Abstract
Diltiazem is a widely prescribed Ca2+ antagonist drug for cardiac arrhythmia, hypertension, and angina pectoris. Using the ancestral CaV channel construct CaVAb as a molecular model for X-ray crystallographic analysis, we show here that diltiazem targets the central cavity of the voltage-gated Ca2+ channel underneath its selectivity filter and physically blocks ion conduction. The diltiazem-binding site overlaps with the receptor site for phenylalkylamine Ca2+ antagonist drugs such as verapamil. The dihydropyridine Ca2+ channel blocker amlodipine binds at a distinct site and allosterically modulates the binding sites for diltiazem and Ca2+. Our studies resolve two distinct binding poses for diltiazem in the absence and presence of amlodipine. The binding pose in the presence of amlodipine may mimic a high-affinity binding configuration induced by voltage-dependent inactivation, which is favored by dihydropyridine binding. In this binding pose, the tertiary amino group of diltiazem projects upward into the inner end of the ion selectivity filter, interacts with ion coordination Site 3 formed by the backbone carbonyls of T175, and alters binding of Ca2+ to ion coordination Sites 1 and 2. Altogether, our results define the receptor site for diltiazem and elucidate the mechanisms for pore block and allosteric modulation by other Ca2+ channel–blocking drugs at the atomic level.
SIGNIFICANCE STATEMENT Calcium antagonist drugs that block voltage-gated calcium channels in heart and vascular smooth muscle are widely used in the treatment of cardiovascular diseases. Our results reveal the chemical details of diltiazem binding in a blocking position in the pore of a model calcium channel and show that binding of another calcium antagonist drug alters binding of diltiazem and calcium. This structural information defines the mechanism of drug action at the atomic level and provides a molecular template for future drug discovery.
Introduction
Benzothiazepines (BZTs), 1,4-dihydropyridines (DHPs), and phenylalkylamines (PAAs) are three major classes of voltage-gated Ca2+ channel blockers, which are widely used in the therapy of cardiovascular disorders, such as hypertension, angina pectoris, and cardiac arrhythmia (Triggle, 2007; Zamponi et al., 2015; Godfraind, 2017). These therapeutic agents were first introduced into clinical practice 40 years ago and are still prescribed to millions of patients. Diltiazem belongs to the BZT class of Ca2+ channel antagonists and has been shown to inhibit the L-type Ca2+ currents conducted by CaV1.2 channels in cardiac and vascular smooth muscle in a voltage- and activity-dependent manner (Lee and Tsien, 1983; Hondeghem and Katzung, 1984; Hockerman et al., 1997; Catterall, 2000; Zamponi et al., 2015), similar to local anesthetics acting on NaV channels (Hille, 1977). The mechanisms of action of representative DHPs and PAAs at two distinct receptor sites on Ca2+ channels have been elucidated at the structural level (Tang et al., 2016). Diltiazem is receiving increased clinical use (Tamariz and Bass, 2004), but how it targets the Ca2+ channel, induces state-dependent block, and interacts allosterically with bound Ca2+, DHPs, and PAAs remains elusive.
Mammalian voltage-gated Ca2+ channels consist of four homologous six-transmembrane domains that form a central pore with four surrounding voltage-sensor modules (Catterall, 2011; Zamponi et al., 2015). These channels most likely evolved from a prokaryotic ancestor, which is exemplified by the homotetrameric bacterial voltage-gated sodium channel, NaVAb (Ren et al., 2001; Payandeh et al., 2011, 2012). By re-engineering the selectivity filter of NaVAb, we constructed a model Ca2+-selective channel, CaVAb, to decipher the structural basis of ion selectivity and conductance and the mechanism of inhibition by DHPs and PAAs (Tang et al., 2014, 2016). We found that CaVAb mimics the Ca2+ selectivity of mammalian cardiac CaV1.2 channels exactly, which allowed us to image bound Ca2+ in the pore and define the conductance mechanism (Tang et al., 2014). We also found that CaVAb is blocked by PAAs and DHPs with similar mechanisms and affinities as mammalian CaV1.2 channels, and we defined the separate binding sites for these two distinct classes of Ca2+ antagonist drugs and elucidated their allosteric interactions (Tang et al., 2016). In our previous work, we were unable to solve the crystal structure of CaVAb with diltiazem bound at high resolution. Here, using improved biochemical and crystallographic methods, we reveal the structural basis for diltiazem block of CaVAb channels by X-ray crystallography and define the molecular mechanism for allosteric coupling among the binding sites for Ca2+, DHPs, and BZTs.
