Calmodulinopathy Variants Impair Ca_V1.3 and Ca_V2.1 Regulation

Ca_V1.2 and CaM D132G

Calmodulinopathy variants have been associated with a range of symptoms and evaluated for effects on a variety of target proteins (Crotti et al., 2013; Hwang et al., 2014; Yin et al., 2014; George, 2015; Jensen et al., 2018; Crotti et al., 2019; Nyegaard and Overgaard, 2019; Clemens et al., 2020; Ledford et al., 2020; Kang et al., 2021). Here, we focus on several variants which have been strongly implicated in LQTS and sudden death, with variable reports of neurological features (Fig. 1 A, B). Mutations D96V, F142L, D130G and D132G reside in the C-lobe of CaM and are strongly associated with LQTS. They have each been documented in patients suffering from ASD, developmental delay, and/or epilepsy, with F142L associated with a broader set of reported neurological deficits (Crotti et al., 2013; Kaplanis et al., 2020; Crotti et al., 2023).(Crotti et al., 2013; Kaplanis et al., 2020; Crotti et al., 2023). While D96V has a reported association with developmental delay (Crotti et al., 2013), the neurological effects appear less consistently reported (Crotti et al., 2013; Crotti et al., 2023) (Crotti et al., 2013; Crotti et al., 2023) and were subsequent to cardiac arrest, making it difficult to rule out hypoxic injury. CaM mutations at residue G114 have previously been associated with sudden death and cardiac arrest, however, neurological symptoms were noted to be absent (Brohus et al., 2021; Crotti et al., 2023). Finally, we include the N-lobe mutation CaM N54I, which has a well-evidenced pathological role in CPVT (Nyegaard et al., 2012) with the effects largely independent of VGCC channels, making it a useful control for nonspecific effects.

In the case of LQTS, CaM mutants are thought to contribute to the disease largely through the disruption of Ca_V1.2 CDI (Limpitikul et al., 2014). The majority of C-lobe mutations focused on in this study have previously been shown to blunt Ca_V1.2 CDI, while the CPVT-associated N54I mutation largely spares CDI (Fig. 1C) (Limpitikul et al., 2014; Brohus et al., 2021). However, the effect of the CaM mutation D132G has yet to be evaluated on Ca_V1.2 despite association with severe LQTS (Zahavich et al., 2018) and developmental delay (Kaplanis et al., 2020). We therefore sought to determine the impact of CaM D132G on Ca_V1.2 CDI. To do so, we co-expressed mutant CaM with Ca_V1.2 in HEK293 cells, such that CDI can be seen as the larger decay of the Ca²⁺ current (red), as compared to the Ba²⁺ current (black) in response to a sustained depolarization from –80 mV to 30 mV (Fig. 1D, top). CDI is then quantified by comparing the ratio of Ca²⁺ current that remains following 300 ms of depolarization (r₃₀₀) with that of Ba²⁺. In this way, Ba²⁺ currents serve to control for the VDI of the channels, which occurs alongside CDI and is minimized in our recording conditions by the use of the β_2a auxiliary subunit. In the presence of CaM D132G, the Ca²⁺ current is largely sustained following the depolarizing step, demonstrating a loss of CDI which persists across a range of test potentials (Fig. 1D, bottom). Thus, CaM D132G displays a similar capacity to reduce Ca_V1.2 CDI as compared to other previously documented LQTS-associated CaM variants (Limpitikul et al., 2014) (Fig. 1C).

