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UNIVERSITY OF BUCHAREST
FACULTY OF PHYSICS Guest
2024-11-23 17:56 |
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Conference: Bucharest University Faculty of Physics 2014 Meeting
Section: Biophysics; Medical Physics
Title:
Complex interactions of diminazene with ASIC1 channels
Authors:
B. AMUZESCU(1), T. KNOTT(2), O. SCHEEL(2), E. RADU(3), Maria MERNEA(1), D. MIHAILESCU (1)
Affiliation:
1) Dept. Biophysics & Physiology, Faculty of Biology, University of Bucharest
2) Cytocentrics Bioscience Gmbh, Rostock, Germany
3) Dept. Histology & Cell Biology, Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
E-mail
bogdan@biologie.kappa.ro
Keywords:
Acid-sensing ion channel, diaryldiamidines, Markov model, open channel
block, automated patch-clamp, molecular docking
Abstract:
Diminazene, a drug belonging to the group of diaryldiamidines, used clinically since the 1940s in the treatment of tropical parasitoses, has been recently found to exert potent and specific blocking effects on acid-sensing ion channels (ASIC) via whole-cell experiments in neurons and heterologous expression systems. Therefore the aim of our study was to gain an in-depth understanding of the details of this complex molecular interaction by combining several powerful biophysical techniques. In whole-cell experiments performed with the CytoPatch™2 equipment, ASIC1 transient currents evoked in HEK293 cells by brief acidic pulses (pH 6.0 for 3 s) were inhibited by diminazene with an IC50 of 0.43 µM and a subunit Hill coefficient (0.79). Single-channel recordings on excised outside-out patches at a holding potential of -70 mV revealed 3 subconductance levels produced by partial occlusion of the ion permeation pathway by calcium, and the two well-known gating modes. Recordings with diminazene at 0.6 or 3 µM indicated a mechanism of open channel block, and a higher affinity for the open conformation of the second gating mode. Single-channel pharmacology data were analyzed either by exponential fits of the distributions of blocked and unblocked periods for each gating mode or by model-dependent fitting with custom hidden Markov model maximum likelihood algorithms, yielding similar results. To explain the increase in apparent Kd at higher diminazene concentration we postulated an accessory binding site that would hamper access of the drug to the main blocking site. Via RT-PCR we proved that both 1a and 1b splice variants are present in HEK293 cells, the relative ratio at mRNA level being 26:1. In docking simulations with diminazene on human ASIC1a and 1b models at atomic resolution in open conformation, we retrieved blocking sites in the transmembrane permeation pathway, with higher affinity for ASIC1b (mean ∆Gbinding of -10.3 kcal/mol for ASIC1b), and an accessory binding site in the lateral fenestrations, specific for ASIC1a, with a mean ∆Gbinding of -8.2 kcal/mol.
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