Materials and Methods
In brief summary, CaVAb and its derivative constructs were expressed in Trichopulsia ni insect cells and purified using anti-Flag resin and size exclusion chromatography, reconstituted into 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate bicelles, and crystallized over an ammonium sulfate solution containing 0.1 M Na-citrate (pH 5.0) in the presence of drugs of interest. The tertiary complex of diltiazem-CavAb-DHP was prepared by addition of diltiazem from the beginning of purification, and addition of the second drug (DHP) before reconstitution into the 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate bicelles. The data sets for the diltiazem-bound complex and the tertiary complexes of diltiazem-CaVAb-DHP were collected at 1.0 Å wavelength at advanced light source. Electrophysiological experiments were performed in Trichopulsia ni cells using standard protocols. Details of our methods are provided online in the Supplemental Material. Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6KE5 and 6KEB.
Results
State-Dependent Inhibition of CaVAb by Diltiazem.
The structure of diltiazem (Fig. 1A) and related benzothiazepines differs substantially from PAAs and DHPs. Nevertheless, ligand-binding and site-directed mutagenesis studies suggest that the receptor sites for PAAs and BZTs overlap at least partially (Kraus et al., 1996; Hockerman et al., 2000; Dilmac et al., 2003). Similar to other Ca2+-channel blockers, diltiazem inhibits CaVAb in a complex state-dependent way. It blocks the channel in its resting state with an IC50 value of 41 μM (Fig. 1, B and D, green). In contrast, its inhibitory effect is strengthened by repetitive depolarizing stimuli that activate and inactivate the channel, yielding an increased potency of 10.4 μM for use-dependent block (Fig. 1, B and D, black). These observations suggest that diltiazem has a higher affinity for the channel in the open and inactivated states, consistent with the modulated receptor hypothesis (Hille, 1977), which postulates that the state of the channel influences drug binding to a site located within the pore in a voltage- and state-dependent manner (Hille, 1977; Lee and Tsien, 1983; Hondeghem and Katzung, 1984). Remarkably, the IC50 value for diltiazem in blocking CaVAb matches its potency against the mammalian cardiac Ca2+ channel CaV1.2 almost exactly, further validating the relevance of CaVAb as a model for studying the structural basis for drug block.
CavAb block by diltiazem. (A) Structural formula of diltiazem. (B) Concentration curves for inhibition of CaVAb by diltiazem in the resting state (green) at a holding potential of −120 mV with IC50 = 41 μM, and use-dependent block (black) with IC50 = 10.4 μM. (C) Use-dependent block following a train of depolarizations applied at 1 Hz (20 pulses) with IC50 = 10.4 μM (black). Blue and red curves represent use-dependent block by T206S and T206A mutants with IC50 = 40 and 60 μM, respectively. (D) Current records for control and resting-state block by 50 or 100 μM diltiazem during a single depolarization, and use-dependent block by 10 or 100 μM diltiazem during trains of depolarizations.
Diltiazem Binding to the Intracellular Side of the Ion Selectivity Filter.
In mammalian CaV1.2 channels, photoaffinity labeling and site-directed mutagenesis studies suggest that diltiazem binds to a receptor site formed by the pore-lining transmembrane segments IIIS6 and IVS6 (Hering et al., 1996; Kraus et al., 1996, 1998; Hockerman et al., 1997, 2000). To map the diltiazem-binding site in three dimensions, we crystallized and determined the structure of the diltiazem-CaVAb complex at 3.2 Å resolution. As expected, the diltiazem-CaVAb complex adopts a nearly 4-fold symmetric structure (Fig. 2, A and B). Diltiazem binds to the intracellular side of the selectivity filter and the nearby walls of the central cavity (Fig. 2, B and C) by displacing lipids present in the apo-CaVAb structure (Payandeh et al., 2011, 2012). The bound drug precisely fits the extra electron density observed in the drug-bound CaVAb complex (Fig. 2C).
