant useful for FRET.
      ) and Venus (
      • Nagai T.
      • Ibata K.
      • Park E.S.
      • Kubota M.
      • Mikoshiba K.
      • Miyawaki A.
      A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.
      ) were used as the donor and acceptor fluorescent proteins instead of eCFP and eYFP, as their optical properties provided more robust and stable FRET signals. Acceptor-centric measurements of FRET were obtained with the 33-FRET algorithm (
      • Erickson M.G.
      • Alseikhan B.A.
      • Peterson B.Z.
      • Yue D.T.
      Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.
      ,
      • Erickson M.G.
      • Liang H.
      • Mori M.X.
      • Yue D.T.
      FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation.
      ), in which the effective FRET efficiency (EEFF) and FRET ratio (FR) are defined as:
      EEff=E×Ab=(FR1)[εVen(440nm)/εCer(440nm)]
      (3)


      where E is the FRET efficiency of a donor–acceptor pair, Ab is the fraction of acceptor molecules bound by a donor, and εVen(440nm)/εCer(440nm) is the approximate molar extinction coefficients of Cerulean and Venus, which was measured as 0.08 on our setup. Intensity measurements at each wavelength were taken from individual cells such that variable expression across the cells enabled population of a binding curve. Binding curves were analyzed using GraphPad software (Prism), providing relative Kd values and standard error based on an unbiased fit to the data. These relative Kd values were then calibrated according to a previously determined calibration factor (
      • Bazzazi H.
      • Ben Johny M.
      • Adams P.J.
      • Soong T.W.
      • Yue D.T.
      Continuously tunable Ca(2+) regulation of RNA-edited CaV1.3 channels.
      ,
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ) and converted to Ka = 1/Kd. Importantly, the previous calibration factor determined for our setup utilized CFP/YFP FRET pairs. In order to account for the difference in FR using Cerulean and Venus, we determined the relative Kd for multiple peptides using both the fluorophore pairs and found that the two data sets differed by a factor of 1.8, which we incorporated into the calibration factor.

      Data availability

      All data is contained within the article.

      Supporting information

      This article contains supporting information (
      • Bazzazi H.
      • Ben Johny M.
      • Adams P.J.
      • Soong T.W.
      • Yue D.T.
      Continuously tunable Ca(2+) regulation of RNA-edited CaV1.3 channels.
      ,
      • Ben Johny M.
      • Yang P.S.
      • Bazzazi H.
      • Yue D.T.
      Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels.
      ,
      • Liu X.
      • Yang P.S.
      • Yang W.
      • Yue D.T.
      Enzyme-inhibitor-like tuning of Ca(2+) channel connectivity with calmodulin.
      ,
      • Hulme J.T.
      • Yarov-Yarovoy V.
      • Lin T.W.
      • Scheuer T.
      • Catterall W.A.
      Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.
      ,
      • Sang L.
      • Dick I.E.
      • Yue D.T.
      Protein kinase A modulation of CaV1.4 calcium channels.
      ,
      • Hulme J.T.
      • Konoki K.
      • Lin T.W.
      • Gritsenko M.A.
      • Camp D.G.
      • Bigelow D.J.
      • Catterall W.A.
      Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle.
      ).

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      This project was initiated under the direction of Dr David Yue, who passed away in 2014. David was a brilliant scientist and exceptional mentor, and we are grateful for his guidance and friendship. We would like to thank Dr Gordon Tomaselli for his guidance and support. We would also like to thank Hojjat Bazzazi for providing FRET plasmids and for useful discussion and advice throughout the project. In addition, we thank Wanjun Yang for dedicated technical support and thank members of the Calcium Signals lab at Johns Hopkins for discussion and support of this project, as well as members of Dr Dick’s lab at the University of Maryland for insightful discussions and editing of the manuscript.

      Author contributions

      L. J. S., D. T. Y., M. B. -J., and I. E. D. designed the study; L. J. S., D. C. O. V., M. B. -J., and I. E. D. performed the experiments; L. J. S., D. C. O. V., D. T. Y., M. B. -J., and I. E. D. analyzed the data and designed the figures; and L. J. S., and I. E. D. wrote the paper with input and edits from all the authors.

      Funding and additional information

      This grant was supported by an NIH/NINDS grant 5R01NS085074 and by an NIH/NHLBI grant 1R01HL149926. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Supporting information

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