Characterizations of the Urate Transporter, GLUT9, and Its Potent Inhibitors by Patch-Clamp Technique

2020 ◽  
pp. 247255522094950
Author(s):  
Yanyu Chen ◽  
Zean Zhao ◽  
Yongmei Li ◽  
Lu Li ◽  
Yu Jiang ◽  
...  
Keyword(s):  
Uric Acid ◽  
Patch Clamp ◽  
Glucose Transporter ◽  
Cell Model ◽  
Outward Current ◽  
External Solution ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Induced Currents

Glucose transporter 9 (GLUT9), which transports urate in an electrogenic and voltage-dependent manner, plays an important role in the maintenance of normal blood uric acid/urate levels. In the present study, we established a cell model based on the single-electrode patch-clamp technique for characterization of GLUT9 and explored the inhibitory effects of benzobromarone (BM) and probenecid (PB) on urate-induced currents in mouse GLUT9a (mGLUT9a)–expressing HEK-293T cells. The results showed that uric acid, rather than glucose perfusion, led to a rapid and large outward current by mGLUT9a in dose-, voltage-, and pH-dependent manners. BM prominently and irreversibly inhibited the uric acid–induced currents through mGLUT9a, and PB weakly and reversibly inhibited mGLUT9a. We found that depletion of K+ in the external solution significantly strengthened the blockade of BM on mGLUT9a. In addition, an enhanced inhibitory rate of BM was detected when the pH of the external solution was changed from 7.4 to 5.5, indicating that BM functions optimally in an acidic environment. In conclusion, the combination of the established cell model with patch-clamp techniques first revealed the function properties of GLUT9 inhibitors and may provide potential benefits to the study of GLUT9 inhibitors as antihyperuricemic or antigout agents.

Gramicidin perforated patch-clamp technique reveals glycine-gated outward chloride current in dissociated nucleus solitarii neurons of the rat

1994 ◽  
Vol 72 (3) ◽  
pp. 1103-1108 ◽  
Author(s):  
J. S. Rhee ◽  
S. Ebihara ◽  
N. Akaike
Keyword(s):  
Patch Clamp ◽  
Hill Coefficient ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Perforated Patch ◽  
Clamp Technique ◽  
Outward Currents ◽  
Patch Technique ◽  
Perforated Patch Clamp

1. The inhibitory response of exogenously applied glycine was investigated in freshly dissociated rat nucleus tractus solitarii neurons under whole cell configuration using new perforated patch-clamp technique termed "gramicidin perforated patch technique," which maintains intact intracellular Cl- concentrations. 2. Using the gramicidin perforated patch technique, at a holding potential (VH) of -45 mV, glycine induced outward currents in a concentration-dependent manner with a EC50 of 4.0 x 10(-5) M and at a Hill coefficient of 1.5. In contrast, using the nystatin perforated patch technique, glycine induced inward currents at the same VH in a concentration-dependent manner with an EC50 of 4.9 x 10(-5) M and at a Hill coefficient of 1.2. 3. The glycine-induced outward currents were blocked by strychnine in a concentration dependent manner with an IC50 of 2.2 x 10(-8) M. The blockade was competitive. 4. The current-voltage relationship for the 10(-5) M glycine response showed a clear outward rectification. 5. Ten-fold change of extracellular Cl- with a large impermeable anion resulted in a 65 mV shift of the reversal potential of glycine-induced currents (EGly), indicating that the membrane behaves like a Cl- electrode in the presence of glycine. 6. The intracellular Cl- activity calculated from the EGly ranged from 7.3 to 18.2 mM, with a mean value of 13.3 mM. 7. The values of EGly in the individual neurons were significantly negative to the resting membrane potentials, suggesting the existence of active transport of Cl-.

