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PubMed1983

Potassium channel distribution in spinal root axons of dystrophic mice.

We have used 4-aminopyridine (4AP), a potassium channel blocker, to assess the presence and distribution of potassium channels in the congenitally abnormally myelinated spinal root axons of dystrophic mice. 1 mM-4AP slightly depressed the amplitude but had no effect on the half-width of the monophasic action potential of normal A fibres, indicating the absence of a significant concentration of potassium channels at normal mouse nodes of Ranvier. By progressively increasing stimulus intensity it was possible to elicit three more or less discrete components of the compound action potential from dystrophic mouse spinal roots, presumably corresponding to myelinated fibres, large diameter bare axons and, in the case of dorsal roots, C fibres. The amplitude and duration of all three components were increased on exposure to 4AP, indicating the presence of potassium channels in all types of dystrophic mouse spinal root axons. Conduction in single fibres was studied using longitudinal current analysis. Both saltatory and continuous conduction were observed corresponding to the myelinated and bare portions of dystrophic mouse spinal root axons. Three types of 'nodal' membrane could be inferred from the membrane current recordings from myelinated dystrophic mouse axons: (1) pure sodium channel membrane, (2) membrane containing both sodium and potassium channels, and (3) membrane containing predominantly, if not exclusively, potassium channels. The large early outward currents at the latter two types of nodes suggested that these nodes were wider than normal. Recordings of continuous conduction indicated that potassium channels were also distributed irregularly along bare portions of the dystrophic mouse axons. These abnormalities of ion channel distribution are interpreted as reflecting failure of normal axon-Schwann cell communication in the dystrophic mouse spinal roots.

Action Potentials↗
PubMed2005

KvAP-based model of the pore region of shaker potassium channel is consistent with cadmium- and ligand-binding experiments.

Potassium channels play fundamental roles in excitable cells. X-ray structures of bacterial potassium channels show that the pore-lining inner helices obstruct the cytoplasmic entrance to the closed channel KcsA, but diverge in widely open channels MthK and KvAP, suggesting a gating-hinge role for a conserved Gly in the inner helix. A different location of the gating hinge and a narrower open pore were proposed for voltage-gated Shaker potassium channels that have the Pro-473-Val-Pro motif. Two major observations back the proposal: cadmium ions lock mutant Val-476-Cys in the open state by bridging Cys-476 and His-486 in adjacent helices, and cadmium blocks the locked-open double mutant Val-474-Cys/Val-476-Cys by binding to Cys-474 residues. Here we used molecular modeling to show that the open Shaker should be as wide as KvAP to accommodate an open-channel blocker, correolide. We further built KvAP-, MthK-, and KcsA-based models of the Shaker mutants and Monte-Carlo-minimized them with constraints Cys-476-Cd(2+)-His-486. The latter were consistent with the KvAP-based model, causing a small-bend N-terminal to the Pro-473-Val-Pro motif. The constraints significantly distorted the MthK-based structure, making it similar to KvAP. The KcsA structure resisted the constraints. Two Cd(2+) ions easily block the locked-open KvAP-based model at Cys-474 residues, whereas constraining a single cadmium ion to four Cys-474 caused large conformational changes and electrostatic imbalance. Although mutual disposition of the voltage-sensor and pore domains in the KvAP x-ray structure is currently disputed, our results suggest that the pore-region domain retains a nativelike conformation in the crystal.

Animals↗
PubMed2001

Role of potassium channels in regulation of brain arteriolar tone: comparison of cerebrum versus brain stem.

BACKGROUND AND PURPOSE: Potassium channels are important regulators of resting tone in large cerebral arteries, but their activity and distribution may vary according to vessel location and species studied. In the cerebral microcirculation in vivo, however, these channels appear to be silent at rest. Our goal was to determine the activity of potassium channels of brain arterioles from 2 origins under basal conditions in vitro. METHODS: Penetrating cerebral (40. 9+/-2.2 microm control diameter) and brain stem (36.2+/-1.2 microm) arterioles of rats were prepared from middle cerebral and basilar arteries, respectively. The internal diameter of cannulated and pressurized vessel was monitored with the inverted microscope before and after administration of potassium channel inhibitors. In addition, we studied the effect of nitric oxide synthase inhibition on potassium channel activity. RESULTS: Cerebral and brain stem arterioles were significantly constricted by 4-aminopyridine and low concentration of BaCl(2) but not by glibenclamide. The addition of N:(omega)-nitro-L-arginine to 4-aminopyridine further decreased diameters of both arterioles. Tetraethylammonium ion caused a significant constriction of brain stem but not cerebral arteriole. The brain stem arteriole was further constricted by additional N:(omega)-nitro-L-arginine. CONCLUSIONS: Voltage-dependent and inward-rectifier, but not ATP-sensitive, potassium channels are active under basal conditions of rat cerebral and brain stem arterioles. There is a regional difference in the activity of calcium-activated potassium channels, which, at rest, are open in brain stem but silent in cerebral arterioles. In addition, basal endogenous nitric oxide may not contribute to the activation of voltage-dependent and calcium-activated potassium channels.

