Identification of Verapamil Binding Sites Within Human Kv1.5 Channel Using Mutagenesis and Docking Simulation.

BACKGROUND/AIMS: The phenylalkylamine class of L-type Ca2+ channel antagonist verapamil prolongs the effective refractory period (ERP) of human atrium, which appears to contribute to the efficacy of verapamil in preventing reentrant-based atrial arrhythmias including atrial fibrillation. This study was designed to investigate the molecular and electrophysiological mechanism underlying the action of verapamil on human Kv1.5 (hKv1.5) channel that determines action potential duration and ERP in human atrium. METHODS: Site-directed mutagenesis created 10 single-point mutations within pore region of hKv1.5 channel. Wholecell patch-clamp method investigated the effect of verapamil on wild-type and mutant hKv1.5 channels heterologously expressed in Chinese hamster ovary cells. Docking simulation was conducted using open-state homology model of hKv1.5 channel pore. RESULTS: Verapamil preferentially blocked hKv1.5 channel in its open state with IC50 of 2.4±0.6 µM (n = 6). The blocking effect of verapamil was significantly attenuated in T479A, T480A, I502A, V505A, I508A, L510A, V512A and V516A mutants, compared with wild-type hKv1.5 channel. Computer docking simulation predicted that verapamil is positioned within central cavity of channel pore and has contact with Thr479, Thr480, Val505, Ile508, Ala509, Val512, Pro513 and Val516. CONCLUSION: Verapamil acts as an open-channel blocker of hKv1.5 channel, presumably due to direct binding to specific amino acids within pore region of hKv1.5 channel, such as Thr479, Thr480, Val505, Ile508, Val512 and Val516. This blocking effect of verapamil on hKv1.5 channel appears to contribute at least partly to prolongation of atrial ERP and resultant antiarrhythmic action on atrial fibrillation in humans.

Deuterium oxide protects against myocardial injury induced by ischemia and reperfusion in rats.

Objectives. Although deuterium oxide (D2O) has preservative property on the extracted organ, whether D2O also protects the in situ myocardial injury remains unknown. Using cardiac microdialysis, local administration of D2O through dialysis probe was applied in situ rat heart. We examined the effect of the D2O on the myocardial injury induced ischemia, reperfusion, and chemical hypoxia. Methodology. We measured dialysate myoglobin levels during 30 min of coronary occlusion and reperfusion in the absence and presence of D2O. Furthermore, to confirm the effect of D2O on NaCN induced myocardial injury, we measured the dialysate myoglobin levels with local perfusion of NaCN in the absence and presence of D2O. Results. The dialysate myoglobin levels increased from 177 ± 45 ng/mL at baseline to 3030 ± 1523 ng/mL during 15-30 min of coronary occlusion and further increased to 8588 ± 1684ng/mL at 0-15 min of reperfusion. The dialysate myoglobin levels with 60 min local perfusion of NaCN increased to 1214 ± 279 ng/mL. D2O attenuated myocardial myoglobin release during 15-30 min of coronary occlusion and 0-30 min of reperfusion and 15-60 min of local perfusion of NaCN. Conclusions. D2O might have a beneficial effect of myocardium against ischemia, reperfusion and chemical hypoxia.

Interactions of Propofol With Human Voltage-gated Kv1.5 Channel Determined by Docking Simulation and Mutagenesis Analyses.

Propofol blocks the voltage-gated human Kv1.5 (hKv1.5) channel by preferentially affecting in its open state. A previous mutational study suggested that several amino acids within the pore region of the hKv1.5 channel are involved in mediating the blocking action of propofol. The present investigation was undertaken to elucidate the predicted binding modes of propofol within the pore cavity of the open-state hKv1.5 channel, using computational docking and mutagenesis approaches. The docking simulation using a homology model of the hKv1.5 channel, constructed based on the crystal structure of the Kv1.2 channel, predicted that propofol was positioned at the base of the pore cavity of hKv1.5 channel, adjacent to 4 amino acids Thr479, Thr480, Val505, and Ile508, and formed arene-H interactions with Val505. The patch-clamp experiments on wild-type and mutant hKv1.5 channels constructed by site-directed mutagenesis revealed that the blocking potency of propofol was significantly reduced in T480A, V505A, and I508A but not in T479A mutants compared with wild-type hKv1.5 channel. These computational docking and experimental mutational analyses suggest that propofol is positioned at the base of the pore cavity and forms functional contact with Thr480, Val505, and Ile508 to directly block the hKv1.5 channel.

