Screening Technologies for Inward Rectifier Potassium Channels: Discovery of New Blockers and Activators

2020 ◽  
Vol 25 (5) ◽  
pp. 420-433 ◽  
Author(s):  
Kenneth B. Walsh
Keyword(s):  
Membrane Potential ◽  
G Protein ◽  
Critical Role ◽  
Inward Rectifier ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Inward Rectification ◽  
Resting Membrane Potential ◽  
Kir Channels ◽  
Inward Rectifier Potassium Channels

K+ channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In nonexcitable cells, K+ channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K+ (Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K+ equilibrium potential (EK). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K+ (GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (KATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

Membrane Resting Potential of Thalamocortical Relay Neurons Is Shaped by the Interaction Among TASK3 and HCN2 Channels

2006 ◽  
Vol 96 (3) ◽  
pp. 1517-1529 ◽  
Author(s):  
Sven G. Meuth ◽  
Tatyana Kanyshkova ◽  
Patrick Meuth ◽  
Peter Landgraf ◽  
Thomas Munsch ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Standard Procedure ◽  
Resting Potential ◽  
Immunocytochemical Staining ◽  
Equilibrium Potential ◽  
Resting Membrane Potential ◽  
Hcn Channels ◽  
Extracellular Acidification ◽  
Knock Out ◽  
Task Channels

By combining molecular biological, electrophysiological, immunological, and computer modeling techniques, we here demonstrate a counterbalancing contribution of TASK channels, underlying hyperpolarizing K+ leak currents, and HCN channels, underlying depolarizing Ih, to the resting membrane potential of thalamocortical relay (TC) neurons. RT-PCR experiments revealed the expression of TASK1, TASK3, and HCN1–4. Quantitative determination of mRNA expression levels and immunocytochemical staining demonstrated that TASK3 and HCN2 channels represent the dominant thalamic isoforms and are coexpressed in TC neurons. Extracellular acidification, a standard procedure to inhibit TASK channels, blocked a TASK current masked by additional action on HCN channels. Only in the presence of the HCN blocker ZD7288 was the pH-sensitive component typical for a TASK current, i.e., outward rectification and current reversal at the K+ equilibrium potential. In a similar way extracellular acidification was able to shift the activity pattern of TC neurons from burst to tonic firing only during block of Ih or genetic knock out of HCN channels. A single compartmental computer model of TC neurons simulated the counterbalancing influence of TASK and HCN on the resting membrane potential. It is concluded that TASK3 and HCN2 channels stabilize the membrane potential by a mutual functional interaction, that the most efficient way to regulate the membrane potential of TC neurons is the converse modulation of TASK and HCN channels, and that TC neurons are potentially more resistant to insults accompanied by extracellular pH shifts in comparison to other CNS regions.

scholarly journals Current Separations in Myxicola Giant Axons

1969 ◽  
Vol 54 (6) ◽  
pp. 741-754 ◽  
Author(s):  
L. Goldman ◽  
L. Binstock
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Reversal Potential ◽  
Transient Current ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Resting Membrane Potential ◽  
Concentration Data ◽  
Giant Axons ◽  
Current Reversal

The effect of reducing the external sodium concentration, [Na]o, on resting potential, action potential, membrane current, and transient current reversal potential in Myxicola giant axons was studied. Tris chloride was used as a substitute for NaCl. Preliminary experiments were carried out to insure that the effect of Tris substitution could be attributed entirely to the reduction in [Na]o. Both choline and tetramethylammonium chloride were found to have additional effects on the membrane. The transient current is carried largely by Na, while the delayed current seems to be independent of [Na]o. Transient current reversal potential behaves much like a pure Nernst equilibrium potential for sodium. Small deviations from this behavior are consistent with the possibility of some small nonsodium component in the transient current. An exact PNa/PK for the transient current channels could not be computed from these data, but is certainly well greater than unity and possibly quite large. The peak of the action potential varied with [Na]o as expected for a sodium action potential with some substantial potassium permeability at the time of peak. Resting membrane potential is independent of [Na]o. This finding is inconsistent with the view that the resting membrane potential is determined only by the distribution of K and Na, and PNa/PK. It is suggested that PNa/PK's obtained from resting membrane potential-potassium concentration data do not always have the physical meaning generally attributed to them.