4-Aminopyridine↗
PubMed2004

Risk of ventricular proarrhythmia with selective opening of the myocardial sarcolemmal versus mitochondrial ATP-gated potassium channel.

Myocardial ATP-gated potassium channels (K-ATPs) are critical in the intracellular signaling cascade resulting in ischemic preconditioning (IP). Mitochondrial K-ATP channels seem to be responsible for IP, whereas the functions of K-ATP channels in the sarcolemmal membrane are less well understood. The proarrhythmic potential of specific versus nonspecific opening of K-ATP channels has not been investigated. In this study, Langendorff-perfused rabbit hearts were exposed to either pinacidil (1.25 microM), a nonselective K-ATP channel agonist, or selective mitochondrial or sarcolemmal K-ATP channel agonists or antagonists. The hearts were then subjected to 12 min of hypoxic perfusion and 40 min of reoxygenation. Hearts were monitored for the induction of ventricular fibrillation (VF). No heart subjected to hypoxia-reoxygenation without drug treatment developed VF (0 of 5). Pinacidil pretreatment induced VF (12 of 14; p = 0.004 versus control). Pinacidil's effect was blocked by HMR-1098 (1-[5-[2-(5-chloro-o-anisamide)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea) (1 microM), a selective sarcolemmal K-ATP channel antagonist (1 of 7; p = 0.007 versus pinacidil; N.S. versus control). Hearts pretreated with 5-hydroxydecanoate (5-HD) (100 microM), a putatively selective mitochondrial K-ATP channel blocker developed VF in one of eight trials (N.S. versus control). 5-HD did not alter the effects of pinacidil (6 of 8; p < 0.05 versus control; N.S. versus pinacidil alone). Selective mitochondrial K-ATP channel activation with [(3R)-trans-4-((4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)dimethyl-2H-1-benzopyran-6-carbonitril monohydrochloride] (BMS-191095) (6 microM) resulted in zero of five hearts developing VF (N.S. versus control). Our data suggest that selective opening of the sarcolemmal K-ATP channel during hypoxia-reoxygenation induced VF, whereas opening of the mitochondrial channel was not associated with VF. The findings suggest that caution should be exercised when developing compounds aimed at inducing IP, and nonspecific opening of the K-ATP channel should be avoided.

Animals↗
PubMed1993

Characterization of large-conductance, calcium-activated potassium channels from human myometrium.

OBJECTIVES: The purpose of our study was to detect and characterize potassium channels in the plasma membrane of smooth muscle cells from human myometrium. STUDY DESIGN: Plasma membrane vesicles were incorporated into lipid bilayers to record single potassium channel activity. RESULTS: We predominantly found a "maxi" calcium-activated potassium channel (261 picosiemens). This channel was calcium (micromoles per liter range) and voltage sensitive, highly selective for K+ over Na+ and Cs+, and was sensitive to external tetraethylammonium (dissociation constant approximately 220 mumol/L) and charybdotoxin (dissociation constant approximately 23 nmol/L). External apamin and 4-aminopyridine had no effect on this channel. Another type of potassium channel that was less frequently observed was also identified. It had a smaller conductance (142 picosiemens) and it seemed to be calcium independent (up to 50 nmol/L). CONCLUSION: Human myometrium possesses abundant "maxi" calcium-activated potassium channels. This channel shares common characteristics with other "maxi" calcium-activated potassium channels, including calcium and voltage gating, high conductance and selectivity, and channel pharmacologic profile.

Calcium↗
PubMed1988

Newly identified brain potassium channels gated by the guanine nucleotide binding protein Go.

