Bilobal architecture is a requirement for signaling to CaV1.3 channels Rahul Banerjeea, Jesse B. Yoderb, David T. Yuea, L. Mario Amzelb, Gordon F. Tomasellic, Sandra B. Gabellib,c,d,1, and Manu Ben-Johnya,e,1

aDepartment of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205; bDepartment of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205; cDivision of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287; dDepartment of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21287; and eDepartment of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032

Edited by William A. Catterall, University of Washington School of Medicine, Seattle, WA, and approved February 13, 2018 (received for review September 17, 2017)

Calmodulin (CaM) regulation of voltage-gated calcium (CaV) channels night blindness (25), dissecting these mechanisms promises to is a powerful Ca2+ feedback mechanism that adjusts Ca2+ influx, lend insights into disease pathogenesis and therapeutics. + affording rich mechanistic insights into Ca2 decoding. CaM possesses We, here, demonstrate that the bilobal architecture of CaM is + a dual-lobed architecture, a salient feature of the myriad Ca2 -sensing a prerequisite for signaling to L-type channels. Channels bound , where two homologous lobes that recognize similar targets to a single lobe of CaM adopt a preinhibited configuration with 2+ hint at redundant signaling mechanisms. Here, by tethering CaM diminished PO and are prohibited from Ca regulation. The lobes, we demonstrate that bilobal architecture is obligatory for sig- reconstitution of two CaM lobes relieves preinhibition and res- 2+ naling to CaV channels. With one lobe bound, CaV carboxy tail rear- cues Ca regulation. Consistent with a baseline change in channel ranges itself, resulting in a preinhibited configuration precluded from gating, small-angle X-ray scattering (SAXS) analysis and molec- Ca2+ feedback. Reconstitution of two lobes, even as separate mole- ular dynamics (MD) simulations suggest a structural rearrange- 2+ ment of the CI when one versus two CaM lobes are bound. These cules, relieves preinhibition and restores Ca feedback. CaV channels + thus detect the coincident binding of two Ca2 -free lobes to promote findings revise the current view of CaM regulation of CaV chan- channel opening, a molecular implementation of a logical NOR oper- nels and bear implications for CaM signaling to its diverse targets. 2+ ation that processes spatiotemporal Ca signals bifurcated by CaM Results lobes. Overall, a unified scheme of CaV channel regulation by CaM now emerges, and our findings highlight the versatility of CaM to Two Lobes of CaM Are Required for CaV Channel Modulation. We perform exquisite Ca2+ computations. sought to evaluate whether a single lobe of CaM by itself could trigger CaV regulation. However, as full-length CaM is ubiqui- tous in eukaryotic cells, probing the effects of individual lobes is ion channels | voltage-gated Ca channels | Ca 1.3 | calmodulin | V challenging. Exogenously expressed CaM lobes may not fully calcium regulation populate the channels and the observed effects may be corrupted by fluctuations in ambient CaM. Similarly, genetic replacement almodulin (CaM) plays a central role in eukaryotic signaling + of endogenous CaM with lobe peptides lacks target specificity Cby decoding cytosolic Ca2 fluctuations to coordinate diverse and impacts cell viability (26). To overcome these limitations, we targets ranging from to ion channels (1, 2). Structurally, utilize a two-prong approach: (i) Full-length or individual CaM CaM possesses a distinctive bilobal architecture: Its two globular 2+ domains, containing paired “EF hand” Ca -binding motifs, ex- Significance hibit high sequence (Fig. 1A) and structural homology (Fig. 1 B and C) (3). The two lobes often recognize similar targets and Calmodulin (CaM) regulation of voltage-gated calcium (CaV) evoke like functional outcomes (3). This apparent redundancy channels constitutes a prototypic biological feedback mecha- + suggests that the bilobal arrangement may be dispensable for nism that contributes prominently toward Ca2 homeostasis in CaM function. Indeed, many CaM targets such as CaM and cardiac myocytes. Here, by partitioning CaM mo- , light-chain , and nitric oxide synthase are lecularly into its two elemental domains or lobes, we uncover a activated by CaM fragments in vitro (4, 5). However, the two- 2+ distinctive nonlinearity in CaM signaling to CaV channels. CaV lobed architecture is a hallmark for many Ca -binding proteins, channels detect the coincident binding of two CaM lobes to up- suggesting it may support latent functions (6). To address this 2+ regulate channel activity. This mechanism elaborates a molecular conundrum, we dissect CaM signaling to voltage-gated Ca logic operation that enables channels to detect combinations of channels (CaV) (7), a prototypic molecular feedback essential for spatiotemporal Ca2+ signals and perform higher-order compu- + cardiac electrical stability (8, 9), neuronal activity (10, 11), and tations on Ca2 signals. These findings uncover the unified coupling to diverse cellular processes (12–14). Functionally, mechanistic basis for CaV channel feedback and, in so doing, CaM exerts two distinct effects on CaV channels (15). First, the 2+ 2+ 2+ shed light on the versatility of CaM in decoding cellular Ca binding of Ca -free CaM (apoCaM) to the Ca -inactivating signals. (CI) module in the channel carboxy terminus (CT) (16) up- regulates baseline open probability (PO)(15)(Fig.1D and E). Author contributions: R.B., D.T.Y., L.M.A., G.F.T., S.B.G., and M.B.-J. designed research; 2+ Second, the binding of Ca to CaM reverses this initial enhance- R.B. and M.B.-J. performed research; R.B., J.B.Y., and M.B.-J. contributed new reagents/ + 2 analytic tools; R.B. and M.B.-J. analyzed data; and R.B. and M.B.-J. wrote the paper. ment in PO, a process termed Ca -dependent inactivation (CDI) (15, 17–20) (Fig. 1 D and E). However, how individual CaM lobes The authors declare no conflict of interest. orchestrate dual regulation and whether the bilobal architecture is a This article is a PNAS Direct Submission. requirement remains critically unknown. As this modulation is Published under the PNAS license. conserved across CaV and voltage-gated sodium (NaV)channels 1To whom correspondence may be addressed. Email: [email protected] or (21), resolving these ambiguities may inform on unified regulatory [email protected]. mechanisms. Moreover, as mutations in conserved CaV and NaV This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CaM-binding interfaces are linked with heritable cardiac arrhythmias 1073/pnas.1716381115/-/DCSupplemental. (22, 23), autism spectral disorder (24), and congenital stationary Published online March 12, 2018.