The Impact of Pathological CaM Mutations on Ca_V1.3 Regulation

Given that regulation of channel gating by CaM extends beyond Ca_V1.2, we investigated the effects of pathological CaM variants on Ca_V1.3 inactivation, starting with the Ca_V1.3_L splice variant. Ca_V1.3_L channels represent a long splice variant (also called Ca_V1.3₄₂ (Bock et al., 2011)), which includes the ICDI/CTM (Singh et al., 2008; Lieb et al., 2012) resulting in a channel with attenuated CDI. Nonetheless, under conditions where exogenous CaM is overexpressed, robust CDI is observed in the presence of CaM WT (Fig. 2A). Strong CDI (red) is observed across a range of depolarizing potentials (Fig. 2A, middle), while Ba²⁺ currents (black) exhibit only modest VDI. While the N-lobe CaM N54I produced no significant deficits in CDI (Fig. 2B), each of the C-lobe CaM mutations imparted significant reduction in CDI as compared to CaM WT (Fig. 2C-G). We also note that the CDI observed in the presence of G114R displayed more rapid inactivation kinetics as compared to the other mutants. As CaM is capable of mediating CDI in Ca_V1.3_L channels through both the C- and N-lobes (Dick et al., 2008), the remaining CDI observed in the calmodulinopathy variants is likely due largely to N-lobe CDI, which is known to exhibit slower inactivation kinetics as compared to C-lobe mediated CDI (Peterson et al., 1999; Yang et al., 2006; Dick et al., 2008). Consistent with this, an artificially mutated CaM₁₂₃₄, in which Ca²⁺ binding is eliminated in both lobes of CaM, results in complete abolition of CDI in Ca_V1.3_L (Fig. 2H). Thus, calmodulinopathy variants can impair Ca_V1.3_L CDI in a manner similar to that observed in Ca_V1.2 (Fig. 1).

Analysis of the voltage dependence of activation (Fig. 2A-H, bottom) revealed no difference across each condition, however evaluation of Ba²⁺ current decay during the depolarization revealed a slight but significant variability in VDI. Specifically, at higher test potential voltages, CaM G114R imparted greater VDI than other mutants or WT CaM as revealed by Ba²⁺ r₁₀₀₀ values (Fig. 2D, black). Additionally, CaM D132G appeared to follow a similar trend, but this effect did not rise to the level of significance (Fig. 2F). To investigate this phenomenon further, these mutants were co-expressed with Ca_V1.3_L in conditions optimized to reveal voltage-dependent effects. Employing the β_1b accessory subunit in the transfection permits robust VDI in Ca_V1.3_L channels (Fig. 3A, top). Once again, the G114R mutant revealed a slight but significantly enhanced VDI as compared to CaM WT or CaM D132G, as revealed by smaller r₁₀₀₀ values following 20 mV depolarizations (Fig. 3A-C, bottom panel). Thus, it appears that VDI is uniquely different in the CaM G114R recordings.

We next considered the impact of the CaM mutants on a short isoform of Ca_V1.3, Ca_V1.3_43s, which lacks the modulatory ICDI/CTM domain and thus displays full CDI (Bock et al., 2011). The CaM mutants had generally similar impacts on CDI in Ca_V1.3_43s as they did in Ca_V1.3_L (Fig. 4A-H). As expected, the N54I N-lobe CPVT mutant was not statistically different from WT (Fig. 4B), while mutations D96V, D130G, D132G, and F142L all significantly impaired CDI (Fig. 4B, 4C, 4E-G). The artificial mutation CaM₁₂₃₄ produced a near total lack of Ca_V1.3_43s CDI (Fig. 4H). In contrast, CaM G114R, which displayed diminished CDI as compared to overexpressed CaM WT in Ca_V1.3_L, showed no effect on Ca_V1.3_43s CDI (Fig. 4D). Additionally, no effects on Ca_V1.3_43s VDI were observed.