Potassium channels in neurons are linked by guanine nucleotide binding (G) proteins to numerous neurotransmitter receptors. The ability of Go, the predominant G protein in the brain, to stimulate potassium channels was tested in cell-free membrane patches of hippocampal pyramidal neurons. Four distinct types of potassium channels, which were otherwise quiescent, were activated by both isolated brain G0 and recombinant Go alpha. Hence brain Go can couple diverse brain potassium channels to neurotransmitter receptors.

Adenylyl Imidodiphosphate↗
PubMed1993

BRL 38227--a potassium channel opener, antagonizes digoxin-induced convulsions.

Experimental evidence suggests that potassium channel openers play an important role in convulsions. In this study, the anticonvulsant activity of BRL 38227, a new potassium channel opener against digoxin-induced convulsions, is reported. Intraventricular administration of digoxin (7.5 micrograms), included "popcorn-type" convulsions in rats. BRL 38227, injected centrally increased the onset time of convulsions and decreased the mortality rate in a dose-dependent manner. Pretreatment with 4-aminopyridine, a potassium channel blocker antagonized the protective effect of BRL 38227. These findings show the involvement of potassium channels in digoxin-induced convulsions. Further these results indicate that in the future potassium channels might be a target for new anticonvulsant drugs.

4-Aminopyridine↗
PubMed2005

[Inhibition of salicylate on potassium channels in rat inferior colliculus neurons].

OBJECTIVE: To understand what role of the transient outward potassium channels and the delayed rectifier potassium channels play in the mechanism of salicylate-induced tinnitus. METHODS: The effects of salicylate on the transient outward potassium channels and the delayed rectifier potassium channels in freshly dissociated inferior colliculus neurons of rats were studied, using the whole-cell voltage clamp method. RESULTS: Salicylate blocked the transient outward potassium current (I(K(A and the delayed rectifier potassium current (I(K(DR in concentration-dependent manner (0.1-1 mmol/L). The IC50 values for the blocking action of salicylate on I(K(A)) and I(K(DR)) were 2.27 and 0.80 mmol/L, respectively. At a concentration of 1 mmol/L, salicylate did not shift the activation and inactivation curves of I(K(A)), but significantly shifted the activation and inactivation curves of I(K(DR)) negatively by approximately 11 mV and 24 mV. CONCLUSIONS: Salicylate inhibits both I(K(A)) and I(K(DR)) in rat inferior colliculus neurons but only significantly affects the activation and inactivation kinetics of I(K(DR)). Effects of I(K(A)) and I(K(DR)), especially I(K(DR)), by salicylate may play an important role in salicylate-induced tinnitus.

Animals↗
PubMed2002

Long-term administration of nicorandil abolishes ischemic and pharmacologic preconditioning of the human myocardium: role of mitochondrial adenosine triphosphate-dependent potassium channels.