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2+ Fig. 1. Bilobal architecture of CaM is necessary for CaV1.3 channel regulation. (A) CaM contains two globular domains each composed of two EF hand Ca - + + binding motifs. The two lobes of CaM share high sequence similarity. (B)Ca2 -dependent conformational changes of CaM. (Left) In the absence of Ca2 , both N and C lobes of CaM adopt a highly similar conformation (PDB ID: 1CDF). (Right) The structural similarity of the CaM lobes persists even when Ca2+-bound

(PDB ID: 1CLL). (C) Schematic summarizes CaM-dependent changes in CaV1.3 gating. (Left) Devoid of CaM, channels adopt a low PO gating configuration. 2+ (Middle) The apoCaM binding switches channels to a high PO gating mode. (Right)Ca binding to CaM relieves the enhancement in PO and switches channels 2+ to a low PO mode. (D)CaV1.3 channels with tethered wild-type CaM (CaV1.3−CaMWT, Top) exhibit robust Ca -dependent regulation. (Middle) Exemplar + + currents. Scale bar pertains to Ca2 currents (red). Ba2 currents (black) are scaled down ∼3× for comparison of decay kinetics. (Bottom) Population data depict 2+ 2+ fraction of peak current after 300-ms depolarization (r300) versus voltage; red, relation for Ca ; black, relation for Ba . Each point is mean ± SEM. (E) Genetic 2+ 2+ fusion of dominant negative mutant CaM1234 to CaV1.3 CT (CaV1.3−CaM1234) abrogates Ca regulation. (F)CaV1.3 fused to CaMC fails to support Ca regulation, suggesting that bilobal architecture of CaM is necessary for functional modulation of CaV channels. (G) Genetic fusion of CaV1.3 with mutant 2+ 2+ CaM12 whose Ca binding is restricted to C lobe alone also supports Ca regulation, suggesting C lobe can trigger channel modulation, provided N lobe is 2+ 2+ also present. (H) Genetic fusion of CaV1.3 with mutant CaM34 whose Ca binding is restricted to N lobe alone also supports Ca regulation. Format for E–H is as in D.

lobes are localized to the channel complex via genetic fusion using (Fig. S1 B and C). Although both N and C lobes of apoCaM bind a flexible linker to the CT (27). (ii) Endogenous free CaM levels to the CI, the C lobe possessed an ∼20-fold higher affinity, with are diminished by overexpressing a CaM sponge derived from the isoleucine–glutamine (IQ) domain as its primary interface unconventional Myosin Va (28, 29). As a single apoCaM binds (Fig. S1). The overall scheme for apoCaM preassociation to the (29) and regulates CaV channels (15, 27), this fusion strategy CaV1.3 (Fig. S1) parallels that for NaV channels (31). Given its satisfies stoichiometric requirements for channel modulation (Fig. high affinity, we probed whether CaM C lobe by itself can sup- 2+ S1A). To validate this approach, we fused full-length CaM to the port Ca regulation by fusing CaMC to the CaV1.3 CT (CaV1.3 CaV1.3 CT (Fig. 1D) and coexpressed CaM sponge. These chan- −CaMC, Fig. 1F). Surprisingly, this maneuver completely dis- nels exhibited robust CDI (Fig. 1D), as evident from the enhanced rupted CDI (P = 5.3E-8) (Fig. 1F). Moreover, Ca 1.3−CaM also + + V C decay kinetics with Ca2 (red) versus Ba2 (black) as charge car- exhibited sharply diminished CDI even in the absence of CaM rier and population data showing fraction of peak current sponge (Fig. S2A; P = 7.5E-6), suggesting the CaM C lobe by itself remaining following 300-ms depolarization (r300). However, when is incapable of supporting CDI. By contrast, fusion of CaV1.3 with 2+ 2+ channels are tethered to mutant CaM1234, with all four Ca - CaM12, a bilobed variant with intact C lobe and Ca -insensitive N binding sites disabled, modulation was abolished (P = 1E-6) (Fig. lobe, sustains CDI (P = 0.02 vs. CaV1.3−CaMC) (Fig. 1G). Like- 1E), further corroborating our strategy (30). wise, tethering CaM34 to CaV1.3, with a functional N lobe and a 2+ To discern the effect of single CaM lobes, we assessed their Ca -insensitive C lobe, also supports CDI (CaV1.3−CaM34,Fig. ability to bind the CI element using FRET two-hybrid assays 1H). Thus, while CaM C lobe binds to CaV1.3, the N lobe is

Banerjee et al. PNAS | vol. 115 | no. 13 | E3027 Downloaded by guest on September 25, 2021 + + necessary for channel modulation even if Ca2 -binding to this lobe the absence and the presence of Ca2 (Fig. S1 C and D). Elevation + is defunct. of cytosolic Ca2 resulted in an increase in the maximal FRET 2+ Thus informed, we sought to determine whether the CaM N efficiency (EA,max), arguing that Ca /CaMC binds to CaV1.3 CI lobe alone can support CDI. As the canonical CaV1.3 IQ domain and elicits a conformational change within the complex. has a high affinity for CaM C lobe, coexpression of CaM sponge Second, channels bound to a single CaM lobe may undergo a + by itself is insufficient to preclude binding of endogenous CaM baseline change in gating even before Ca2 influx, yielding a (32). Consequently, we assessed CDI of an RNA-edited channel, “nonpermissive” configuration incapable of CDI. Intriguingly, CaV1.3MQDY, with a single amino acid substitution in its IQ previous studies have shown that apoCaM binding itself tunes domain that weakens CaM binding (33). Furthermore, to attain CaV gating, with the loss of preassociated CaM resulting in a high local concentrations, we tethered CaM variants to the β2A- throttling of channel openings (15). Could the binding of CaM C subunit (30, 33). In the presence of β2A−CaMWT,CaV1.3MQDY lobe alone also alter the channel PO as expected with a change in exhibited strong CDI establishing baseline CDI for this channel gating? Accordingly, we undertook low-noise single-channel variant (Fig. S2B). By contrast, coexpression of β2A−CaMN, electrophysiology to compare the PO of CaV1.3 fused to CaM composed of a single N lobe alone, elicited a marked reduction C lobe (CaV1.3−CaMC) with those (i) fused to full-length CaM 2+ in CDI (P = 7E-8) (Fig. S2C). Even so, CaV1.3MQDY exhibits or (ii) devoid of CaM altogether. To isolate Ca -independent 2+ robust CDI in the presence of β2A−CaM34, a bilobed CaM var- changes in channel gating, Ba was chosen as the charge carrier, 2+ iant with an intact N lobe and a Ca -insensitive C lobe (Fig. as it binds poorly to CaM (36). Moreover, as CaV1.3 exhibits S2D)(P = 1E-4 compared with CaV1.3MQDY/β2A−CaMN). Thus, minimal voltage-dependent inactivation, a slow voltage ramp was a single N lobe alone in the channel complex is also insufficient utilized to elicit stochastic channel openings that reflect near−steady- to trigger CDI; rather, the presence of the C lobe is necessary to state PO at each voltage. With this experimental framework, we 2+ elicit channel modulation even if Ca -binding to this lobe is undertook recordings of CaV1.3−CaMWT with a CaM sponge defunct. Together, these results illustrate that the dual-lobe ar- overexpressed to obviate any confounding effects of endogenous chitecture of CaM is a core requirement for CaV regulation. CaM. Fig. 2A, Middle displays exemplary stochastic records, where channel closures correspond to the zero-current portions of the Channels Bound to a Single CaM Lobe Are Preinhibited. The striking trace (horizontal gray lines) and openings correspond to downward 2+ difference in Ca responsiveness of CaV channels in the presence of deflections to the open level (slanted gray curves). Averaging many one versus two lobes of CaM may arise from two distinct possibilities. such records yields a mean current that can be divided into the First, when CaM C lobe alone is preassociated to the channel, open level (slanted gray curve) to obtain the steady-state P as a + O it may either fail to bind Ca2 and/or trigger downstream con- function of voltage (sigmoidal trace at the bottom). The maximal formational changes necessary for channel modulation. Bio- PO of the CaM-fused channel was found to be PO,max = 0.41 ± 0.05 2+ chemically, individual CaM lobes in solution can bind Ca ions (n = 8 patches and 850 records; mean ± SEM). To estimate the PO with a similar affinity, and the resultant conformational changes of CaV devoid of CaM, we analyzed the single-molecule behavior are nearly identical to that of intact CaM (34, 35). To explicitly of the RNA-edited CaV1.3MQDY (11). Once again, we resorted to test this possibility, we use live-cell FRET two-hybrid assay to studying this variant, as the canonical CaV1.3 short variant has a compare the binding of YFP-tagged CaV1.3 CI to CFP−CaMC in high affinity for apoCaM such that strong overexpression of CaM