The Effect of CaM Mutations on Ca_V1.3 Association

While most C-lobe CaM mutants conferred Ca_V1.3_L and Ca_V1.3_43s CDI deficits in a similar fashion to their action on Ca_V1.2, data from CaM G114R diverges from this paradigm. It is therefore reasonable to question how CaM G114R interacts with Ca_V1.3. Notably, the results for each Ca_V1.3 splice variant would be consistent with a lack of CaM G114R binding to the channel. Thus, the possibility emerges that CaM G114R does not interact with either Ca_V1.3 variant, and the results demonstrate the expected CDI under endogenous CaM WT levels (Liu et al., 2010). Moreover, any mutant CaM must bind to the channel at least as well as CaM WT in the apo state in order for the variant CaM to preassociate with a significant portion of channels in the context of mixed WT and mutant CaM expression (Erickson et al., 2003; Limpitikul et al., 2014; Badone et al., 2018; Hussey et al., 2023). We therefore sought to explore whether each mutant CaM is capable of binding the channel with a similar affinity as compared to CaM WT utilizing Förster resonance energy transfer (FRET). To quantify binding activity, we employed a previously validated (Ben-Johny et al., 2016; Lee et al., 2016; Rivas et al., 2021) live-cell flow cytometric methodology to construct FRET-based binding curves and estimate the effective binding affinity (K_D,Eff) for each channel/CaM pair (Table 1). A peptide spanning known C-terminal CaM binding regions of Ca_V1.3 was ligated to a donor fluorophore, while CaM was joined to an acceptor fluorophore (Fig. 5A), and the two were expressed in HEK293 cells under basal Ca²⁺ conditions. Exemplar binding curves (Fig. 5D-I) of CaM variants N54I, D96V, D130G, D132G, and F142L show broad similarity with the binding curve of CaM WT, and the average results from each of these mutants demonstrate no significant alterations to K_D,Eff relative to WT (Fig. 5B, Table 1). However, CaM G114R demonstrates a profoundly diminished K_D,Eff (Fig. 5B, F, Table 1), where maximal saturation could not be achieved under the experimental condition. Thus, CaM G114R appears to exhibit reduced preassociation with the channel in the apo state.

Given that CaM binding can be impacted by its association with Ca²⁺ (Brohus et al., 2021), we investigated whether CaM G114R/Ca_V1.3 binding could be recovered under conditions sufficient to calcify CaM. Subsequent testing revealed that, in the presence of an ionophore and high external Ca²⁺, binding activity could be partially rescued (Fig. 6A-C). These data indicate that calcified CaM G114R maintains the ability to bind Ca_V1.3 at high Ca²⁺, but preassociation in the apo-state is likely reduced. Given this result, the modified CDI observed in Ca_V1.3_L (Fig. 2D) as compared to overexpressed CaM WT (Fig. 2A) is likely due to the known competition between CaM and ICDI/CTM for binding to the Ca_V1.3 IQ (Fig. 6D). At low, endogenous levels of CaM WT, ICDI/CTM is able to out-compete CaM for binding to the channel, resulting in a significant reduction of CDI as previously described (Singh et al., 2006; Wahl-Schott et al., 2006; Liu et al., 2010; Lieb et al., 2012; Sang et al., 2021). Upon overexpression of CaM WT, CDI is restored due to mass action. Without sufficient apoCaM binding, CaM G114R does not alter this scheme. Moreover, in Ca_V1.3_43s lacking ICDI/CTM, endogenous CaM is sufficient to drive robust CDI, and the lack of CaM G114R binding causes no apparent difference in CDI.