BACKGROUND: Acute administration of mitochondrial adenosine triphosphate-dependent potassium channel openers preconditions the heart, but whether their long-term administration induces a permanent state of protection is unknown. These studies investigate the effect of long-term treatment with the mitochondrial adenosine triphosphate-dependent potassium channel opener nicorandil on the response of the human myocardium to ischemia and preconditioning. METHODS: Right atrial tissue obtained from patients regularly treated with or without nicorandil (mean of 20 mg/d for 18.6 +/- 2.5 months) and undergoing cardiac surgery was sliced and equilibrated for 30 minutes and then subjected to 90 minutes of simulated ischemia, followed by 120 minutes of reoxygenation. In study 1 the following groups were studied to investigate the effect of nicorandil on the susceptibility of the myocardium to ischemia and on the protective effect of ischemic and pharmacologic preconditioning: (1) aerobic control; (2) simulated ischemia and reoxygenation alone; (3) ischemic preconditioning with 5 minutes of simulated ischemia and 5 minutes of reoxygenation; and (4) phenylephrine (0.1 micromol/L) for 5 minutes and 5 minutes' washout before simulated ischemia and reoxygenation. In study 2 the following groups were studied to investigate the effect of nicorandil on the responsiveness of mitochondrial adenosine triphosphate-dependent potassium channels: (1) aerobic control; (2) simulated ischemia and reoxygenation; (3) ischemic preconditioning; (4) diazoxide (100 micromol/L) for 10 minutes before simulated ischemia and reoxygenation, and (5) 5-hydroxydecanoate (1 mmol/L) for 10 minutes before simulated ischemia and reoxygenation. In study 3 the following groups were included to investigate the effect of the long-term administration of nicorandil on the kinase pathway involved in preconditioning: (1) aerobic control; (2) simulated ischemia and reoxygenation alone; (3) ischemic preconditioning; (4) phorbol 12-myristate 13-acetate (1 micromol/L), a protein kinase C activator, for 10 minutes before simulated ischemia and reoxygenation; and (5) anisomycin (1 nmol/L), a p38 mitogen-activated protein kinase activator, for 10 minutes before simulated ischemia and reoxygenation. At the end of each protocol, the leakage of creatine kinase (in units per gram wet weight) and the reduction of 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide into insoluble formazan dye (in millimoles per gram wet weight) were measured. RESULTS: In study 1 the leakage of creatine kinase and the reduction of 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide induced by simulated ischemia and reoxygenation were similar in the groups with or without nicorandil (creatine kinase, 3.4 +/- 0.1 and 3.5 +/- 0.2, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 74.6 +/- 3.9 and 67.9 +/- 7.3, respectively; P >.2 in each instance). Ischemic preconditioning and pharmacologic preconditioning protected the myocardium from patients without nicorandil (creatine kinase, 2.3 +/- 0.1 and 2.4 +/- 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 131.4 +/- 4.9 and 128.4 +/- 5.6, respectively; P < 0.001 vs simulated ischemia and reoxygenation alone in each instance) but not the myocardium from patients receiving nicorandil (creatine kinase, 3.3 +/- 0.1 and 3.3 +/- 0.2, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 89.7 +/- 6.5 and 86.4 +/- 5.2, respectively; P >.2 vs simulated ischemia and reoxygenation alone in each instance). In study 2 the administration of diazoxide had identical protection to that of ischemic preconditioning in the myocardium of patients not receiving nicorandil (creatine kinase, 2.1 +/- 0.2 and 2.3 +/- 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 141.4 +/- 7.4 and 131.4 +/- 4.9, respectively; P < 0.001 vs simulated ischemia and reoxygenation alone in each instance) but failed to precondition the myocardium from patients treated with nicorandil (creatine kinase, 3.3 +/- 0.2 and 3.4 +/- 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 90.1 +/- 7.2 and 86.4 +/- 5.2, respectively; P > 0.2 vs simulated ischemia and reoxygenation alone in each instance). In study 3 phorbol 12-myristate 13-acetate or anisomycin given for 10 minutes before simulated ischemia and reoxygenation afforded similar protection to that of ischemic preconditioning in the myocardium from patients with (creatine kinase, 1.5 +/- 0.3 and 1.4 +/- 0.1, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 147.0 +/- 4.9 and 160.0 +/- 16.1, respectively; P < 0.001 vs simulated ischemia and reoxygenation alone in each instance) and without nicorandil (creatine kinase, 1.7 +/- 0.4 and 1.4 +/- 0.2, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 160.3 +/- 13.6 and 158.3 +/- 11.8, respectively; P <.001 vs simulated ischemia and reoxygenation alone in each instance). CONCLUSION: The myocardium of patients chronically treated with nicorandil cannot be preconditioned either by ischemia or pharmacologically, and this is because of unresponsive mitochondrial adenosine triphosphate-dependent potassium channels. However, protection can be obtained by protein kinase C and p38 mitogen-activated protein kinase activation, which are downstream of mitochondrial adenosine triphosphate-dependent potassium channels in the signaling transduction pathway of preconditioning.

Adenosine Triphosphate↗
PubMed2005

Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels.

ATP-sensitive potassium (K(ATP)) channels of pancreatic beta-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane K(ATP) channels determines the sensitivity of beta-cells to glucose stimulation. The K(ATP) channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of K(ATP) channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of K(ATP) channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic beta-cell line, INS-1, that express endogenous K(ATP) channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of beta-cell K(ATP) channels.

ATP-Binding Cassette Transporters↗
PubMed2005

A gate in the selectivity filter of potassium channels.