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Fig. 2. Channels bound to a single CaM lobe adopt a preinhibited configuration. For experiments in A–D, CaM sponge was coexpressed. (A)CaV1.3 fused to 2+ CaMWT (CaV1.3−CaMWT) exhibits high baseline PO, with Ba as charge carrier, consistent with channels in gating configuration A (Fig. 1D). (Top) Exemplary current records show response to a voltage-ramp protocol. (Bottom) PO–voltage (V) relationship determined from the ensemble average of single-channel recordings. (B)CaV1.3 channels lacking prebound CaM exhibit diminished baseline PO. Here, we used the CaV1.3 MQDY variant with a low CaM binding affinity. (C) When CaV1.3 is fused to CaMC (CaV1.3−CaMC) alone, the baseline PO is substantially diminished, consistent with channels adopting a preinhibited configuration. (D) Reconstitution of N lobe of CaM via attachment to the auxiliary CaVβ2A subunit (β2A−CaMN) potently up-regulates the maximal PO of CaV1.3−CaMC channels (red curve). Format for B−D is as in A.

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Fig. 3. Binding of CaM lobes evoke discrete CaV1.3 gating modes. CaM sponge was present for A–L.(A) Sequential single-channel trials of CaV1.3−CaMWT in  response to a voltage ramp (187 records). Diary plot displays single-trial average PO computed for −30 mV ≤ V ≤ +25 mV (PO). Dashed line discriminates low  (red area) and high PO (gray area) traces. (B) Histogram shows number of sweeps with PO(−30 ≤ V ≤ 25) for given range. (C) Average PO at each voltage calculated for high PO traces estimates PO−V relationship for mode A.(D) Cumulative open duration distribution [green bars; P(TO > t)] follows a single- −1 exponential decay (green fit) consistent with a single open state in mode A with an exit rate, kOCjA = 3.3 ms .(E–H)CaV1.3S/MQDY with CaM sponge ap-  proximates behavior of CaM-less channels (117 records). Openings are brief and sparse. Single-trial PO distribution is unimodal in low PO range. Maximal PO is −1 reduced. Open-duration P(TO > t) is single-exponential with kOCjE = 9.3 ms > kOCjA. Format is as in A–D.(I–L) Analysis of CaV1.3−CaMC (141 records) shows  similarity to CaM-less channels with brief and sparse openings and PO histogram and PO−V relation reminiscent of mode E. P(TO > t) is single exponential with rate constant ≈ kOCjE.(M–P) With β2A−CaMN,CaV1.3−CaMC PO is enhanced in a quantized manner (139 records). Channels switch between low and high  activity epochs with bimodal PO distribution. P(TO > t) distribution is biexponential consistent with rate constants kOCjE and kOCjA.

Banerjee et al. PNAS | vol. 115 | no. 13 | E3029 Downloaded by guest on September 25, 2021 chelator is insufficient to fully preclude CaM binding (32). By decay, suggesting a distinct open state associated with mode E ≈ −1 contrast, the CaM binding status of CaV1.3MQDY can be readily (OE) with an exit rate of kOCjE 9.3 ms (Fig. 3H and Fig. S3C). tuned by the same maneuver (33). Under low levels of CaM, we With CaM-dependent gating modes quantified, we next un- − found the steady-state maximal PO of CaV1.3MQDY to be drastically dertook in-depth analysis of CaV1.3 CaMC. Qualitatively, chan- = ± ± =  diminished, with PO,max 0.08 0.01 (mean SEM; n 4patches nel openings are sparse as with mode E, and the diary plot of PO with 539 records) (Fig. 2B). With these two limits firmly estab- further confirms their low activity (Fig. 3I). In fact, the P dis- − O lished, we probed the function of CaV1.3 CaMC.Remarkably, tribution shows a single peak restricted to low PO limits (Fig. 3J) these channels also open sparsely with their baseline PO sub- like mode E but not A (P < 0.05, KS test). The steady-state PO−V = ± stantially diminished (Fig. 2C), yielding PO,max 0.06 0.02 relationship also exhibits a diminished PO,max (Fig. 3K) identical to ± = (mean SEM; n 5 patches with 492 records). Thus, channels mode E (15) (Fig. 3G), and analysis of open durations (TO) bound to a single CaM lobe undergo a baseline change in gating revealed a monoexponential distribution with decay constant iden- 2+ even before Ca entry, akin to channels that lack prebound tical to kOCjE deduced from mode E openings (Fig. 3L and Fig. CaM altogether. S3D). These uncanny similarities suggest the functional equiva- Assured of the functional necessity for the bilobal architecture lence of channels bound to a single lobe of CaM to those devoid of CaM, we considered whether the binding of the two lobes is of CaM altogether. sufficient to restore channel modulation. More specifically, could If CaV1.3 bound to CaM C lobe is restricted to mode E, then the reconstitution of the N lobe of CaM as a distinct molecular the binding of CaM N lobe may reenable sojourns into mode A. entity enhance the PO of CaV1.3−CaMC? Practically, simple Remarkably, examination of single-channel trials showed that overexpression of the CaM N lobe is insufficient for reliable CaV1.3−CaMC in the presence of β2A−CaMN appeared to switch reconstitution given its weak affinity for the Ca channel CI between epochs of low and high activity, as confirmed from the V  module (Fig. S1B). Consequently, for effective delivery of CaM diary plot of P for individual trials from the same channel (Fig.  O N lobe to the channel complex, we utilized an alternate strategy 3M). The PO distribution obtained from all trials was bimodal, whereby this lobe is fused to the auxiliary β2A subunit (β2A− fitting with channels transitioning between mode E and mode A CaMN), an obligatory component for functional CaV channel behaviors (Fig. 3N). In fact, categorizing trials into high and low complexes (37, 38). Remarkably, single-channel recordings of PO groups revealed conditional PO−V relations with a fivefold CaV1.3−CaMC in the presence of β2A−CaMN revealed enhanced difference in the asymptotic PO,max values for the two categories openings, and the ensemble average showed a partial rescue of (Fig. 3O). Given this mode-switching behavior, channel openings the baseline gating (Fig. 2D)withPO,max = 0.24 ± 0.03 (mean ± may represent sojourns to open states in modes E and A (i.e., SEM; n = 4 patches with 538 records). These findings suggest that either OE or OA). Fitting with this scheme, the open durations the restoration of the complementary lobe may reverse the change in now follow a biexponential distribution well approximated as a channel gating associated with the binding of a single lobe of CaM. weighted sum of exponentials with decay rates, kOCjE and kOCjA, observed for mode E and A openings, respectively (Fig. 3P and

