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...