The selectivity filter of potassium channels is the structural element directly responsible for the selective and rapid conduction of K+, whereas other parts of the protein are thought to function as a molecular gate that either permits or blocks the passage of ions. However, whether the selectivity filter itself also possesses the ability to play the role of a gate is an unresolved question. Using free energy molecular dynamics simulations, it is shown that the reorientation of two peptide linkages in the selectivity filter of the KcsA K+ channel can lead to a stable nonconducting conformational state. Two microscopic factors influence the transition toward such a conformational state: the occupancy of one specific cation binding site in the selectivity filter (S2), and the strength of intersubunit interactions involving the GYG signature sequence. These results suggest that such conformational transitions occurring in the selectivity filter might be related to different K+ channel gating events, including C-type (slow) inactivation.

Ion Channel Gating↗
PubMed1993

The effects of the ATP-dependent potassium channel antagonist, glyburide, on coronary blood flow and susceptibility to ventricular fibrillation in unanesthetized dogs.

Extracellular potassium rapidly increases during myocardial ischemia and has been implicated in the development of ventricular fibrillation (VF). Recent evidence suggests that ischemically induced potassium efflux results from the opening of ATP-dependent potassium channels. If extracellular potassium accumulation contributes to VF, one would predict that drugs that block the ATP-dependent potassium channels should protect against these arrhythmias. Therefore, VF was induced in 15 mongrel dogs with healed myocardial infarctions by a 2-min coronary occlusion during exercise. This exercise (Ex) plus ischemia test consistently induced VF during each control (vehicle) presentation. However, glyburide, a sulfonylurea drug that selectively blocks the ATP-dependent potassium channel, prevented VF in 13 of 15 animals tested (p < 0.001, chi 2). Glyburide (G) elicited significant reductions in left ventricular dP/dtmax (Ex: control, 5,031 +/- 386.4; G, 3,286 +/- 116.6 mm Hg/s) and mean coronary blood flow (Ex: control, 49.6 +/- 8.7; G, 31.3 +/- 5.5 ml/min) both before and during exercise. The heart rate responses to exercise and coronary occlusion were also reduced. Since heart rate reductions could contribute to the cardioprotection, the exercise plus ischemia test was repeated with the heart rate held constant by ventricular pacing. Glyburide still protected four of five animals under these conditions. These data indicate that glyburide, a drug that blocks the ATP-dependent potassium channels, can prevent VF independently of changes in the heart rate. The data further suggest that these channels may contribute to the coronary vasodilation elicited by an increase in metabolic demand during exercise.

Adenosine Triphosphate↗
PubMed1994

Modulation by protein kinase A of a cloned rat brain potassium channel expressed in Xenopus oocytes.

A potassium channel from rat brain was expressed in Xenopus oocytes in order to study modulation of channel function by phosphorylation via protein kinase A. Application of 8-Br-cAMP to oocytes expressing the drk1 channel (with the first 139 amino acids of the N terminus deleted, delta Ndrk1) caused a voltage-independent elevation of current amplitude, which was not seen for endogenous currents or for wild-type full-length drk1 channel. This effect on delta Ndrk1 was blocked by pre-injection of oocytes with Walsh-peptide protein kinase A inhibitor, suggesting mediation via protein kinase A. The protein kinase inhibitor also reduced both delta Ndrk1 and full-length drk1 currents. Substitution of the serine residues by alanine at one or both of the two consensus protein kinase A phosphorylation sites on the C terminus (residues 440 and 492) of delta Ndrk1 resulted in a loss of function of the expressed channels. These results indicate that phosphorylation via protein kinase A modulates drk1 channel function and that both consensus phosphorylation sites seems to be essential for channels to function.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Single potassium channels of human glioma cells.

Single potassium channels in the membrane of human malignant glioma cells U-118MG were studied using the technique of patch clamp in cell-attached and inside-out configurations. Three types of potassium channels were found which differed from each other under conditions close to physiological in their conductance and gating characteristics. The lowest-conductance channel (20 pS near the reversal potential) showed a mild outward rectification up to 45 pS at positive voltages and spontaneous modes of high and low activity. At extreme values of potentials its activity was generally low. The intermediate conductance channel had an S-shaped I-V curve, giving a conductance of 63 pS at reversal, and a low and voltage independent opening probability. The high-conductance (215 pS) channel was found to be activated by both membrane potential and Ca2+ ions and blocked by internal sodium at high voltages. The current-voltage curves of all three channel types displayed saturation.

Calcium↗
PubMed2000

Energetic and structural interactions between delta-dendrotoxin and a voltage-gated potassium channel.