Binding of CaM Lobes Switches PO in Quantized Manner. That said, Fig. S3E). the observed changes in channel PO may result from multiple Thus, while channels bound to the C lobe alone are restricted indirect mechanisms that are unrelated to CaM lobe interac- to mode E gating, the binding of the N lobe, even as a distinct tions. However, if changes in channel PO, in fact, result from molecular entity, is sufficient to switch channels into the high PO CaM lobes engaging distinct regulatory interfaces, then they are gating mode A. The efficacy of mode switching observed here expected to be quantized in agreement with transitions between likely depends on the propensity of both N and C lobes of unique channel conformations (39). Indeed, changes in channel apoCaM to be bound to their respective molecular interfaces on gating associated with apoCaM binding follow this paradigm (15, the channel CT. Thus, the CI module serves as a coincidence 39, 40). More specifically, channels devoid of CaM adopt gating detector that stabilizes channel openings. mode E marked by low PO, while apoCaM binding switches channels into gating mode A with a high P as schematized in Two Detached CaM Lobes Support Channel Regulation. If coexpression O β − − Fig. S3A (15). With CaM C lobe bound, we hypothesize that of 2A CaMN restores mode A gating for CaV1.3 CaMC,then channels may be restricted to mode E, while restoration of the N channels are no longer delimited to a nonpermissive configuration, lobe may reenable sojourns into mode A. suggesting that this maneuver may also restore CDI. Of note, while both N and C lobes of CaM are no longer part of a single mo- To test this hypothesis, we sought to discern the core features + lecular entity, both lobes are capable of binding Ca2 ions (34, 35). of gating modes A and E. Fusion of CaMWT would ensnare Ca 1.3 into mode A (Fig. S3B). Fig. 3A displays 10 sequential Remarkably, both exemplary whole-cell recordings and population V data illustrate the reemergence of CDI, evident as the enhanced trials of the Ca 1.3−CaM single-channel activity evoked by + + V WT decay kinetics of Ca2 versus Ba2 currents (Fig. 4A). The partial voltage ramps introduced at 12-s intervals. The activity of the 2+ channel appears uniformly high, as confirmed by the diary plot of rescue of Ca regulation observed here accords well with the partial restoration of mode A gating following reintroduction of average P within individual trials (P , Fig. 3A) and a unimodal O O theCaMNlobe(Fig.3M–P). Furthermore, the targeted delivery of  + PO distribution obtained from a larger set of trials (Fig. 3B). The 2 CaMN12 with Ca binding to N lobe impaired also sufficed to steady-state PO−V relationship shown in Fig. 3C illustrates the rescue CDI to the CaV1.3−CaMC channels (Fig. 4B). Similarly, the high maximal open probability (PO,max) characteristic of mode A combination of β2A−CaMN and CaV1.3−CaMC34 also resulted in gating. We further assessed the distribution of open durations low levels of CDI, revealing the N-lobe component of CDI for the (TO) in our single-channel trials during epochs where the reconfigured channel arrangement (Fig. 4C). Reassuringly, coex- membrane potential ranged between −30 mV and +20 mV. The pression of β2A−CaMN12 with CaV1.3−CaMC34 resulted in strong open durations largely followed a single-exponential decay con- reduction of CDI (Fig. 4D) in comparison with all three conditions sistent with a single open state in gating mode A (OA) with an above (Ca 1.3−CaM /β −CaM , P = 2E-5; Ca 1.3−CaM /β − ≈ −1 V C 2A N V C 2A exit rate, kOCjA 1.7 ms (Fig. 3D and Fig. S3B) (40). By CaM , P = 2E-3; Ca 1.3−CaM /β −CaM , P = 5E-3). Thus, N12 V C34 2A N + contrast, for CaV1.3MQDY deprived of CaM, openings are uni- both N and C lobes of CaM are capable of decoding Ca2 signals as formly sparse, with low single-trial activity (P ) (Fig. 3E). The  O disparate molecular entities; however, channel modulation ensues overall PO distribution remains unimodal but is now restricted to if and only if both lobes are present in the same complex. low activity limits (Fig. 3F), distinct from CaV1.3−CaMWT [P < A corollary to this principle is that, if two C lobes were teth- − 0.05, Kolmogorov Smirnov (KS) test]. Thus, CaV1.3MQDY ered (CaMCC), the bilobal requirement would be satisfied and adopts, almost exclusively, mode E with the steady-state PO−V the channels would be able to elicit channel regulation. Re- relationship exhibiting a drastically reduced P (Fig. 3G and markably, coexpression of CaM with Ca 1.3 demonstrated O,max + CC V Fig. S3C). The open durations also followed a single-exponential robust functional Ca2 modulation (Fig. 4E), unlike channels