Structural basis of CavAb block by diltiazem. (A) Overall structure of CavAb illustrated with each subunit distinctly colored (PD, pore domain; VSD, voltage-sensing domain). (B) A cross-sectional view of CavAb in complex with diltiazem. (C) A close-up view of diltiazem binding at the intracellular side of the selectivity filter. Diltiazem is shown in stick model fitting into an Fo-Fc omit map colored in magenta and contoured at 3σ. Nearby side chains are highlighted and shown as sticks. An arrow is shown to mark the position of the fenestration in CavAb (S6, S6-helix; P, P-helix).
As initially revealed for NaVAb (Payandeh et al., 2011), fenestrations penetrate the sides of the central cavities in both NaV and CaV channels and connect them to the surrounding lipid bilayer membrane in situ or to the bicelle lipid phase in our crystals (Wu et al., 2016; Pan et al., 2018). When bound to CaVAb, the methoxybenzene ring of diltiazem points toward the fenestration formed between two adjacent S6 helices, making hydrophobic contacts with M209 on S6 of one subunit and T206 on S6 of the neighboring subunit (Fig. 2C; see also Fig. 4B). By bridging two subunits, this binding pose may induce allosteric interactions. M174 and T175 from the P-loop of the neighboring subunit are also in close proximity to the methoxy-aromatic ring and contribute to its binding and to allosteric interactions between subunits (Fig. 2C). The central 1,5-benzothiazepine scaffold of diltiazem lies horizontally beneath the P-helix of one subunit in parallel to the lipid bilayer, with its sulfur atom positioned near the central axis of the pore (Fig. 2, B and C). The tertiary amino group of bound diltiazem is positioned on the intracellular side of the bound drug molecule, facing the central cavity, and the acetyl branch of diltiazem is oriented toward the innermost Ca2+-binding site in the selectivity filter (Site 3), which is unoccupied. T175 and L176 of one P-loop and L176 of an adjacent P-loop appear to hold the 1,5-benzothiazepine moiety in place via hydrophobic interactions from the top (Fig. 2, B and C). Thus, diltiazem occupies the top half of the central cavity of CaVAb and effectively blocks the exit of Ca2+ from the intracellular side of the narrow selectivity filter.
Comparison of the structures of CaVAb in complex with diltiazem and Br-verapamil reveals that the diltiazem-binding site partially overlaps with that of the PAA (Fig. 3), consistent with competitive binding interactions observed in ligand-binding studies (Kraus et al., 1998). Interestingly, the monomethoxybenzene group of diltiazem and the dimethoxybenzene group of Br-verapamil share a similar binding pose in the CaVAb central cavity and both directly contact T206, a key residue in channel inactivation (Gamal El-Din et al., 2019). Mutation of T206 not only impairs Br-verapamil block of CaVAb, but also increases the IC50 value of diltiazem for use-dependent inhibition of the channel (Fig. 1C). Mutation to Ser increases the IC50 value of diltiazem by 4-fold, and mutation to Ala further increases the IC50 value by 6-fold (Fig. 1C), indicating an important role of this residue in determining state-dependent block by both of these classes of drugs (Lee and Tsien, 1983).
Comparison of CavAb block by diltiazem and verapamil. (A) Side view of CaVAb with diltiazem (sticks in green) bound underneath the selectivity filter. Ca2+ is shown as green spheres. The three calcium-binding sites are indicated by the numbers 1, 2, and 3. Portions of the channel are omitted for clarity. (B) Side view of CaVAb with Br-verapamil (sticks in pink) bound reveals overlap between the binding sites of the PAA drug and diltiazem. (C) Side view of CavAb as in (A and B), with superposition of bound diltiazem (green sticks) and verapamil (pink sticks). (D) Orthogonal view of the central cavity of CavAb, showing the overlapping PAA/BZT-binding sites as if one were standing at the bottom of the central cavity and looking upward at the selectivity filter.
Allosteric Interaction between Diltiazem and DHPs on CaVAb.