In addition to investigating the interaction between CaM and the Ca_V1.3 IQ in low and high Ca²⁺ conditions, we explored the Ca²⁺-dependence of the interaction of each CaM variant with the channel domain. This was done by monitoring the change in fluorescence anisotropy of the 5-TAMRA-labeled Ca_V1.3 IQ during titration with CaM at eight different Ca²⁺-concentrations (Fig. 7), as previously done for the Ca_V1.2 IQ (Wang 2018; Wang 2020; Brohus 2021). The affinity of CaM WT for Ca_V1.3 IQ dramatically increased as a function of Ca²⁺-concentration (Fig. 7A). Likewise, the affinity of all other CaM variants increased with Ca²⁺-concentration (Fig. 7B-G). As expected, the Ca²⁺-insensitive variant, CaM₁₂₃₄, retained an apo-affinity for the Ca_V1.3 IQ across the entire range of Ca²⁺ concentrations tested, demonstrating its inability to bind Ca²⁺-ions (Fig. 7H). To summarize the effects of the CaM mutations on the interaction with the Ca_V1.3 IQ, the complex affinity in apo- and Ca²⁺-saturating conditions, and its Ca²⁺-sensitivity were estimated from the EC₅₀ fits (Fig. 7A, bottom panel dashed lines). First, the complex affinities in apo- and Ca²⁺-saturating conditions were estimated by the initial and final plateaus, respectively (Fig. 7A, horizontal dashed lines and Fig. 7I-J). In support of the FRET experiments, and the hypothesis of reduced channel preassociation, CaM G114R had the largest effect on the apo-affinity (defined by the initial plateau), causing a significant 10-fold reduction compared to CaM WT (Fig. 7I). CaM D130G and D132G significantly reduced the apo-affinity by 2-fold compared to CaM WT. Of note, only CaM F142L caused an increase in the apo-affinity (13-fold compared to CaM WT), in agreement with what has been observed for the Ca_V1.2 IQ (Wang et al., 2018). In Ca²⁺ saturating conditions, defined by the final plateau of the EC₅₀ curve, the generally high affinity caused a more variable determination of K_D values, in turn causing a less well-defined final plateau compared to the initial plateau (apo-conditions). Despite the higher variability at high Ca²⁺-concentrations, we tested for significant differences in the CaM/Ca_V1.3 IQ-domain affinities. CaM D130G reduced the IQ-domain affinity 5.5-fold compared to CaM WT whereas CaM D132G and F142L caused a 3.5- and 2-fold increase, respectively (Fig. 7J). As a measure of the Ca²⁺-sensitivity of CaM when in complex with the Ca_V1.3 IQ domain, we determined the Ca²⁺-concentration causing a half-maximal change in log(K_D) (representing binding energy) between the apo- and Ca²⁺-bound states (EC₅₀) (Fig. 7A, dotted vertical line and Fig. 7K). The EC₅₀-value for CaM WT/Ca_V1.3 IQ is 400 nM free Ca²⁺. All C-lobe mutations impaired the Ca²⁺- sensing of CaM when complexed to the Ca_V1.3 IQ (2–11-fold) in line with their reduction of C-lobe Ca²⁺-affinity (Fig. 7K), and consistent with their effect in impairment of Ca_V1.3 CDI.

The Impact of Pathological CaM Mutations on Ca_V2.1 Regulation

Having evaluated the impacts of pathological CaM mutants on Ca_V1.3, we next turned to investigate the P/Q-type channel, Ca_V2.1. To do so, we utilized a similar experimental setup as before with Ca_V2.1 transfected into HEK293 cells along with the mutant CaMs. For Ca_V2.1, C-lobe mediated CaM regulation takes the form of CDF (DeMaria et al., 2001). To evaluate CDF, we employed a step depolarization, with and without a prepulse at varying potentials. Ca²⁺ entry during the prepulse initiates CDF, resulting in a larger initial current during the test pulse. To quantify the amount of CDF, the area between the two curves is integrated to give the relative facilitation (RF) value (Fig. 8) (DeMaria et al., 2001; Chaudhuri et al., 2004). As expected for a C-lobe mediated process, the N-lobe CaM mutant N54I did not significantly impair Ca_V2.1 CDF as compared to WT (Fig. 8B). On the other hand, the C-lobe mutants G114R, D130G, D132G, and F142L all significantly reduced CDF, with G114R and F142L displaying somewhat more moderate effects (Fig. 8D-G). The artificial mutant CaM₁₂₃₄ demonstrates the impact of a complete loss of Ca²⁺ binding to CaM (Fig. 8H). Interestingly, the C-lobe mutant D96V did not display impaired CDF (Fig. 8C) despite the fact that the mutated residue resides in the third Ca²⁺ binding EF hand and has been shown to dramatically reduce Ca²⁺ binding (Limpitikul et al., 2014; Sondergaard et al., 2015b). Finally, evaluation of the normalized current-voltage relationship demonstrated small (<10 mV) shifts in the half maximal voltage of activation of Ca²⁺ currents for G114R, D132G, and F142L variants relative to WT (Fig. 8, bottom panels).