E3030 | www.pnas.org/cgi/doi/10.1073/pnas.1716381115 Banerjee et al. Downloaded by guest on September 25, 2021 + bound to CaM (Fig. 1F). In comparison, coexpression of CaM lobes of Ca2 -free CaM is necessary to stabilize the Ca CI in the C + CC1234 V with all four EF hands Ca2 -insensitive results in a dramatic re- extended conformation. The switching between the extended and duction in CDI, further corroborating the ability of CaMCC to bent conformations appears to correlate with channels transition- trigger CDI (P = 1E-4; Welch’s t test) (Fig. 4F). Thus, while a ing between gating modes A and E,respectively. single C lobe alone is incapable of supporting channel regulation, To experimentally corroborate this conformational change, we two of the same lobes together elicit robust modulation. Thus, the purified recombinant CaV1.3 CI module in complex with CaMWT bilobal architecture of CaM is both necessary and sufficient for and CaMC alone (Fig. S6A), and utilized SAXS analysis to probe + signaling to CaV channels. their overall molecular shapes under low Ca2 conditions. Both CaMWT- and CaMC-bound complexes eluted as monodisperse CaV CI Module Rearranges Itself if Only CaM C-Lobe Is Bound. With entities in size exclusion chromatography with elution volumes the functional requirement for bilobal CaM established, we consistent with a 1:1 stoichiometry (Fig. S6 B and C). Experimental probed whether discrete changes in channel gating results from parallel conformational rearrangements of the CI module. As solution scattering profiles and P(r) distributions revealed key dif- ferences between the CaMWT-andtheCaMC-bound complexes currently available atomic structures of CaV−CaM complex are limited to short segments (e.g., the IQ domain) and only in the (Fig. 5E). Furthermore, ab initio simulations (47) revealed distinct + presence of Ca2 (41–45), we constructed a homology model of coarse-grain molecular envelopes for the CI in complex with CaMWT F CaV1.3 CI bound to apoCaM based on known structures of and CaMC (Fig. 5 ). Interestingly, the molecular shape of the CaV1.1 (46) and NaV1.5 (31) (Fig. S4). The CI adopts an ex- CI/CaMWT complex fits well with the extended conformation as tended conformation with an apoCaM preassociation interface in our homology model before (Fig. 5F, Left and Fig. S6D)and that aligns well with hotspots identified from systematic func- after MD relaxation (Fig. S6 E and F). By contrast, the shape of tional analysis (Fig. S4D) (17). CI/CaMC complex overlaid closely with only the bent confor- Given this overall agreement, we performed explicit-solvent mation with the IQ reoriented (Fig. 5F, Right and Fig. S6G) MD simulations of the homology model to probe CaM-dependent and not the extended conformation (Fig. S6H). Overall, these structural rearrangements of CaV1.3 CI. Analysis of 100-ns tra- results substantiate a marked conformational change of the jectory showed equilibration of CaMWT-bound CI with minor Ca CI depending on the coincident binding of two CaM lobes. “ ” V deviations from its initial extended conformation (Fig. 5A and Ultimately, these conformational changes may be allosterically Fig. S5 A and B). Devoid of CaM, however, the CI undergoes a coupled to the channel pore to diminish openings. Recent dramatic rearrangement resulting in a marked increase in the Cα structures of CaV1.1 and NaV1.4 show that the CI module is in- rmsd and the reorientation of the IQ toward the EF1,2 segments, timately associated with the III−IV linker (46, 48). Accordingly, termed a “bent” conformation (Fig. 5B and Fig. S5C). Of note, one possibility is that rearrangement of the CI module between the IQ and EF1,2 subdomains themselves remain stable (Fig. S5D). Strikingly, this change in CI conformation with the loss of the extended and bent conformations may preferentially bias the orientation of the III−IV linker and the propensity of the inner prebound CaM parallels functional changes in channel PO,sug- gesting that the transitions between gating modes A and E may be S6 gates to twist open (46, 48). It is noteworthy that CaV1.1 have related to changes in CI conformation. Fitting with this hypothesis, poor apoCaM binding (49), suggesting that the currently available the CaMC-bound CI again reorients into the bent conformation, structures of CaV1.1 may correspond to a preinhibited confor- PHYSIOLOGY with the IQ deflected toward the EF1,2 (Fig. 5C and Fig. S5 E and mation (46). Alternatively, CI changes may alter the folding of F). In sharp contrast, when CaMCC (i.e., with two identical C distal S6 segments and change channel activation (50, 51). Re- lobes) is bound, the CI maintains its extended conformation (Fig. solving the mechanisms by which CaM/CI module conformations 5D and Fig. S5G). These results suggest that the binding of two couple to the pore domain remains an important frontier.

ABCD EF

2+ Fig. 4. Reconstitution of detached CaM hemilobes is sufficient to restore CaV regulation. (A) Coexpression of β2A−CaMN partially restores Ca regulation to 2+ CaV1.3−CaMC channels, suggesting that the presence of two lobes of CaM is sufficient to evoke channel Ca feedback regulation. Format is as in Fig. 1A.(B) 2+ 2+ Reconstitution of CaM N lobe with its Ca binding disabled (β2A−CaMN12) is also sufficient to partially rescue Ca regulation of CaV1.3−CaMC. Format is as in 2+ A.(C) Reconstitution of β2A−CaMN with CaV1.3−CaMC34 results in small residual CDI. (D)Ca regulation is absent following reconstitution of β2A−CaMN12 to CaV1.3−CaMC34.(E) Exemplary current records show robust CDI of CaV1.3 mediated by CaMCC, CaM variant with two identical C lobes. Population data confirms 2+ strong CDI of CaV1.3 when bound to CaMCC.(F) Coexpression of CaMCC1234,withCa binding disabled to its two C -lobes, strongly reduces CDI of CaV1.3.

Banerjee et al. PNAS | vol. 115 | no. 13 | E3031 Downloaded by guest on September 25, 2021 AD+CaMWT B -CaM C +CaMC +CaMCC

CaV1.3 CI

CaM N-lobe CaM Fig. 5. Binding of CaM lobes evokes distinct con- CaM CaM C-lobe C-lobe formational rearrangements for CaV1.3 CI. (A)MD C-lobe simulation shows the relative stability of the CaV1.3 CI bound to CaM. (Top)Cα rmsd for Ca 1.3 CI region ‘extended’ ‘bent’ ‘bent’ ‘extended’ V for 100-ns trajectory. (Bottom) Structural model fol- 12 lowing 40-ns equilibration. The angle between the

IQ domain and a helix within EF1,2 is shown, to fa- cilitate comparison of conformational changes. (B) 1.3 CI

V MD simulation shows the dramatic conformational RMSD (A) a  C  rearrangement of CaV1.3 CI module devoid of CaM.

C 10 ns Format is as in A.(C)CaV1.3 CI module bound to CaM 0 0ns 0ns 0ns C lobe alone also undergoes a conformational rear- 0ns rangement. Format is as in A.(D)CaV1.3 CI bound to CaMCC undergoes minor structural reorientations. EFFormat is as in A.(E) Small-angle X-ray solution 103 CaV1.3CI 0.025 SAXS CaM CaV1.3CI scattering profile of the CaV1.3 CI module in complex WT envelope CaMC with CaMWT (black) or CaMC (red). (Left) Scattering P(r) intensity is plotted as a function of momentum transfer. (Right) Radial pair-distribution function [P(r)] intensity is computed for radial vectors (r) and describes the set CaM CaM C-lobe CaM of all paired distances within the structure. (F)(Left) 1 0 N-lobe C-lobe Ab initio molecular envelope of CaV1.3 CI/CaM 0 q (A-1) 0.3 0 r (A) 70 CaV1.3CI complex overlaid on a homology model (Fig. S4D). (Right) Ab initio molecular envelope of CaV1.3 CI/ apoCaM CaMC complex overlaid on the MD-relaxed model. Ca2+/CaM lobes bind G 1-lobe bound 2-lobes bound (G) Schematic shows a unifying model of CaM regu- multiple effectors lation of CaV1 channels. Devoid of CaM, the channel CI adopts a bent conformation, with the IQ reor- `` `` `` `` iented toward the EF domains, while openings are 12CaM 3 Ca2+ 4 1,2 allosterically diminished. If only one apoCaM lobe is bound, the bent conformation of the CI persists, and channel openings remain diminished. Binding of two apoCaM lobes switches the CI into an extended CaM CaM conformation, and this rearrangement enhances + channel openings. On Ca2 binding, one or two lobes CI ‘bent’ ‘bent’ ‘extended’ ‘bent’ of CaM depart from its preassociation interface, and pore mode E mode E mode A mode E the CI module reverts to a bent conformation and low PO low PO high PO low PO channel openings are diminished.