Dendrotoxin proteins isolated from Mamba snake venom block potassium channels with a high degree of specificity and selectivity. Using site-directed mutagenesis we have identified residues that constitute the functional interaction surfaces of delta-dendrotoxin and its voltage-gated potassium channel receptor. delta-Dendrotoxin uses a triangular patch formed by seven side-chains (Lys3, Tyr4, Lys6, Leu7, Pro8, Arg10, Lys26) to block K(+) currents carried by a Shaker potassium channel variant. The inhibitory surface of the toxin interacts with channel residues at Shaker positions 423, 425, 427, 431, and 449 near the pore. Amino acid mutations that interact across the toxin-channel interface were identified by mutant cycle analysis. These results constrain the possible orientation of dendrotoxin with respect to the K(+) channel structure. We propose that dendrotoxin binds near the pore entryway but does not act as a physical plug.

Amino Acid Sequence↗
PubMed1994

Use of cultured human neuroblastoma cells in rapid discovery of the voltage-gated potassium-channel blockers.

Depolarization of human neuroblastoma cells by high concentrations of extracellular potassium ions, leads to the activation of the voltage-gated potassium channels. The activity of such potassium channels can be effectively and rapidly monitored by tracking the efflux of 86Rb from pre-loaded target cells in response to the depolarizing stimulus. The inclusion of compounds with unknown activity in the assay medium, can result in the identification of novel blockers of the voltage-gated potassium channels. Since this functional assay is performed in 96-well microtitre plates, it represents a rapid and high-volume primary screening method for the detection and identification of the voltage-gated potassium-channel blockers, which may have therapeutic utility in several indications including memory degeneration and cardiac arrhythmias.

4-Aminopyridine↗
PubMed2007

Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes.

Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.

Animals↗
PubMed1993

Potassium channel blockade of atrial negative inotropic responses to P1-purinoceptor and muscarinic receptor agonists and to cromakalim.

The negative inotropic responses of guinea pig isolated left atria to the P1-receptor agonists adenosine and L-N6-phenylisopropyladenosine (L-PIA), the muscarinic receptor agonist carbachol, and the potassium channel activator cromakalim were examined. The potassium channel blocker 4-aminopyridine (4-AP, 10 mM) decreased the concentration-response curve for L-N-6-phenylisopropyladenosine (L-PIA) so that the maximum reduction in atrial tension was significantly reduced from 77.8 to 61.9%. The concentration-response curve for the negative inotropic response to carbachol was displaced to the right by a substantially greater amount (521-fold), partly due to the muscarinic receptor blocking activity of 4-AP. The response to a single submaximum dose of L-PIA (520 nM) was slow in onset and monophasic, whereas adenosine induced a rapid reduction in tension followed by a gradual return toward the resting level. In the presence of 4-AP (10 mM), the peak response to L-PIA was significantly reduced from a 59.7 to 31.0% inhibition of tension. In addition, the rate of development of the response was significantly slowed. The peak inhibition of tension by adenosine (112 microM) (60.0%) was also significantly reduced to 37.4% in the presence of 4-AP (10 mM). Furthermore, there was no rapid decrease in tension but a gradual decrease to a peak effect which was no different from that in the absence of 4-AP. These results suggest that the P1-receptor agonists exert negative inotropy through a K+ channel-dependent component which is blocked by 4-AP and a slower-onset K+ channel-independent component. The potassium channel blocker, bromobenzoylmethyladamantylamine (BMA 100 microM) also shifted (11-fold) the concentration-response curve for the negative inotropic response to adenosine, and to a lesser extent that to cromakalim (2.1-fold) and carbachol. Glibenclamide (0.3, 3, and 30 microM), a selective antagonist of ATP-regulated potassium channels, was a potent antagonist of the negative inotropic responses to cromakalim. However, 30 microM failed to antagonize the responses to L-PIA, adenosine, or carbachol, indicating that they do not mediate their responses through ATP-regulated potassium channels. 4-AP (10 mM) caused a slight shift (2.3-fold) of the concentration-response curve for cromakalim, indicating weak activity against ATP-regulated potassium channels in the left atrium. This study showed that P1-receptor agonists exert negative inotropic responses in guinea pig left atria only in part through activation of potassium channels. These channels differ from the ATP-regulated potassium channels activated by cromakalim.

4-Aminopyridine↗