Given the lack of CDF effect for D96V, we wondered if the affinity of the D96V mutant for Ca_V2.1 might be impaired as seen for G114R for Ca_V1.3. As before, we evaluated the capacity of mutant CaMs to bind via FRET. Using a C-terminal fragment of Ca_V2.1 coupled to a cerulean fluorophore, the donor molecule of the FRET pair was constructed. Similarly, each mutant CaM was ligated to Venus as the acceptor fluorophore. Evaluation of the FRET binding curves to estimate K_D,eff under basal Ca²⁺ conditions revealed that CaM G114R alone displayed significantly altered binding affinity (Fig. 9A-G, Table 2), although the effect was much milder as compared to the binding deficits observed in Ca_V1.3. Interestingly, CaM₁₂₃₄ displayed a similarly mild but significant increase in K_D,eff (Fig. 9J, Table 2), implying that even in the apo state, this form of CaM has diminished binding affinity.

To explore the Ca²⁺-dependency of the CaM/Ca_V2.1 IQ interaction, we once again used the fluorescence anisotropy assay (Fig. 10). The affinity of CaM WT for the Ca_V2.1 IQ dramatically increased with increasing Ca²⁺-concentration (Fig. 10A). All other CaM variants, except CaM₁₂₃₄, followed the same pattern of increasing complex affinity with Ca²⁺-concentration (Fig. 10B-H). In the low Ca²⁺ apo-state, all CaM C-lobe mutations displayed a small but significant reduction in binding affinity for the Ca_V2.1 IQ domain (1.2- to 2.7-fold compared to CaM WT) (Fig. 10I). Of note, the magnitude of reduction in apo-affinity for CaM G114R towards the Ca_V2.1 IQ was less than that towards the Ca_V1.3 IQ (2.7-fold reduction for Ca_V2.1 IQ vs 10-fold reduction for Ca_V1.3 IQ). This difference in binding affinity between the two channels supports a lack of apoCaM G114R preassociation with Ca_V1.3, and thus a lack of effect on CDI, while retaining an effect on Ca_V2.1 CDF. Similarly to the observation for the apo-affinities, all C-lobe mutations caused a reduction in the affinity of the Ca²⁺-saturated complex (2- to 14-fold compared to CaM WT) (Fig. 10J). All C-lobe mutations reduced the Ca²⁺-sensitivity of the complex (1.8- to 4.3-fold compared to CaM WT), in line with their reduction of C-lobe Ca²⁺- affinity (Fig. 7K), and consistent with their general impairment of Ca_V2.1 CDF. These results, however, do not explain the distinct effects of D96V observed in functional assays. It is possible that the relatively small Ca_V2.1 peptide used here and in our FRET assay is not sufficient to elucidate all CaM binding effects.

Effect of Mixed Expression of WT and Mutant CaM

While our experiments utilize overexpression, physiologically each calmodulinopathy mutation would be expressed in a single of six CALM genes, resulting in a mix of WT and mutant CaM protein. In order to address the question of whether a mutant CaM would modulate the channel in the context of mixed expression with CaM WT, we co-expressed CaM D130G in a 1:1 ratio with CaM WT and evaluated the CDI and CDF of Ca_V1.3 and Ca_V2.1 respectively (Fig. 11). Indeed, this configuration led to an intermediate decrease in CDI of Ca_V1.3_L (Fig. 11A) and Ca_V1.3_43S (Fig. 11B), as compared to overexpressed D130G (Figs. 2, 4). Likewise, mixed expression of CaM WT and D130G resulted in intermediate deficits in CDF of Ca_V2.1 (Fig. 11C vs. Fig. 8). These results are consistent with prior data showing that the effect of mutant CaM on Ca_V1.2 scaled with the expression of mutant to WT protein (Limpitikul et al., 2014).