Discussion regulation of CaV channels whereby the dislodging of a lobe of The dual-lobe architecture is a salient feature of many cytosolic CaM from its preassociation interface is sufficient to trigger + Ca2 signaling proteins, including CaM (3, 52, 53), channel regulation (Fig. 5G) as follows: (i) Devoid of CaM, the (54), various neuronal calcium sensors (55, 56), and members of CaV CI module adopts a bent conformation, while channels the S100 family that function as dimers (57). Here, we find that exhibit a low PO mode E gating behavior characterized by sparse the bilobal nature of CaM is a fundamental requirement for its and brief openings (15). (ii) Following the binding of a single ability to regulate Ca channels, a biologically vital feedback loop apoCaM lobe, the CI module continues to be destabilized in the V + (7–9, 12), and a prototype for deducing general Ca2 -decoding bent conformation, and channels continue to reside in mode E. principles (58, 59). Ca channels bound to a single CaM lobe alone (iii) However, the concurrent binding of both N and C lobes of V + entirely fail to be Ca2 -modulated, and instead adopt a pre- apoCaM stabilizes the CI module in the extended conformation and channels switch into mode A marked by more frequent and inhibited configuration like channels that lack CaM altogether. + Strikingly, reconstitution of the complementary CaM lobe (N lobe) longer channel openings. (iv) Following Ca2 influx, one or two as a disparate molecular entity relieves channel preinhibition and lobes of CaM may dislodge en route to its binding interface, thus + restores Ca2 feedback. These changes in function parallel a CaM- destabilizing the CI module into the bent configuration and dependent conformational rearrangement of the CI module be- reverting channels into mode E. For example, for CaV1.3 and 2+ tween extended and bent conformations that, when transduced to CaV1.2, the N-terminal spatial Ca transforming element the channel pore domain, switches channels between permissive (NSCaTE) loci on the channel amino terminus serves as an ef- and preinhibited modes. These findings revise long-held mecha- fector domain for N-lobe CDI (58, 67). Mechanistically, N-lobe 2+ nistic views of CaV regulation. CDI results from a multistep process where Ca -free N lobe Traditional models of Ca regulation have centered on the prebinds the CI module. Following transient dissociation and + V + Ca2 -dependent association of CaM with multiple effector do- binding of Ca2 , the N lobe interacts with the NSCaTE interface mains embedded across various cytosolic loops (7, 17, 60–67). to trigger CDI (58, 67). Subsequently, this process may couple to Although disparate, these binding events are all thought to the pore domain via formation or disruption of bridge between communicate to the transmembrane pore domain to throttle the channel amino termini and carboxy termini mediated by + channel openings. How do Ca2 /CaM interactions with unique CaM (68) or via direct interaction (69). Our present findings molecular interfaces all support the same end-stage outcome? suggest that the disengagement of apo N lobe from the CI in- + Our findings here point to a unifying release model for Ca2 terface and subsequent conformational rearrangement of the CI

E3032 | www.pnas.org/cgi/doi/10.1073/pnas.1716381115 Banerjee et al. Downloaded by guest on September 25, 2021 + module triggers CDI. More broadly, Ca2 /CaM interaction with Materials and Methods 2+ any of the multiple Ca /CaM effector interfaces encoded within Expression and Purification. The cDNA corresponding to the CT the channel cytosolic domains would evoke channel regulation. fragment of the rat Cav1.3 channel α-subunit (amino acids 1473 to 1629) was More broadly, our results highlight the versatility of CaM to cloned into the pGEX-6-P1 vector carboxyl terminal to GST with a PreScission 2+ process Ca signals and thereupon perform complex molecular site encoded in the linker. Homo sapiens CaM gene was cloned into computations. Canonical CaM signaling entails the interaction of the pET24b vector using the BamHI and NdeI sites following PCR amplifi- 2+ Ca /CaM lobes with a target, such that individual lobes are cation, yielding CaMWT/pET24b vector. BL21-CodonPlus RIL (DE3) cells were often sufficient to activate the target, while the two lobes to- transformed with both the Cav1.3-containing and CaM-containing plasmids + gether merely enhances the overall affinity and Ca2 sensitivity simultaneously. The cells were grown overnight at 37 °C in 8 L of LB medium + (70). Prominent examples include Ca2 /CaM-dependent kinases, supplemented with 100 μg/mL of kanamycin, 100 μg/mL of ampicillin, and μ myosin light chain kinase, and nitric oxide synthase (4, 5). For 100 g/mL of chloramphenicol until they attained an optical density (OD600) Ca channels, previous studies have argued for the canonical of 0.9. Subsequently, protein expression was induced with 0.1 mM Isopropyl V β mode of CaM function based on the finding that channel run- -D-1-thiogalactopyranoside for 16 h to 18 h at 18 °C. The cells were har- vested by centrifugation and resuspended in lysis buffer (12 mM Tris·HCl + down can be slowed via reconstitution of individual CaM lobes + (71). In sharp contrast, our results suggest that CaM implements 150 mM NaCl 5 mM DTT, pH 7.5). Cell lysis was performed by micro- fluidization. Following removal of cell debris, the supernatant was collected a novel molecular operation—a logical NOR gate that decodes 2+ and incubated with GST beads for 2 h at 4 °C to facilitate complete binding Ca signals—with high levels of CaV1.3 activity occurring only 2+ of GST-fused proteins to the beads. The beads were washed with wash on the coincident binding of two Ca -free CaM lobes. The · + + + 2+ buffer (25 mM Tris HCl 150 mM NaCl 10 mM MgCl2 5 mM DTT) thrice nonlinearity in Ca sensing is illustrated by the ability of two to remove any unbound proteins or nonspecific binding to the column. tethered C lobes to support channel modulation even while a Cav1.3 CT region in complex with CaM was then eluted by incubating the single C lobe fails to impart even a partial regulatory effect. beads overnight with PreScission protease. The supernatant yielded the 2+ Importantly, the two lobes of CaM possess distinct Ca -binding desired Ca 1.3−CT/CaM complex. 2+ V kinetics that enables them to differentially decode spatial Ca The complex eluted as a monodispersed peak when run through HiLoad signals (58). For example, the C lobe senses local, large- 26/600 Superdex 200-pg size exclusion column. The isolated complex was 2+ + amplitude Ca signals, while the N lobe preferentially decodes concentrated and dialyzed with Ca2 -free solution (25 mM Tris·HCl + 150 mM 2+ lower-amplitude global signals. As spatial Ca selectivity is NaCl + 5 mM DTT + 2% glycerol + 10 mM MgCl2 + 5 mM EGTA) to ensure tunable in an analog fashion (58), its conjunction with digital CaM is in its apo form. A similar protocol was followed for isolating CaV1.3 logic operations enables a wide repertoire of molecular compu- CT peptide in complex with CaM C lobe alone. Here, the CaM C lobe (resi- 2+ tations to process and interpret cellular Ca signals. The CaV dues 77 to 148) was cloned in place of full length CaM in the CaMWT/pET24b channel CI module serves as an exemplary that vector described above using BamH1 and NdeI restriction sites. explicitly performs such computations. As this module is con- Small-Angle X-Ray Scattering Data Collection. Twenty-five microliters of protein served across CaV and NaV channel families, these principles 2+ may elaborate general algorithms of Ca computing. In this solution containing Cav1.3 CT peptide in complex with either CaMWT or CaM C regard, the NOR gate is considered “universal” in digital elec- lobe were pipetted into 96-well PCR plates (VWR catalog 10011-228; Corning tronics, as its combination can implement any Boolean opera- Axygen) at 2.5 mg/mL and 5.0 mg/mL each. The trays were sealed with a silicone tion. Thus, generalization of the CI module may enable a wide lid (VWR catalog number 10011-130; Corning Axygen), flash frozen, and sent to range of related logical operations that allow cells to extract the Advanced Light Source (ALS) Structurally Integrated Biology for Life Sci- PHYSIOLOGY + complex spatial and temporal features of Ca2 signals (72). ences (SIBYLS) beam line (12.3.1) at Lawrence Berkeley Lab. SAXS data were collected by the beam line staff through the SIBYLS beam line mail-in program. These findings also bear important biophysical and physio- For each protein concentration, the sample was exposed for 0.3 s within 10-s logical implications for CaV channel function. First, as CaM acquisition blocks, resulting in 33 frames per sample. Buffer subtractions and possesses a high affinity for CaV channels, disengaging it from subsequent analysis were performed with the program ScÅtter 3.0 (www. the channel complex either pharmacologically or via regulatory bioisis.net/scatter). Ab initio molecular shapes were determined using the proteins may be a formidable task. However, our results suggest DAMMIF module (47) in ATSAS package. Average molecular shape was de- that dislodging a single lobe of CaM, with a far weaker affinity termined with 10 solutions obtained from DAMMIF using the DAMAVER for the channel, suffices to diminish channel activity. Antipsy- module (83) in the ATSAS suite. chotics and CaM antagonists such as trifluoperazine and calm-