Dendritic excitability and a voltage-gated calcium current in locust nonspiking local interneurons

1993 ◽  
Vol 69 (5) ◽  
pp. 1484-1498 ◽  
Author(s):  
G. Laurent ◽  
K. J. Seymour-Laurent ◽  
K. Johnson
Keyword(s):  
Patch Clamp ◽  
Resting Potential ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Inward Current ◽  
Time To Peak ◽  
Clamp Technique ◽  
Voltage Dependent

1. The active properties of axonless nonspiking interneurons in the thoracic nervous system of the locust Schistocerca americana were studied in vivo with the switched current-clamp technique from dendritic impalements, and in vitro with the whole-cell variation of the patch-clamp technique. 2. In 20% of in vivo recordings, depolarization of a dendrite to potentials more positive than about -40 mV evoked resonant behaviour and/or regenerative potentials. The latter were slow (half width: 20-30 ms), small (base-to-peak amplitude: 25-35 mV), and were often followed by a pronounced after hyperpolarization (AHP). 3. The slow regenerative potentials sometimes had multiple peaks separated by incomplete repolarizations. The voltage envelope of such potentials was always broader than that of spikes with single peaks. In other recordings, a same depolarizing pulse could evoke several regenerative potentials with different waveforms. These results suggested the presence of multiple dendritic initiation sites separated by regions of inexcitable membrane, allowing decremental conduction and the passive fusion of spike envelopes. 4. Graded active responses could also be evoked on rebound from short hyperpolarizations such as inhibitory postsynaptic potentials (IPSPs) provided that the membrane was already depolarized to about -40 mV. IPSPs evoked by several presynaptic interneurons differed in their ability to evoke rebound potentials suggesting that some synaptic sites were electrically closer than others to regions of active membrane. 5. Patch-clamp recordings from somata of nonspiking neurons isolated from 75% embryos and grown in culture medium for 1-2 days revealed the presence of an inactivating inward current resistant to 0.5-1 microM tetrodotoxin (TTX). The inward current was carried equally well by Ba2+, and sensitive to blockade by Cd2+ (0.5 mM), Ni2+ (0.75 mM), or Co2+ (2.5 mM). 6. The current activated around -40 mV, with voltage-dependent activation (time-to-peak approximately 20 ms at -35 mV and 1-2 ms at 0 mV). Tail currents evoked upon repolarization were well fitted by a single exponential (tau = 1-2 ms). Deactivation time constants shorter than 300 microseconds, however, could not be measured. 7. The current inactivated rapidly in a voltage-dependent manner, following two-exponential kinetics. A very small persistent component could be explained by the overlap between activation and inactivation curves, greatest at approximately -20 mV. The voltage of half-inactivation was about -25 mV. At a resting potential of -58 mV, 90% of the current was available for activation. Recovery from steady-state inactivation followed the sum of two or more exponential processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Vasorelaxing Activity of R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol from Dalbergia tonkinensis: Involvement of Smooth Muscle CaV1.2 Channels

Planta Medica ◽  
2020 ◽  
Vol 86 (04) ◽  
pp. 284-293 ◽  
Author(s):  
Nguyen Manh Cuong ◽  
Ninh The Son ◽  
Ngu Truong Nhan ◽  
Pham Ngoc Khanh ◽  
Tran Thu Huong ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Antihypertensive Drugs ◽  
Docking Simulation ◽  
Mechanical Activity ◽  
Dependent Manner ◽  
Vascular Effects ◽  
Patch Clamp Technique ◽  
Spasmolytic Activity ◽  
Cav1.2 Channels ◽  
Dalbergia Tonkinensis

Abstract Dalbergia species heartwood, widely used in traditional medicine to treat various cardiovascular diseases, might represent a rich source of vasoactive agents. In Vietnam, Dalbergia tonkinensis is an endemic tree. Therefore, the aim of the present work was to investigate the vascular activity of R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol isolated from the heartwood of D. tonkinensis and to provide circular dichroism features of its R absolute configuration. The vascular effects of R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol were assessed on the in vitro mechanical activity of rat aorta rings, under isometric conditions, and on whole-cell Ba2+ currents through CaV1.2 channels (IBa1.2) recorded in single, rat tail main artery myocytes by means of the patch-clamp technique. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol showed concentration-dependent, vasorelaxant activity on both endothelium-deprived and endothelium intact rings precontracted with the α 1 receptor agonist phenylephrine. Neither the NO (nitric oxide) synthase inhibitor Nω-nitro-L-arginine methyl ester nor the cyclooxygenase inhibitor indomethacin affected its spasmolytic activity. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol-induced vasorelaxation was antagonized by (S)-(−)-Bay K 8644 and unaffected by tetraethylammonium plus glibenclamide. In patch-clamp experiments, R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol inhibited IBa1.2 in a concentration-dependent manner and significantly decreased the time constant of current inactivation. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol likely stabilized the channel in its closed state, as suggested by molecular modelling and docking simulation to the CaV1.2 channel α 1c subunit. In conclusion, D. tonkinensis species may represent a source of agents potentially useful for the development of novel antihypertensive drugs.

Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells

2003 ◽  
Vol 285 (3) ◽  
pp. H1347-H1355 ◽  
Author(s):  
Jin Han ◽  
Nari Kim ◽  
Hyun Joo ◽  
Euiyong Kim
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Cerebral Arteries ◽  
Muscle Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Arterial Smooth Muscle ◽  
Clamp Technique ◽  
Arterial Smooth Muscle Cells

Although ketamine and Ca2+-activated K+ (KCa) channels have been implicated in the contractile activity regulation of cerebral arteries, no studies have addressed the specific interactions between ketamine and the KCa channels in cerebral arteries. The purpose of this study was to examine the direct effects of ketamine on KCa channel activities using the patch-clamp technique in single-cell preparations of rabbit middle cerebral arterial smooth muscle. We tested the hypothesis that ketamine modulates the KCa channel activity of the cerebral arterial smooth muscle cells of the rabbit. Vascular myocytes were isolated from rabbit middle cerebral arteries using enzymatic dissociation. Single KCa channel activities of smooth muscle cells from rabbit cerebral arteries were recorded using the patch-clamp technique. In the inside-out patches, ketamine in the micromolar range inhibited channel activity with a half-maximal inhibition of the ketamine conentration value of 83.8 ± 12.9 μM. The Hill coefficient was 1.2 ± 0.3. The slope conductance of the current-voltage relationship was 320.1 ± 2.0 pS between 0 and +60 mV in the presence of ketamine and symmetrical 145 mM K+. Ketamine had little effect on either the voltage-dependency or open- and closed-time histograms of KCa channel. The present study clearly demonstrates that ketamine inhibits KCa channel activities in rabbit middle cerebral arterial smooth muscle cells. This inhibition of KCa channels may represent a mechanism for ketamine-induced cerebral vasoconstriction.

Molecular mechanisms of action for CFTR potentiators

2019 ◽  
Author(s):  
◽  
Han-I Yeh
Keyword(s):  
Cystic Fibrosis ◽  
Patch Clamp ◽  
Binding Site ◽  
Molecular Mechanisms ◽  
Mechanisms Of Action ◽  
Drug Binding ◽  
Patch Clamp Technique ◽  
Chemical Structures ◽  
Clamp Technique ◽  
Sites Of Action

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] As the culprit behind cystic fibrosis (CF) is the dysfunction of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR), pharmacological reagents targeting CFTR may hold the key to the ultimate cure of CF. In this thesis, we present the studies in the mechanisms of action for CFTR potentiators, the small molecules that enhance the functions of CFTR. Using the patch-clamp technique, we demonstrated that the permeant anion nitrate modulates CFTR gating through a mechanism similar to the FDA-approved CFTR potentiator VX-770 (ivacaftor). Via separate sites of action, VX-770 and nitrate stabilize the open channel conformation of CFTR in an energetically additive manner. Next, we investigated the action of a novel CFTR potentiator, GLPG1837, and showed that despite their different chemical structures, GLPG1837 and VX-770 share the same mechanisms of action on CFTR gating and compete for a common binding site in the transmembrane domains of CFTR. An allosteric modulation model is further proposed to explain how the affinity and efficacy of both potentiators are determined by the energetic coupling between drug binding and channel gating. Finally, we combined molecular docking and patch-clamp technique to identify the binding site(s) for GLPG1837 and VX-770.

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