Vasa recta pericytes express a strong inward rectifier K+ conductance

2006 ◽  
Vol 290 (6) ◽  
pp. R1601-R1607 ◽  
Author(s):  
Chunhua Cao ◽  
Jae Hwan Goo ◽  
Whaseon Lee-Kwon ◽  
Thomas L. Pallone
Keyword(s):  
Smooth Muscle ◽  
Binding Constants ◽  
Inward Rectifier ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Inward Rectifier Potassium Channels ◽  
Whole Cell Patch Clamp ◽  
Vasa Recta ◽  
Voltage Dependent ◽  
Inward Currents

Strong inward rectifier potassium channels are expressed by some vascular smooth muscle cells and facilitate K+-induced hyperpolarization. Using whole cell patch clamp of isolated descending vasa recta (DVR), we tested whether strong inward rectifier K+ currents are present in smooth muscle and pericytes. Increasing extracellular K+ from 5 to 50 and 140 mmol/l induced inward rectifying currents. Those currents were Ba2+ sensitive and reversed at the K+ equilibrium potential imposed by the electrode and extracellular buffers. Ba2+ binding constants in symmetrical K+ varied between 0.24 and 24 μmol/l at −150 and −20 mV, respectively. Ba2+ blockade was time and voltage dependent. Extracellular Cs+ also blocked the inward currents with binding constants between 268 and 4,938 μmol/l at −150 and −50 mV, respectively. Ba2+ (30 μmol/l) and ouabain (1 mmol/l) depolarized pericytes by an average of 11 and 24 mV, respectively. Elevation of extracellular K+ from 5 to 10 mmol/l hyperpolarized pericytes by 6 mV. That hyperpolarization was reversed by Ba2+ (30 μmol/l). We conclude that strong inward rectifier K+ channels and Na+-K+-ATPase contribute to resting potential and that KIR channels can mediate K+-induced hyperpolarization of DVR pericytes.

scholarly journals Ca2+-activated K+ Channels in Murine Endothelial Cells: Block by Intracellular Calcium and Magnesium

2008 ◽  
Vol 131 (2) ◽  
pp. 125-135 ◽  
Author(s):  
Jonathan Ledoux ◽  
Adrian D. Bonev ◽  
Mark T. Nelson
Keyword(s):  
Endothelial Cells ◽  
Membrane Potential ◽  
Intracellular Calcium ◽  
Membrane Current ◽  
Mesenteric Arteries ◽  
Equilibrium Potential ◽  
Inward Rectification ◽  
Resting Membrane Potential ◽  
Additional Increase ◽  
K Channels

The intermediate (IKCa) and small (SKCa) conductance Ca2+-sensitive K+ channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IKCa and SKCa channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 μM free [Ca2+]i and 1 mM free [Mg2+]i, membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca2+-sensitive potassium (BKCa) and strong inward rectifier potassium (Kir) channels did not affect the membrane current. However, blockers of IKCa channels, charybdotoxin (ChTX), and of SKCa channels, apamin (Ap), significantly reduced the whole-cell current. Although IKCa and SKCa channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg2+ significantly increased these currents. Moreover, concomitant reduction of the [Ca2+]i to 1 μM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IKCa and SKCa channels caused a significant endothelial membrane potential depolarization (≈11 mV) and decrease in [Ca2+]i in mesenteric arteries in the absence of an agonist. These results indicate that [Ca2+]i can both activate and block IKCa and SKCa channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium.

Inwardly rectifying K+ channels in esophageal smooth muscle

2000 ◽  
Vol 279 (5) ◽  
pp. G951-G960 ◽  
Author(s):  
Junzhi Ji ◽  
Anne Marie F. Salapatek ◽  
Nicholas E. Diamant
Keyword(s):  
Membrane Potential ◽  
Longitudinal Muscle ◽  
Muscle Cells ◽  
Equilibrium Potential ◽  
Inward Rectification ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Inward Current ◽  
Inwardly Rectifying

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K+(Kir) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K+ equilibrium potential ( E k) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential ( E rev) of −76.5 mV. Elevation of external K+ increased the inward current amplitude and positively shifted its E rev after the E k, suggesting that potassium ions carry this current. External Ba2+ and Cs+ inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC50 for Ba2+ and Cs+ at −60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba2+ of 10 μM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that Kir channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.

Descending vasa recta endothelia express inward rectifier potassium channels

2007 ◽  
Vol 293 (4) ◽  
pp. F1248-F1255 ◽  
Author(s):  
Chunhua Cao ◽  
Whaseon Lee-Kwon ◽  
Kristie Payne ◽  
Aurélie Edwards ◽  
Thomas L. Pallone
Keyword(s):  
Endothelial Cells ◽  
Membrane Potential ◽  
Potassium Channels ◽  
Inward Rectifier ◽  
Equilibrium Potential ◽  
Dependent Manner ◽  
Membrane Hyperpolarization ◽  
Inward Rectifier Potassium Channels ◽  
Vasa Recta ◽  
Inwardly Rectifying