idazolium may utilize such a mechanism to inhibit CaV channels Molecular Modeling. Homology models of CaV1.3CT in complex with CaM 2+ independent of Ca ions (73, 74). Similarly, auxiliary signaling (CaV1.3CT/CaM) were made using MODELLER (84). As templates, we utilized the proteins such as the family of neuronal CaBPs may utilize a atomic structure of NaV1.5 CT in complex with CaM (PDB ID: 4OVN) and the EF similar strategy and allosterically alter CaM binding to shunt hand and preIQ segments of the cryoelectron microscopy structure of CaV1.1 CaM signaling to CaV channels (30). For NaV channels, the (PDBID:5GJV).TomodeltheCaV1.3CT peptide bound to CaM C lobe, we de- binding of fibroblast homologous factor (FHF) leted the N lobe of CaM (residues 1 to 78) from the initial CaV1.3CT/CaM model. dislodges the N lobe of CaM from its preassociation interface of Na channels and alters channel function (31, 75). Second, as MD Simulations. MD simulations in the presence of explicit water molecules V + CaM may engage in local signaling to downstream Ca2 -dependent were performed using Amber14 (85). The ff12SB force field was used for the simulation. The systems were minimized first using a combination of enzymes (76–78)recruitedtotheCaV channel complex, our results highlight the possibility that a single CaM could multiplex channel steepest descent and conjugated gradient methods for 2,000 steps. A posi- regulation and activation of downstream enzymes. Specifically, tional restraint (10 kcal/mol) was applied on all protein atoms except hy- while apoCaM prebound to the channel up-regulates its activity, drogen atoms during the minimization step. The whole system was + Ca2 /CaM migrating to a downstream target enzyme may dually minimized again using a combination of steepest descent and conjugated gradient methods for 20,000 steps without any positional restraint. A time activate the target and diminish channel activity. Third, our re- step of 2 fs was used for all subsequent heating, equilibration, and pro- sults suggest an allosteric linkage between the conformation duction runs with the SHAKE option on all bonds containing H atoms. of the CI/CaM complex and distinct channel gating modes. As Langevin dynamics was used for temperature control in the heating, equil- transitions between these modes (39) are also governed by a ibration, and production steps. The minimized system was heated from 0 K multitude of factors, including dihydropyridines (79), voltage to 300 K in 0.5 ns. Weak positional restraints (2 kcal/mol/Å2) on all protein depolarization (80), phosphorylation (81), and G proteins atoms were applied during the heating cycle. Constant pressure equilibra- (82), resolving how the CaV channels integrate these disparate tion was done at 300 K for 1 ns, and positional restraint was applied on all signaling modalities represents an exciting new frontier. backbone atoms in the protein. Finally, a trajectory of 100 ns was generated In all, our findings unravel the sophisticated mechanisms by during the production run. All of the subsequent analysis of the MD tra- which CaM exerts potent feedback control of its targets. jectory was done using Ambertools14.

Banerjee et al. PNAS | vol. 115 | no. 13 | E3033 Downloaded by guest on September 25, 2021 Molecular Biology. All engineering of CaV1.3 was performed with a truncated ramps between −80 mV and +50 mV (currents between −50 and 40 mV variant of rat α1D (AF3070009), CaV1.3Δ1626 as previously described (17, 30), displayed and analyzed). For each patch, more than 100 to 200 sweeps were containing a unique XbaI site immediately upstream of its stop codon. recorded with a repetition interval of 12 s. Patches with one to three − − − CaV1.3 CaMWT,CaV1.3 CaM1234, and CaV1.3 CaM12 constructs were con- channels were analyzed as follows: (i) The leak for each sweep was fitted structed as described previously (30). The CaV1.3 MQDY variant was also as and subtracted from each trace. (ii) The unitary current relation, i(V), was described previously (57) with I[1608] substituted with methionine. For fitted to the open-channel current level using the following equation (15) − constructing CaV1.3 CaMC, we PCR-amplified wild-type CaM residues 79 to (Fig. 2 A–D, slanted gray line): 148 and ligated it into CaV1.3Δ1626 following restriction digest with SpeI and