Transfection and molecular biology for electrophysiology

For electrophysiological experiments, a plasmid encoding a human α_1C channel variant containing exon 8a was employed for Ca_V1.2 (Z34810) (Dick et al., 2008). Plasmids encoding a human α_1D channel variant containing exons 8a and 42 was employed for Ca_V1.3_L (EU363339) and a variant terminating in exon 43 was employed for Ca_V1.3 43s (NM_001128839) (Sang et al., 2021). These plasmids were generously provided by Dr. Jörg Striessnig (University of Innsbruck). A plasmid encoding an α_1A channel variant including exons 43 and 44 and excluding exon 47, originally provided by Dr. Terrance Snutch (University of British Columbia), was employed for Ca_V2.1 (NM_001127221) (Lee et al., 2015). Each of these was within a pcDNA3 expression vector. For these same experiments, human CALM1 cDNA within the pIRES2-EGFP vector was utilized for WT CaM (Clontech Laboratories). From this WT CaM-pIRES-EGFP construct, pathogenic CaM mutations were generated using QuikChange Lightning^™ site-directed mutagenesis (Agilent). 8 μg each of human α₁ cDNA, rat brain β_2a (M_80545) or rat brain β_1b (X61394), rat brain α_2δ (NM_012919.3) subunits, and WT or mutant CaM were heterologously co-expressed. For all recordings, β_2a was used unless otherwise specified. Expression was enhanced with 2 μg of simian virus 40 T antigen (Tag) cDNA. Expression of all plasmids was under the control of a cytomegalovirus (CMV) promoter. HEK293 cells were cultured in 10-cm dishes on glass coverslips, and channels were transfected using an established Ca²⁺ phosphate method (Peterson et al., 1999). Data from each experimental condition were gathered from a minimum of two independent transfections.

Patch clamp recordings

Whole-cell voltage-clamp recordings of HEK 293 cells were carried out 1–2 days post-transfection at ambient temperature. An Axopatch 200B amplifier (Axon Instruments) was utilized to generate the recordings. Noise in these recordings was minimized via a lowpass filter at 2 kHz, followed by digital sampling at 10 kHz. Pipette resistances were 1.5–5 MΩ and P/8 leak subtraction was used. Internal solutions contained (in mM): CsMeSO₃, 114; CsCl, 5; MgCl₂, 1; MgATP, 4; HEPES (pH 7.4), 10; and BAPTA, 10; at 295 mOsm adjusted with CsMeSO₃. External solutions contained (in mM): TEA-MeSO₃, 140; HEPES (pH 7.4), 10; and CaCl₂ or BaCl₂, 40; at 300 mOsm, adjusted with TEA-MeSO3. These solutions produced the following uncorrected junction potentials: 10 BAPTA/40 Ca²⁺: 10.5 mV; 10 BAPTA/40 Ba²⁺: 10.2 mV. Note that the use of 10 BAPTA limits Ca²⁺ to only the local signal due to entry through the channel (Dick et al., 2008). For Ca_V2.1, this manipulation restricts CaM regulation to C-lobe mediated CDF (Ben-Johny and Dick, 2022), reducing the confounding effect of N-lobe mediated CDI in our CDF measurements. However, it should be noted that this manipulation also reduces the ability to observe N-lobe effects, thus the N54 mutation is used largely as a negative control under these conditions.

Quantification of patch clamp data

For r₃₀₀ Ca²⁺ data in Ca_V1.2/CaM experiments, a ratio of peak current during a step depolarization to 30 mV to the current remaining following 300 ms was employed for each CaM variant. For each cell, this value was subtracted from the analogous value for Ba²⁺ at 30 mV, resulting in an f₃₀₀ value that quantified CDI. For both sets of Ca_V1.3/CaM datasets, similar comparisons were employed, with the following differences: currents were compared at 10 mV step depolarizations, and the 1000 ms depolarization timepoint was used resulting in f₁₀₀₀ values. For experiments investigating Ca_V1.3/CaM VDI, r₁₀₀₀ values acquired during Ba²⁺ depolarizations were used to estimate VDI.