Descending vasa recta (DVR) are capillary-sized microvessels that supply blood flow to the renal medulla. They are composed of contractile pericytes and endothelial cells. In this study, we used the whole cell patch-clamp method to determine whether inward rectifier potassium channels (KIR) exist in the endothelia, affect membrane potential, and modulate intracellular Ca2+ concentration ([Ca2+]cyt). The endothelium was accessed for electrophysiology by removing abluminal pericytes from collagenase-digested vessels. KIR currents were recorded using symmetrical 140 mM K+ solutions that served to maximize currents and eliminate cell-to-cell coupling by closing gap junctions. Large, inwardly rectifying currents were observed at membrane potentials below the equilibrium potential for K+. Ba2+ potently inhibited those currents in a voltage-dependent manner, with affinity k = 0.18, 0.33, 0.60, and 1.20 μM at −160, −120, −80, and −40 mV, respectively. Cs+ also blocked those currents with k = 20, 48, 253, and 1,856 μM at −160, −120, −80, and −40 mV, respectively. In the presence of 1 mM ouabain, increasing extracellular K+ concentration from 5 to 10 mM hyperpolarized endothelial membrane potential by 15 mV and raised endothelial [Ca2+]cyt. Both the K+-induced membrane hyperpolarization and the [Ca2+]cyt elevation were reversed by Ba2+. Immunochemical staining verified that both pericytes and endothelial cells of DVR express KIR2.1, KIR2.2, and KIR2.3 subunits. We conclude that strong, inwardly rectifying KIR2.x isoforms are expressed in DVR and mediate K+-induced hyperpolarization of the endothelium.

scholarly journals The Influence of H+ on the Membrane Potential and Ion Fluxes of Nitella

1968 ◽  
Vol 52 (1) ◽  
pp. 60-87 ◽  
Author(s):  
Hiroshi Kitasato
Keyword(s):  
Membrane Potential ◽  
Membrane Conductance ◽  
Potential Difference ◽  
Potential Change ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Resting Membrane Potential ◽  
Ph Change ◽  
Ph Changes ◽  
External Ph

The resting membrane potential of the Nitella cell is relatively insensitive to [K]o, but behaves like a hydrogen electrode. K+ and Cl- effluxes from the cell were measured continuously, while the membrane potential was changed either by means of a negative feedback circuit or by external pH changes. The experiments indicate that PK and PCl are independent of pH but are a function of membrane potential. Slope ion conductances, GK, GCl, and GNa were calculated from efflux measurements, and their sum was found to be negligible compared to membrane conductance. The possibility that a boundary potential change might be responsible for the membrane potential change was considered but was ruled out by the fact that the peak of the action potential remained at a constant level regardless of pH changes in the external solution. The conductance for H+ was estimated by measuring the membrane current change during an external pH change while the membrane potential was clamped at K+ equilibrium potential. In the range of external pH 5 to 6, H+ chord conductance was substantially equal to the membrane conductance. However, the [H]i measured by various methods was not such as would be predicted from the [H]o and the membrane potential using the Nernst equation. In artificial pond water containing DNP, the resting membrane potential decreased; this suggested that some energy-consuming mechanism maintains the membrane potential at the resting level. It is probable that there is a H+ extrusion mechanism in the Nitella cell, because the potential difference between the resting potential and the H+ equilibrium potential is always maintained notwithstanding a continuous H+ inward current which should result from the potential difference.

scholarly journals WIN55,212-2, a Dual Modulator of Cannabinoid Receptors and G Protein-Coupled Inward Rectifier Potassium Channels

Biomedicines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 484
Author(s):  
Dongchen An ◽  
Steve Peigneur ◽  
Jan Tytgat
Keyword(s):  
Potassium Channels ◽  
Potassium Channel ◽  
G Protein ◽  
Cannabinoid Receptors ◽  
Inward Rectifier ◽  
Xenopus Laevis Oocyte ◽  
Inward Rectifier Potassium Channels ◽  
Wide Range ◽  
Cb2 Receptors ◽  
G Protein Coupled