General anesthetic binding mode via hydration with weak affinity and molecular discrimination: General anesthetic dissolution in interfacial water of the common binding site of GABAA receptor.

The GABAA receptor (GABAAR) is a target channel for the loss of awareness of general anesthesia. General anesthetic (GA) spans a wide range of chemical structures, such as monatomic molecules, barbital acids, phenols, ethers, and alkanes. GA has a weak binding affinity, and the affinity has a characteristic that correlates with the solubility in olive oil rather than the molecular shape. The GA binding site of GABAAR is common to GAs and exists in the transmembrane domain of the GABAAR intersubunit. In this study, the mechanism of GA binding, which allows binding of various GAs with intersubunit selectivity, was elucidated from the hydration analysis of the binding site. Regardless of the diverse GA chemical structures, a strong correlation was observed between the binding free energy and total dehydration number of the binding process. The GA binding free energy was more involved in the binding dehydration and showed molecular recognition that allowed for the binding of various GA structures via binding site hydration. We regarded the GA substitution for the interfacial water molecule of the binding site as a dissolution into the interfacial hydration layer. The elucidation of the GA binding mechanism mediated by hydration at the GABAAR common binding site provides a rationale for the combined use of anesthetics in medical practice and its combination adjustments via drug interactions.

Noble gas binding to human serum albumin using docking simulation: nonimmobilizers and anesthetics bind to different sites.

BACKGROUND: Nonimmobilizers are structurally similar to anesthetics, but do not produce anesthesia at clinically relevant concentrations. Xenon, krypton, and argon are anesthetics, whereas neon and helium are nonimmobilizers. The structures of noble gases with anesthetics or nonimmobilizers are similar and their interactions are simple. Whether the binding site of anesthetics differs from that of nonimmobilizers has long been a question in molecular anesthesiology. METHODS: We investigated the binding sites and energies of anesthetic and nonimmobilizer noble gases in human serum albumin (HSA) because the 3D structure of HSA is well known and it has an anesthetic binding site. The computational docking simulation we used searches for binding sites and calculates the binding energy for small molecules and a template molecule. RESULTS: Xenon, krypton, and argon were found to bind to the enflurane binding site of HSA, whereas neon and helium were found to bind to sites different from the xenon binding site. Rare gas anesthetic binding was dominated by van der Waals energy, while nonimmobilizer binding was dominated by solvent-effect energy. Binding site preference was determined by the ratios of local binding energy (van der Waals energy) and nonspecific binding energy (solvent-effect energy) to the total binding energy. van der Waals energy dominance is necessary for anesthetic binding. CONCLUSIONS: This analysis of binding energy components provides a rationale for the binding site difference of anesthetics and nonimmobilizers, reveals the differences between the binding interactions of anesthetics and nonimmobilizers, may explain pharmacological differences between anesthetics and nonimmobilizers, and provide an understanding of anesthetic action at the atomic level.

[New theories in molecular mechanisms of general anesthesia].

The controversy of the specific and non-specific theories of general anesthesia continues. Recently, non-specific theory began to have the persuasive power again. Molecular dynamics of target protein of the anesthesia and hydrophobic dehydration due to anesthetic binding to the protein have been drawing researcher's attention. Clarification of relation between the action site and the molecular dynamics modification of target protein has been an increasingly important problem. With these contexts, I believe that the research in anesthetic mechanisms will shift to a new paradigm.

Three-dimensional distribution function theory for the prediction of protein-ligand binding sites and affinities: application to the binding of noble gases to hen egg-white lysozyme in aqueous solution.