For Ca_V2.1/CaM experiments, CDF was characterized by comparing Ca²⁺ currents during depolarizing voltage steps to 10 mV in the presence and absence of a prepulse which was sufficient to partially facilitate the channel (DeMaria et al., 2001; Chaudhuri et al., 2004). To adjust for variations in current density each sweep is normalized relative to the end of the 50 ms test pulse. This timepoint is chosen such that CDF is complete and CDI is minimal. ΔQ is quantified as the area between the currents with and without the pre-pulse. In order to normalize for the time course of facilitation, ΔQ is divided by the time constant of the curve without the prepulse τ, giving an approximation of the degree of CDF triggered by the prepulse, termed relative facilitation (RF). In cases where the lack of CDF led to an absence of a monoexponential curve to derive τ from, τ was set as the average value from WT dataset (8.5). Data from prepulses to 20 mV were used for statistical comparisons.

To evaluate voltage-dependence of activation, Ca²⁺ current densities of each variant were plotted as function of depolarization voltage. Data for each cell was normalized to peak value and averaged across cells. Data points were fit by the curve Y=G_max*(X-V_rev)/(1+exp((V₅₀-X)/Slope)). The half maximal voltage of activation was calculated from these curves.

Generation of FRET constructs

The principles underlying flow cytometric FRET having been previously established (Ben-Johny et al., 2016; Lee et al., 2016; Rivas et al., 2021). Venus-CaM plasmids correspond to those described previously (Ben-Johny et al., 2016; Lee et al., 2016; Niu et al., 2018). QuikChange Lightning mutagenesis (Aligent) was employed to create the Venus-CaM mutants of interest. For Ca_V1.3, the C-terminal fragment (DNFDY…VGKYP) was ligated into a pcDNA3 expression vector containing an upstream Cerulean fluorophore. The DNA template encoding the Ca_V1.3 fragment as well as XbaI and NotI restriction digest sites at positions flanking the coding sequence was purchased (Twist Bioscience). Using this template, digestion with these enzymes was carried out, followed by ligation into the expression vector using T3 DNA ligase (New England Biolabs). To ensure expression and read-through, the Cerulean fluorophore was preceded by a CMV promotor, and an in-frame short linker sequence of alanine was present between the end of the Cerulean sequence and before the Ca_V1.3 fragment. At the end of the Ca_V1.3 fragment was a stop codon to create the fusion protein suitable for FRET. The same procedure was employed to generate the Cerulean/Ca_V2.1 construct, with the Ca_V2.1 sequence (DNFEY…EOGPP) inserted into the vector. Dimers linking Cerulean and Venus of various lengths (C5V, C32V, C40V, CTV) were utilized as previously described (Ben-Johny et al., 2016; Lee et al., 2016; Rivas et al., 2021). All constructs were verified by sequencing (Plasmidsaurus).

Transfection of FRET constructs

Cells were seeded in 6-well plates and transfected with FRET constructs and fluorophore pairs using a PEI protocol (Longo et al., 2013). 48 hours post-transfection, cells were treated with 100 μM cycloheximide for 2–4 hours to reduce incidence of immature fluorophores, and were then harvested and resuspended in Tyrode’s solution with minimal Ca²⁺. For experiments involving calcified CaM, 10 mM CaCl₂ was added to equal volumes of cell suspension along with 4 μM ionomycin (Sigma-Aldrich I0634).