The coupling of cannabinoid receptors, CB1 and CB2, to G protein-coupled inward rectifier potassium channels, GIRK1 and GIRK2, modulates neuronal excitability in the human brain. The present study established and validated the functional expression in a Xenopus laevis oocyte expression system of CB1 and CB2 receptors, interacting with heteromeric GIRK1/2 channels and a regulator of G protein signaling, RGS4. This ex vivo system enables the discovery of a wide range of ligands interacting orthosterically or allosterically with CB1 and/or CB2 receptors. WIN55,212-2, a non-selective agonist of CB1 and CB2, was used to explore the CB1- or CB2-GIRK1/2-RGS4 signaling cascade. We show that WIN55,212-2 activates CB1 and CB2 at low concentrations whereas at higher concentrations it exerts a direct block of GIRK1/2. This illustrates a dual modulatory function, a feature not described before, which helps to explain the adverse effects induced by WIN55,212-2 in vivo. When comparing the effects with other typical cannabinoids such as Δ9-THC, CBD, CP55,940, and rimonabant, only WIN55,212-2 can significantly block GIRK1/2. Interestingly, the inward rectifier potassium channel, IRK1, a non-G protein-coupled potassium channel important for setting the resting membrane voltage and highly similar to GIRK1 and GIRK2, is not sensitive to WIN55,212-2, Δ9-THC, CBD, CP55,940, or rimonabant. From this, it is concluded that WIN55,212-2 selectively blocks GIRK1/2.

Inward rectification in rat nucleus accumbens neurons

1989 ◽  
Vol 62 (6) ◽  
pp. 1280-1286 ◽  
Author(s):  
N. Uchimura ◽  
E. Cherubini ◽  
R. A. North
Keyword(s):  
Steady State ◽  
Nucleus Accumbens ◽  
Time Course ◽  
Potassium Conductance ◽  
Resting Potential ◽  
Inward Rectification ◽  
Resting Membrane Potential ◽  
Potential Range ◽  
Inward Current ◽  
Rat Nucleus Accumbens

1. Intracellular recordings were made from neurons in slices cut from the rat nucleus accumbens septi. Membrane currents were measured with a single-electrode voltage-clamp amplifier in the potential range -50 to -140 mV. 2. In control conditions (2.5 mM potassium), the resting membrane potential of the neurons was -83.4 +/- 1.1 (SE) mV (n = 157). Steady state membrane conductance was voltage dependent, being 34.8 +/- 1.7 nS (n = 25) at -100 mV and 8.0 +/- 0.7 nS (n = 25) at -60 mV. 3. Barium (1 microM) markedly reduced the inward rectification and caused a small inward current (40.6 +/- 8.7 pA, n = 8) at the resting potential. These effects became larger with higher barium concentrations, and, in 100 microM barium, the current-voltage relation was straight. 4. The block of the inward current by barium (at -130 mV) occurred with an exponential time course; the time constant was approximately 1 s at 1 microM barium and less than 90 ms with 100 microM. Strontium had effects similar to those of barium, but 1000-fold higher concentrations were required. Cesium chloride (2 mM) and rubidium chloride (2 mM) also blocked the inward rectification; their action reached steady state within 50 ms. 5. It is concluded that the nucleus accumbens neurons have a potassium conductance with many features of a typical inward rectifier and that this contributes to the potassium conductance at the resting potential.

Beta-adrenergic actions on membrane electrical properties of dissociated smooth muscle cells

1988 ◽  
Vol 254 (3) ◽  
pp. C423-C431 ◽  
Author(s):  
H. Yamaguchi ◽  
T. W. Honeyman ◽  
F. S. Fay
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Electrical Properties ◽  
Smooth Muscle Cells ◽  
Input Resistance ◽  
Muscle Cells ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Dependent Manner ◽  
Beta Adrenergic

Studies were carried out to determine the effects of the beta-adrenergic agent, isoproterenol (ISO), on membrane electrical properties in single smooth muscle cells enzymatically dispersed from toad stomach. In cells bathed in buffer of physiological composition, the average resting potential was -56.4 +/- 1.4 mV (mean +/- SE, n = 35). The dominant effect of exposure to ISO was hyperpolarization. The hyperpolarization was apparent in all cells studied and averaged 11.6 +/- 1.2 mV (n = 27). In the majority of the cells, hyperpolarization was accompanied by a decreased input resistance (Rin). Often the change in resistance appeared to lag behind the change in membrane potential. The lack of coincident changes in membrane potential and resistance may reflect a superposition of the outward rectification properties of the membrane on beta-adrenergic-induced increases in ionic conductance. In about half of the cells, an initial small depolarization (3.1 +/- 0.3 mV, n = 14) was accompanied by a small but distinct increase in Rin (12 +/- 2.5%). When membrane potential was made more negative than the estimated equilibrium potential for K+ (EK) by injection of current, ISO also produced biphasic effects, an initial hyperpolarization which reversed to a sustained depolarization to a value (-90 mV) near the estimated EK. The hyperpolarization by ISO could be diminished in a time-dependent manner by previous exposure to ouabain. The inhibition by ouabain, however, appeared to be a fortuitous result of glycoside-induced positive shifts in EK. These observations indicate that the dominant electrophysiological effect of beta-adrenergic stimuli is to hyperpolarize the cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

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