The three-dimensional distribution function theory of molecular liquids is applied to lysozyme in mixtures of water and noble gases. The results indicate that the theory has the capability of predicting the protein-ligand binding sites and affinities. First, it is shown that the theory successfully reproduces the binding sites of xenon found by X-ray crystallography. Then, the ability of the theory to predict the size selectivity of noble gases is demonstrated. The effect of water on the selectivity is clarified by a theoretical analysis. Finally, it is demonstrated that the dose-response curve, which is employed in experiments for examining the binding affinity, is realized by the theory.

Theoretical study of volume changes accompanying xenon-lysozyme binding: implications for the molecular mechanism of pressure reversal of anesthesia.

The change in partial molar volume (PMV) accompanying the xenon-lysozyme binding was investigated for elucidating the molecular mechanism of the pressure reversal of general anesthesia, using the three-dimensional reference interaction site model theory of molecular solvation. An increase of the PMV from xenon binding to the substrate binding site of lysozyme was found, and the binding is suppressed by pressure, while the internal site binding did not change the PMV. The PMV change was analyzed by decomposing it into several contributions from geometry and hydration. We also analyzed the hydration change due to the binding. From the results, we draw a molecular picture of the PMV change accompanying xenon-lysozyme binding, which gives a possible mechanism of pressure reversal of anesthesia.

Putative binding sites for arachidonic acid on the human cardiac Kv 1.5 channel.

BACKGROUND AND PURPOSE: In human heart, the Kv 1.5 channel contributes to repolarization of atrial action potentials. This study examined the electrophysiological and molecular mechanisms underlying arachidonic acid (AA)-induced inhibition of the human Kv 1.5 (hKv 1.5) channel. EXPERIMENTAL APPROACH: Site-directed mutagenesis was conducted to mutate amino acids that reside within the pore domain of the hKv 1.5 channel. Whole-cell patch-clamp method was used to record membrane currents through wild type and mutant hKv 1.5 channels heterologously expressed in CHO cells. Computer docking simulation was conducted to predict the putative binding site(s) of AA in an open-state model of the Kv 1.5 channel. KEY RESULTS: The hKv 1.5 current was minimally affected at the onset of depolarization but was progressively reduced during depolarization by the presence of AA, suggesting that AA acts as an open-channel blocker. AA itself affected the channel at extracellular sites independently of its metabolites and signalling pathways. The blocking effect of AA was attenuated at pH 8.0 but not at pH 6.4. The blocking action of AA developed rather rapidly by co-expression of Kv ß1.3. The AA-induced block was significantly attenuated in H463C, T480A, R487V, I502A, I508A, V512A and V516A, but not in T462C, A501V and L510A mutants of the hKv 1.5 channel. Docking simulation predicted that H463, T480, R487, I508, V512 and V516 are potentially accessible for interaction with AA. CONCLUSIONS AND IMPLICATIONS: AA itself interacts with multiple amino acids located in the pore domain of the hKv 1.5 channel. These findings may provide useful information for future development of selective blockers of hKv 1.5 channels.

Anesthetic management of an extremely obese patient.

We present the case of a morbidly obese woman, with a body mass index (BMI) of 73.7 kg.m(-2), who had a gynecological operation under combined general and epidural anesthesia. The patient's trachea was intubated, using a fiberscope, while she was breathing spontaneously after the intravenous injection of fentanyl and propofol as sedatives. Anesthesia was maintained with intravenous propofol and epidural mepivacaine. When the gynecologist placed a sponge in the abdominal cavity to retract the bowel, the patient experienced severe arterial deoxygenation and mild hypotension, due to massive atelectasis of the left lung. Both oxygenation and perfusion were corrected by the removal of the sponge and with the placement of a pillow under the patient's left shoulder. The atelectasis resulted from compression of the left lung by the fatty mediastinum and by the diaphragm being pushed up by the sponge. The hypotension resulted from impaired venous return and hypoxia. The patient suffered no perioperative complications other than atelectasis and a surgical-site infection. Key factors that contributed to the favorable outcome of this patient included a careful tracheal intubation technique, the choice and dose of anesthetic agents, immediate correction of the factors leading to atelectasis, early ambulation, and prophylaxis for deep vein thrombosis.