Previous mutagenesis studies indicated partial overlap of IIIS6 and IVS6 residues that are important for DHP binding with those that are important for diltiazem binding (Kraus et al., 1996, 1998; Hockerman et al., 2000; Dilmac et al., 2003). Diltiazem inhibits PAA binding but stimulates DHP binding to CaV1.2 channels by allosteric mechanisms (Murphy et al., 1983; Goll et al., 1984; Striessnig et al., 1986). Therefore, BZTs and DHPs most likely target Ca2+ channels at distinct, but possibly overlapping, binding sites. By comparison with our previous structure of CaVAb in complex with the DHP amlodipine, the diltiazem-binding site is indeed physically separate from the DHP-binding site, which is located at an intersubunit crevice on the lipid-facing surface of the pore module. To investigate the structural basis for the allosteric interactions between these sites, we determined the crystal structure of CaVAb with both diltiazem and amlodipine bound. Consistent with the structural results obtained with the two antagonists individually, diltiazem and amlodipine are engaged with their respective binding sites on two sides of the S6 segments that form the wall of the CaVAb pore module (Fig. 4, A and B). Bound diltiazem is located inside the pore, whereas amlodipine is more than 11 Å away, docking at the outer lipid-facing surface of the pore, which is separated from the diltiazem site by the S6 segments.
Structural basis for allosteric interactions between diltiazem and dihydropyridines bound to CavAb. (A) Surface representation of CaVAb in complex with diltiazem and amlodipine. (B) Zoom-in view of diltiazem binding at the intracellular side of the selectivity filter. Diltiazem is shown in stick format, along with an Fo-Fc omit map contoured at 3σ. Nearby side chains are highlighted and shown in stick format. (C) Comparison of diltiazem binding to CaVAb alone (green) or in the presence of amlodipine (yellow). Dashed arrows indicate the differences between diltiazem positions. (D) Orthogonal view of (C) with calcium bound to the selectivity filter shown in green spheres.
The structure of the diltiazem-CaVAb in complex with amlodipine reveals changes in the binding pose of diltiazem (Fig. 4, C and D). The distinct binding poses of diltiazem in the absence and presence of amlodipine are illustrated at higher resolution with the associated electron density maps in Supplemental Fig. 1. Similar to its binding mode without the DHPs, the central 1,5-benzothiazepine scaffold of diltiazem lies parallel to the lipid bilayer underneath the selectivity filter. However, compared with the CaVAb complex with diltiazem alone, the 1,5-benzothiazepine scaffold is flipped by ∼180° and the methoxylbenezene moiety is inserted deeper into the fenestration (Fig. 4, C and D; Supplemental Fig. 1). As a result, the positively charged tertiary amino group extends toward the intracellular end of the narrow passage through the selectivity filter and approaches the backbone carbonyl group of Thr175. This backbone carbonyl contributes to the inner Ca2+ coordination site (Site 3) in the selectivity filter, which has the lowest affinity of the three Ca2+-binding sites and is unoccupied by Ca2+ in the presence of diltiazem in our crystals.
Comparison between the diltiazem-CaVAb-amlodipine and CaVAb-amlodipine structures shows that the C-terminal regions of two CaVAb subunits become partially ordered when diltiazem is present, suggesting a global conformational change (Fig. 5). We speculate that the structure of diltiazem bound with amlodipine represents a higher affinity binding pose with the tertiary amino group inserted into the inner end of the ion selectivity filter to engage Ca2+-binding Site 3 formed by the backbone carbonyls of Thr175 (Figs. 4 and 5). This change in binding pose is induced by conformational changes caused by amlodipine binding, which may be similar to the structural changes in the inactivated state to which both amlodipine and diltiazem bind preferentially. The pose of diltiazem bound alone might represent an intermediate state of diltiazem binding, in which the drug has not yet taken its final high-affinity position to plug the selectivity filter. Thus, our structures reveal plasticity of diltiazem binding induced by allosteric interactions with DHP binding and potentially by the conformational transition to the inactivated state.
Structural basis for inhibition of CaVAb by amlodipine and diltiazem in combination. (A) Surface representation of diltiazem and amlodipine bound to CaVAb reveals that these drugs bind to different sides of S6. (B) Overall structure of diltiazem-CavAb-amlodipine shown as ribbons.
Diltiazem and DHPs Alter Ca2+ Binding.
