Molecular Modeling of Benzothiazepine Binding in the L-type Calcium Channel

Abstract

In our open channel model, key functional groups of BTZs interact with BTZ-sensing residues, which were identified in previous mutational experiments. The bulky tricyclic moiety occupies interface between domains III and IV, while the ammonium group protrudes into the inner pore, where it is stabilized by nucleophilic C-ends of the pore helices. In the closed channel model, contacts with several ligand-sensing residues in the inner helices are lost, which weakens ligand-channel interactions. An important feature of the ligand-binding mode in both open and closed channels is an interaction between the BTZ carbonyl group and a Ca 2؉ ion chelated by the selectivity filter glutamates in domains III and IV. In the absence of Ca 2؉ , the tricyclic BTZ moiety remains in the domain interface, while the ammonium group directly interacts with a glutamate residue in the selectivity filter. Our model suggests that the Ca 2؉ potentiation involves a direct electrostatic interaction between a Ca 2؉ ion and the ligand rather than an allosteric mechanism. Energy profiles indicate that BTZs can reach the binding site from the domain interface, whereas access through the open activation gate is unlikely, because reorientation of the bulky molecule in the pore is hindered. Benz(othi)azepines (BTZs)2 represent one of three main classes of ligands of L-type calcium channels (1, 2). Tonic (resting) block of Ca 2ϩ channels is usually measured by applying infrequent depolarization stimuli. Frequency-dependent block is measured during more rapid trains of depolarization stimuli. Increasing the stimulation frequency generally leads to more effective inhibition by BTZs (3-5). Voltage and activation dependence of the block are consistent with the idea that different functional states of the channel have different affinities for the drug in the order inactivated Ͼ open Ͼ closed state. These observations are consistent with the "modulated receptor" hypothesis (6), which was initially proposed to explain the action of local anesthetics on Na ϩ channels. The effect of BTZs also depends on the ionic environment. Raising concentrations of Ca 2ϩ or Ba 2ϩ antagonize diltiazem block (3). However, the block is more pronounced when Ca 2ϩ rather than Ba 2ϩ is used as a charge carrier (3, 7), and thus the block is considered potentiated by Ca 2ϩ (7). The conductance with Ba 2ϩ is higher than with Ca 2ϩ , because Ca 2ϩ binds more tightly to the outer-pore glutamates (8). These experiments suggest that the high affinity binding of BTZs requires a cation binding in the channel.Extracellular applications of both the tertiary and quaternary BTZ analogs effectively block Ca 2ϩ channels. In contrast, intracellular application of the same compounds does not result in significant block, leading to the conclusion that BTZs block the channels via an extracellular pathway (9 -11).Mutational studies have largely delimited BTZ-sensing residues to the inner helices and P-loops of domains III and IV (see Table 1). However, applying experimental data for mapping the BTZ...

Overlapping Binding Sites of Structurally Different Antiarrhythmics Flecainide and Propafenone in the Subunit Interface of Potassium Channel Kv2.1*

“…From this niche, the drugs expose their ammonium groups in the inner pore to block the current. The interface between pore-forming subunits was proposed as the binding site of some ligands of Ca 2ϩ channels (47,48). Our present study provides evidence that some open K ϩ channel blockers can also bind mainly in the interface of the pore-forming subunits.…”

Structural Model for Phenylalkylamine Binding to L-type Calcium Channels

“…However, it should be emphasized that we did not modify the template structure to satisfy the proposed interactions. The same model based on the same sequence alignment was used previously for analyses of binding of DHPs and BTZs to LTCC (19,20). It should be noted that despite the obvious differences in the structure and mechanism of action of these three classes of LTCC ligands, two important features of ligand-channel interactions are common in our models.…”

“…However, E 3p50 and E 4p50 , whose mutations significantly affect the PAA block (10), are bound in our model to the Ca 2ϩ ion that directly interacts with the nitrile group of potent PAAs. Calcium coordination by E 3p50 and E 4p50 is not an ad hoc feature of the current model; it is also proposed in our LTCC models with BTZs (19) and DHPs (20).…”

“…In the BTZ-LTCC models (19), the main body of the ligands binds in the repeat interface, whereas the amino group protrudes into the inner pore, where it is stabilized by nucleophilic C-terminal ends of the pore helices. In the DHP-LTCC models (20), the ligands also bind in the interface between repeats III and IV, whereas the moieties that differ between agonist and antagonists extend to the pore.…”

A Pore-blocking Hydrophobic Motif at the Cytoplasmic Aperture of the Closed-state Nav1.7 Channel Is Disrupted by the Erythromelalgia-associated F1449V Mutation

“…Moreover, although differences in prokaryotic and eukaryotic bilayer thickness and the presence of the S1-S4 voltage sensor may alter pore structure, numerous studies have used an homology model generated from the open-state MthK channel (e.g. Cronin et al (24)), and KcsA is the more common template for homology modeling studies of closed state eukaryotic sodium (25,26), potassium (27,28), and calcium (29,30) channel pores, demonstrating the suitability of potassium channels as structural templates for homology modeling.…”