FRET data collection

To estimate binding curves and K_D,eff for each of the donor-acceptor pairs, fluorescence measurements are acquired from three filters within the flow cytometer, specifically selected to detect donor, acceptor, and FRET signals. This technique, termed 3³-FRET, permits the isolation of FRET signal from donor and acceptor signals (Erickson et al., 2001). This methodology required evaluation of instrument-specific properties during each experimental run, evaluated using a series of linked Cerulean/Venus dimers such that a 1:1 stoichiometry of each fluorophore was ensured (Ben-Johny et al., 2016). Moreover, Venus and Cerulean fluorophores, individually and co-expressed, were measured to permit evaluation of non-specific fluorescence that could confound FRET analysis. Cells were kept in Normal Tyrodes solution for recording containing (in mM): NaCl, 138; KCl, 4; MgCl₂, 1; HEPES (pH 7.4), 10 at 300 mOsm adjusted with NaOH. External solutions contained (in mM): TEA-MeSO₃, 140; HEPES (pH 7.4), For high-Ca²⁺ conditions, external Ca²⁺ was increased to 4 mM and 10 µM Ionomycin was added to facilitate Ca²⁺ entry into cells prior to recording.

Fluorescence Anisotropy Binding Assays

Peptides representative of the Ca_V1.3 and Ca_V2.1 IQ-domains were N-terminally labeled with a 5-TAMRA fluorophore (Ca_V1.3: 5-TAMRA- DEVTVGKFYATFLIQDYFRKFKKRKEQGLVGKYP-NH2, Ca_V2.1:_5-TAMRA-TDLTVGKIYAAMMIMEYYRQSKAKKLQAMREEQD-NH2; Proteogenix, purity > 95%). Using a previously described 384-well plate-based assay (Brohus et al., 2019), CaM was titrated across the 24 columns at 8 different free calcium concentrations (3 nM to 400 µM), one per row (Brohus et al., 2021). The change in fluorescence anisotropy (FA) of the TAMRA-labeled IQ-domains, a measure of their tumbling rate, was monitored as a function of CaM concentration. This setup resulted in 8 CaM/IQ-domain binding curves, each representing binding at a specific free Ca²⁺ concentration.

A stoichiometric binding model was fitted to each binding curve to determine the CaM/IQ-domain binding affinity (represented by the dissociation constant, K_D) at each Ca²⁺ concentration (Jensen et al., 2024):

Where K_D is the dissociation constant, and [IQ-domain] and [CaM] is the total concentration of the 5-TAMRA-labeled IQ-domain and CaM, respectively. All model parameters (FA_initial, FA_span, K_D, and [IQ domain]) were fitted with constraints that all parameters be above zero. In the case of Ca_V1.3 IQ, binding curves for CaM G114R did not reach an upper plateau at low Ca²⁺- concentrations; thus, curve spans from CaM WT were assumed for these curves to determine the apo-affinity (i.e. the FA_span parameter for these binding curves was kept constant equal to 583 the WT parameter).

Affinities and Ca²⁺ concentrations were log10-transformed before fitting an EC₅₀ curve:

Due to the high CaM/Ca_V1.3 IQ-domain affinity at high Ca²⁺-concentrations, K_D values were difficult to determine robustly between replicates. For this reason, all determined K_D values from all replicates were combined to provide enough data points to fit the EC₅₀ model. From the EC₅₀ model, the fitted initial plateau (at low Ca²⁺-concentrations) was extracted to represent the apo-affinity, and the fitted final plateau (at high Ca²⁺-concentrations) was extracted to represent the CaM/Ca_V IQ-domain affinity at saturating Ca²⁺-concentrations (Fig. 7A, lower panel horizontal stapled lines). The fitted EC₅₀ values were extracted to represent the Ca²⁺-concentration that provides the half-maximal change in logK_D (binding energy) between the apo- and Ca²⁺-bound states – a measure of the Ca²⁺-sensitivity of the interaction (Fig. 7A, lower panel vertical stapled line). In summary, the assay provides information on the Ca²⁺-dependency of the CaM/IQ-domain interaction across the physiologically relevant Ca²⁺-concentration span.