XbaI enzymes. This maneuver yielded CaM C lobe fused to the CaV1.3 with iðVÞ = −g · ðV − VSÞ · expð−ðV − VSÞ · z · F=ðR · TÞÞ=ð1 − expð−ðV − VSÞ · z · F=ðR · TÞÞÞ a linker SSGGGGSGGG. To generate CaV1.3−CaMC34, we similarly PCR-amplified CaM1234 residues 79 to 148 using identical primers, and the resultant product where g is the single-channel conductance (∼0.02 pA/mV), z is the apparent was ligated into CaV1.3Δ1626 following SpeI/XbaI restriction digest. This con- valence of permeation (∼2.1), F is Faraday’s constant, R is the gas constant, struct also utilized the linker (SSGGGGSGGG). For constructing β −CaM ,we 2a N and T is the temperature in degrees Kelvin. All these parameters were held first engineered rat β (NM_053851.1) pcDNA3 construct such that its stop 2a constant for all patches, except for slight variations in the voltage-shift pa- codon was flanked by BamHI and XbaI restriction sites. Subsequently, CaM N rameter V ≈ 36 mV, as detailed below. (iii) All leak-subtracted traces for lobe (residues 1 to 78) was PCR-amplified and ligated into the engineered β − S 2a each patch were averaged (and divided by the number of channels in the pcDNA3 using restriction sites BamHI and XbaI. In so doing, we generated the patch) to yield the current−voltage (I–V) relationship for that patch. Since fused β2a−CaMN with a linker sequence GSGGGGGGGG. We used a similar slight variability in VS was observed among patches, we calculated an av- strategy to construct β2a−CaMN12 whereby the N lobe of CaM1234 (residues 1 to erage V for each construct, V . The data from each patch were then 78) was PCR-amplified and inserted into the β −pcDNA3 construct, again uti- S S,AVE 2a Δ = − Δ lizing enzymes BamHI and XbaI. CaM was constructed such that residues 10 to shifted in voltage by an amount V VS,AVE VS, with V typically CC ± 76 of wild-type CaM are replaced with residues 83 to 148 of CaM, all within about 5 mV. This maneuver allowed all patches for a given construct to share a common open-channel GHK relation. Thus, shifted, the I–V relations the pcDNA3 vector. CaMCC1234 was synthesized by alanine substitution of resi- obtained from different patches for each construct were then averaged dues D[21], D[57], D[94], and D[130] of CaMCC. For FRET experiments, CFP-tagged together. (iv) P at each voltage was determined by dividing the average I CaM was constructed as previously described (86). For CFP–CaMN or CFP–CaMC O constructs, we replaced CaM in CFP–CaM with PCR-amplified CaM N lobe or C (determined in step iii above) into the open-channel GHK relation. lobe, respectively, using unique restriction sites NotI and XbaI. YFP–CI and YFP–IQ

constructs were obtained by fusing enhanced YFP to either the α1D carboxy FRET Two-Hybrid Analysis. We conducted FRET two-hybrid experiments in terminus or IQ domain, respectively, as previously described (17, 33). HEK293 cells cultured on glass-bottom dishes using an inverted fluorescence microscope as extensively described by our laboratory (29). Fluorescence Whole-Cell Recording. Whole-cell recordings were obtained using an Axo- measurements were obtained from cells cotransfected with ECFP- and EYFP- patch 200A amplifier (Axon Instruments). Electrodes were made from bo- tagged FRET partners bathed in Tyrode’s solution [138 mM NaCl, 4 mM KCl,

rosilicate glass capillaries (MTW 150-F4; World Precision Instruments) yielding 1 mM MgCl2, 2 mM CaCl2, 10 mM Hepes (pH 7.4 using NaOH), 0.2 mM Ω 1- to 2-M resistances, which were, in turn, compensated for series resistance NaHPO4,and5mMD-glucose]. Fluorescence measurements were obtained from by 70%. Currents were low-pass filtered at 2 kHz before digital acquisition at single cells using CFP, YFP, and FRET fluorescent cubes, and 33-FRET efficiencies 10 kHz. A P/8 leak-subtraction protocol was used. The internal solution at were estimated as previously described (29). Binding curves were determined by 300 mOsm, adjusted with TEA-MeSO3, contained the following: CsMeSO3, least-squares error minimization of data from multiple cells, with relative affinity

114 mM; CsCl, 5 mM; MgCl2, 1 mM; MgATP, 4 mM; Hepes (pH 7.4), 10 mM; and (Kd,EFF = 1/Ka,EFF) and maximal FRET efficiency allowed to vary. BAPTA [1,2-bis(o-aminophenoxy)ethane- N,N,N0,N0-tetraacetic acid], 10 mM; at 290 mOsm adjusted with glucose. The bath solution at 300 mOsm, adjusted Transfection of HEK293 Cells. HEK293 cells were cultured on glass coverslips in with TEA-MeSO3, was as follows: TEA-MeSO3, 102 mM; Hepes (pH 7.4), 10 mM; a 10-cm dish and transfected using a calcium phosphate method (20). Typ- CaCl2 or BaCl2, 40 mM. Data for each construct were obtained from two to four ically, we transfected 8 μgofCaV1.3 α1 subunit and variants, 8 μg from independent transfections. For statistical comparison of CaV1.3 inactivation β2a (87) or β2a fused to single or bilobal CaM, 8 μg from rat α2δ (88) under various conditions (Figs. 1 and 4), we performed Welch’s t test to com- (NM012919.2), and 4 μg of YFP-tagged Myosin Va IQ as CaM sponge (29), pare the magnitude of CDI quantified as 1 − r /r obtained at +10 mV. 2-6 Ca Ba and 2 μg of SV40 T antigen was also cotransfected to enhance expression.

For experiments in Fig. 4F,8μg of CaMCC or CaMCC1234 were added, as ap- Single-Channel Recording. Cell-attached single-channel recordings were per- propriate. For FRET two-hybrid experiments, HEK293 were cultured on glass- formed at room temperature, using previously established methods from our bottom dishes and transfected using a standard polyethylenimine protocol laboratory (15) (Axopatch 200A; Axon Instruments). Patch pipettes (8 MΩ to (89). Epifluorescence was collected 1 d to 2 d after transfection. 10 MΩ) were pulled from ultra-thick-walled borosilicate glass (BF200-116-10; Sutter Instruments) further coated with Sylgard. Currents were filtered at ACKNOWLEDGMENTS. We thank G. L. Hura, K. Burnett, and the staff of 2 kHz to 5 kHz. The pipette solution contained 140 mM tetraethylammo- SIBYLS beam line 12.3.1 at the Advanced Light Source (ALS) and Wanjun nium methanesulfonate, 10 mM Hepes, and 40 mM BaCl , at 300 mOsm 2 Yang for dedicated technical support. We thank Ivy Dick and Christian adjusted with tetraethylammonium methanesulfonate, and pH 7.4 adjusted Wahl-Schott for insightful comments and for gifting β2A−CaM34 construct. with tetraethylammonium hydroxide. To zero membrane potential in all + This work is supported by National Heart, Lung, and Blood Institute Grant single channel experiments, the bath contained 132 mM K -glutamate, HL128743 and National Institute for Mental Health Grant MH065531. SAXS 5 mM KCl, 5 mM NaCl, 3 mM MgCl2, 2 mM EGTA, 10 mM glucose, and 20 mM experiments were carried out using the mailing-in procedure at the ALS Hepes, at 300 mOsm adjusted with glucose, and pH 7.4 adjusted with NaOH. 12.3.1 SIBYLS beam line supported by US Department of Energy program Cell-attached single-channel currents were measured during 200-ms voltage Integrated Diffraction Analysis Technologies Grant DEAC02-05CH11231.

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