Besides physically blocking the ion-conduction pathway, diltiazem also alters the interactions between Ca2+ and the selectivity filter of CaVAb (Fig. 6). In the diltiazem-CaVAb structure, we observed electron densities at both Sites 1 and 2, which most likely represent bound Ca2+ (Tang et al., 2014). Strikingly, unlike Ca2+ bound to the unblocked channel, these bound ions are off the central axis of the pore when diltiazem is bound (Fig. 6, A and C; see also the electron density map in Supplemental Fig. 1). Ca2+ in Site 1 interacts directly with the carboxyl group of one of four D178 residues, suggesting that it is in a partially dehydrated state. This blocker-induced direct interaction between the Ca2+ at Site 1 and the selectivity filter is very similar to the proposed mechanism by which DHPs allosterically block CaVAb (Tang et al., 2014). These changes in Ca2+ binding provide a plausible explanation for potentiation of diltiazem binding by Ca2+ (Dilmac et al., 2003) and for the allosteric interactions between amlodipine and diltiazem. Consistent with the notion that the diltiazem-CaVAb-amlodipine structure has captured diltiazem transitioning into its high-affinity bound form, Ca2+ bound at Site 1 interacts with a D178 carboxyl side chain that has rotated around one torsion angle to form a hydrogen-bonding network with neighboring side chains (Fig. 6B). This side chain rotation resembles the dunking motion of E177 side chains of NaVAb as they interact with entering Na+ ions (Chakrabarti et al., 2013); therefore, it is likely to represent a normal conformational transition of the pore.
Diltiazem binding modifies Ca2+ binding in the selectivity filter of CavAb. (A and B) Comparisons of selectivity filter ions in the presence of diltiazem (left) and diltiazem + amlodipine (right). Selectivity filter residues 175–179 and diltiazem are shown in stick format, with Ca2+ shown as green spheres and hydrogen bonds shown as dashed yellow lines. (C and D) Comparisons of Ca2+ bound in Site 1 between diltiazem (left) and diltiazem + amlodipine (right). Side chains from D178 and N181 are shown in each case, along with Ca2+ (green spheres), hydrogen bonds (dashed yellow lines), and estimated intermolecular distances (dashed gray lines).
Discussion
Diltiazem Binds in the Central Cavity and Physically Blocks Ca2+ Permeation.
Our structural analysis of diltiazem block of CaVAb has mapped its binding site on the Ca2+ channel in three dimensions in detail. The BZT receptor site is located in the central cavity of the pore, just on the intracellular side of the ion selectivity filter. In this position, diltiazem would prevent conductance of Ca2+ by physically blocking it. This binding position is consistent with electrophysiological results, which show that diltiazem completely blocks Ca2+ current.
Diltiazem Binding Overlaps the PAA Receptor Site.
Radioligand-binding studies have suggested a complex allosteric/competitive binding interaction between BZTs and PAAs. Binding of PAAs was inhibited by diltiazem but substantial PAA binding remained at apparently saturating concentrations of PAAs, consistent with an indirect negative allosteric interaction (Goll et al., 1984). On the other hand, extensive molecular mapping studies using photoaffinity labeling and site-directed mutagenesis revealed overlapping amino acid residues in the BZT and PAA receptor sites (Kraus et al., 1996; Dilmac et al., 2003). Our results are consistent with both aspects of this previous work. On one hand, we show that the diltiazem-binding site clearly overlaps with the PAA-binding site. On the other hand, our results reveal complexities of diltiazem binding that may lead to allosteric/competitive interactions. We find that diltiazem has two binding poses that can be allosterically modulated by amlodipine binding and potentially by voltage-dependent inactivation. The lower affinity binding pose of diltiazem may leave room for PAA binding in their overlapping sites, thereby inducing a mixed allosteric/competitive mode of inhibition in ligand-binding studies.
Diltiazem Interacts Allosterically with Amlodipine.
We previously found that binding of amlodipine induces a global conformational change in CaVAb, which alters its quaternary structure (Tang et al., 2016). Our present studies reveal the structural basis for allosteric interaction between diltiazem and dihydropyridines, consistent with the allosteric binding interactions observed in classic ligand-binding studies of CaV channels (Murphy et al., 1983; Striessnig et al., 1986). Amlodipine binding also modifies the coordination of Ca2+, bringing one Ca2+ ion close to the carboxylate side chain of the D178 residue in the CaVAb subunit that binds amlodipine (Tang et al., 2016). In this study, we also found that drug binding alters the coordination of Ca2+; that is, diltiazem binding to its site in the central cavity induces an allosteric change in Ca2+ coordination. As for amlodipine, it is likely that this change in Ca2+ coordination greatly reduces or blocks ion conductance through the pore.
