Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154620/1/fsb2fj201700349r.pd

Non-Specific Inhibition of Ischemia- and Acidosis-Induced Intracellular Calcium Elevations and Membrane Currents by α-Phenyl-N-tert-butylnitrone, Butylated Hydroxytoluene and Trolox

Ischemia, and subsequent acidosis, induces neuronal death following brain injury. Oxidative stress is believed to be a key component of this neuronal degeneration. Acute chemical ischemia (azide in the absence of external glucose) and acidosis (external media buffered to pH 6.0) produce increases in intracellular calcium concentration ([Ca2+]i) and inward membrane currents in cultured rat cortical neurons. Two α-tocopherol analogues, trolox and butylated hydroxytoluene (BHT), and the spin trapping molecule α-Phenyl-N-tert-butylnitrone (PBN) were used to determine the role of free radicals in these responses. PBN and BHT inhibited the initial transient increases in [Ca2+]i, produced by ischemia, acidosis and acidic ischemia and increased steady state levels in response to acidosis and the acidic ischemia. BHT and PBN also potentiated the rate at which [Ca2+]i increased after the initial transients during acidic ischemia. Trolox inhibited peak and sustained increases in [Ca2+]i during ischemia. BHT inhibited ischemia induced initial inward currents and trolox inhibited initial inward currents activated by acidosis and acidic ischemia. Given the inconsistent results obtained using these antioxidants, it is unlikely their effects were due to elimination of free radicals. Instead, it appears these compounds have non-specific effects on the ion channels and exchangers responsible for these responses

An ATP-sensitive potassium conductance in rabbit arterial endothelial cells

1. Whole-cell patch clamp recording was used to study an ATP-sensitive, sulphonylurea-inhibitable potassium (K+) conductance in freshly dissociated endothelial cells from rabbit arteries. 2. The ATP-sensitive K+ conductance was activated by micromolar concentrations of the K+ channel opener, levcromakalim, and by metabolic inhibition of endothelial cells using dinitrophenol and iodoacetic acid. The current-voltage (I-V) relationship obtained in isotonic K+ solutions was linear between -150 and -50 mV and had a slope conductance of approximately 1 nS. 3. The permeability of the ATP-sensitive K+ conductance determined from reversal potential measurements exhibited the following ionic selectivity sequence: Rb+ \u3e K+ \u3e Cs+ ≥ Na+ \u3e NH4 + \u3e Li+. 4. Membrane currents activated by either levcromakalim or metabolic inhibition were inhibited by the sulphonylurea drugs, glibenclamide and tolbutamide, with half-maximal inhibitory concentrations of 43 nM and 224 μM and Hill coefficients of 1.1 and 1.2, respectively. Levcromakalim-induced currents were also inhibited by millimolar concentrations of Ba2+ or tetraethylammonium ions in the external solution. 5. Levcromakalim (3 μM) and metabolic inhibition hyperpolarized endothelial cells by approximately 10-15 mV in normal physiological salt solutions. The hyperpolarization induced by levcromakalim or metabolic inhibition was inhibited by bath application of 10 μM glibenclamide. 6. Internal perfusion of the cytosol of whole-cell voltage-clamped endothelial cells with an ATP-free pipette solution activated a membrane current which was reversibly inhibited by internal perfusion with a 3 mM MgATP pipette solution. This current was insensitive to other adenine and guanine nucleotides in the pipette solution. The inward current evoked in a nominally ATP-free internal solution was further increased by bath application of levcromakalim. 7. Levcromakalim (25 μM) did not induce a change in the intracellular Ca2+ concentration of fura-2-loaded endothelial cells, whereas metabolic inhibition caused a slow and sustained increase in intracellular Ca2+ concentration, which was attenuated by 10 μM glibenclamide applied externally. 8. ATP-sensitive K+ channel activation in arterial endothelial cells may contribute to endothelium-dependent vascular changes in response to ischaemia-induced hypoxia producing membrane hyperpolarization

Afobazole nanoparticles formulation for enhanced therapeutics

A novel nanoparticle drug composition and method of use thereof is presented herein. The nanoparticle drug composition is comprised of at least one nanoparticle carrier, formed from the conjugation of PLGA and PEG, which encapsulates a drug such as afobazole and its derivatives, in a pharmaceutically acceptable carrier. The nanoparticle drug composition may be used to treat various diseases of the central nervous system involving excessive neuronal activity and inflammation such as stroke, Alzheimer\u27s disease and anxiety

ACh- and caffeine-induced Ca2+ mobilization and current activation in rabbit arterial endothelial cells

Fura 2 microfluorometry and perforated-patch whole cell recording were carried out simultaneously to investigate the relationship between intracellular free Ca2+ concentration ([Ca2+](i)) and membrane current activation in response to ACh and caffeine in freshly dissociated arterial endothelial cells. ACh and caffeine evoked transient increases in [Ca2+](i). The initial increase in [Ca2+](i) was accompanied by a transient outward current, which caused membrane hyperpolarization. The amplitudes of the [Ca2+](i) transient and outward current were dependent on caffeine concentration (EC50 ~ 1 mM). Cyclopiazonic acid raised resting [Ca2+](i) levels by ≥50 nM and failed to completely block caffeine- or ACh-induced [Ca2+](i) transients but slowed [Ca2+](i) recovery fourfold. The reversal potential of caffeine-induced currents was dependent on external K+ and Cl- concentrations. Caffeine-induced current amplitudes, but not [Ca2+](i) responses, were attenuated by external tetraethylammonium, Zn2+, and La3+. A consistent temporal relationship between agonist- activated membrane current and [Ca2+](i) increases was not observed, and, in some cases, time differences were greater than expected for simple diffusion of Ca2+ throughout the cell. These results suggest that Ca2+- dependent current activation monitors local [Ca2+](i) changes adjacent to the plasmalemma, whereas single-cell photometry provides a measure of global changes in [Ca2+](i)

Afobazole nanoparticles formulation for enhanced therapeutics

A novel nanoparticle drug composition and method of use thereof is presented herein. The nanoparticle drug composition is comprised of at least one nanoparticle carrier, formed from the conjugation of PLGA and PEG, which encapsulates a drug such as afobazole and its derivatives, in a pharmaceutically acceptable carrier. The nanoparticle drug composition may be used to treat various diseases of the central nervous system involving excessive neuronal activity and inflammation such as stroke, Alzheimer\u27s disease and anxiety