Diltiazem Binding May Be a Two-Step Process.
Our structures reveal diltiazem in two binding poses. In the absence of other drugs, diltiazem binds loosely to the upper walls of the central cavity, in what appears to be a low-affinity binding mode, but it does not penetrate the ion selectivity filter. In the presence of amlodipine, diltiazem binding appears tighter, and its tertiary amino group extends upward into the inner end of the selectivity filter and interacts with Site 3 formed by the backbone carbonyls of T175. We speculate that this binding pose may be favored by voltage-dependent inactivation, which is also favored by amlodipine binding. Detailed studies of the kinetics of diltiazem binding have also suggested the possibility of two distinct binding poses and partial stepwise binding interactions (Prinz and Striessnig, 1993). Thus, in the absence of other drugs, diltiazem may enter the pore, form a loose channel-blocking complex, and then rearrange to a tighter-binding, more stably blocked complex with bound diltiazem projecting into the selectivity filter from the central cavity upon voltage-dependent inactivation. Conformational changes in the ion selectivity filter that we observed upon inactivation of NaVAb may be responsible for this change in binding of diltiazem.
Diltiazem Binding Modulates Ca2+ Binding in the Selectivity Filter.
Allosteric interactions of bound diltiazem induce high-affinity binding of Ca2+ in the pore, as judged by close the interaction of bound Ca2+ with one D178 side chain. This allosteric change in Ca2+ binding may contribute to pore block and to the energetics of allosteric interactions between the two drug-binding sites. Thus, our structures unveil, at the atomic level, the mechanism of pore block and allosteric interactions of this important class of Ca2+-channel blockers and provide guidance and strategy for developing next-generation BZTs with improved potency and specificity.
Comparison with Ca2+ Antagonist Receptor Sites on Mammalian CaV Channels.
As we prepared this paper for submission, a cryoelectron microscopy study of skeletal muscle CaV1.1 channels revealed the structures of the receptor sites for PAAs, DHPs, and BZTs at high resolution in that channel type (Zhao et al., 2019). Although the CaV1.1 channels are not themselves a pharmacological target for Ca2+ antagonists, they are modulated by these drugs in a similar manner as the cardiac/vascular smooth muscle CaV1.2 channels that are the in vivo drug targets; however, CaV1.1 channels typically have lower affinity for Ca2+ antagonist drug binding. As we show here, diltiazem bound to a site in the central cavity of CaV1.1, just on the intracellular side of the ion selectivity filter overlapping the PAA-binding site. The binding pose of diltiazem in CaV1.1 is most similar to the pose we observe in the presence of amlodipine, which is potentially a higher affinity binding configuration stimulated by transition to an inactivated state. Overall, there is remarkable agreement between the BZT binding to bacterial and mammalian CaV channels, suggesting that the details of allosteric modulation of amlodipine and Ca2+ binding that we have observed here in CaVAb may also be relevant for mammalian CaV channels.
Acknowledgments
We are grateful to the beamline staff at the Advanced Light Source (BL8.2.1 and BL8.2.2) for assistance during data collection and to Dr. Jin Li for technical and editorial support.
Authorship Contributions
Participated in research design: Tang, Gamal El-Din, Zheng, Catterall.
Conducted experiments: Tang, Gamal El-Din.
Performed data analysis: Tang, Gamal El-Din, Lenaeus.
Wrote or contributed to the writing of the manuscript: Tang, Zheng, Gamal El-Din, Lenaeus, Catterall.
Footnotes
- Received June 13, 2019.
- Accepted August 2, 2019.
↵1 W.A.C. and N.Z. are co-senior authors.
The research reported in this publication was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Award R01HL112808] (to W.A.C. and N.Z.) and National Institute of Neurologic Disorders and Stroke [Award R01NS015751] (to W.A.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the Howard Hughes Medical Institute (to N.Z.).
The authors declare no competing financial interests.
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This article has supplemental material available at molpharm.aspetjournals.org.
Abbreviations
- BZT
- benzothiazepine
- DHP
- dihydropyridine
- PAA
- phenylalkylamine
- Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics