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PubMed2006

The functional HERG variant 897T is associated with Conn's adenoma.

OBJECTIVE: Aldosterone secreting adenomas (aldosteronomas) have an unknown molecular origin. Ion channel currents are involved in signal transduction leading to aldosterone synthesis and secretion. HERG (human-ether-a-go-go-related gene) encodes for a potassium channel responsible for the outward rectifying delayed current and it is mutation prone. When mutated it causes most of the familial forms of both long QT and short QT syndromes. Abnormal repolarization in glomerulosa cells might increase aldosterone secretion or induce a proliferative advantage. The aims of this study were to: (1) evaluate HERG expression in aldosteronomas; (2) search for HERG somatic mutations; and (3) determine whether there is any relationship between the common HERG functional variant (A2690C, leading from lysine 897 to threonine, K897T) and aldosteronoma. DESIGN AND METHODS: Aldosteronoma and blood samples from 17 patients were studied to evaluate HERG expression, full-length HERG complementary DNA sequencing, and genotyping for K897T alleles. The prevalence of HERG 897 alleles was also tested in a control population and a population consisting entirely of hypertensive individuals. RESULTS: HERG was expressed in all aldosteronomas analysed. HERG somatic mutations were not detected. The 897T variant of HERG was significantly more common among patients with aldosteronoma (897T allele 41%) than in patients with moderate-severe essential hypertension (897T allele 20%, P = 0.007) or in the control population (897T allele 12%, P < 0.0001). The 897T/T genotype was present in 24% of the aldosteronoma patients versus 7% (P = 0.040) and 3% (P = 0.001) in essential hypertension and in the control population, respectively. When the chi test was performed considering the three groups together, the significance was similar (for alleles P < 0.0001 and for genotypes P = 0.004). CONCLUSION: The common functional HERG variant 897T may predispose to the development of aldosteronoma.

Adrenocortical Adenoma↗
PubMed1995

Cholinergic inhibition of slow delayed-rectifier K+ current in guinea pig sino-atrial node is not mediated by muscarinic receptors.

We studied the effects of cholinergic agonists on slow delayed-rectifier K+ current (IKs) in isolated cells from the sino-atrial node (SAN) region of guinea pig heart, using patch-clamp procedures. Carbachol (5 nM to 10 microM) inhibited IKs in guinea pig SAN cells in the absence of previous beta-adrenergic stimulation and in cells pretreated with 8-(4-chlorophenylthio)-cAMP. Neither the muscarinic antagonist atropine nor the nicotinic antagonist hexamethonium antagonized carbachol inhibition of the current. Similar results were obtained with other cholinergic agonists. Cholinergic stimulation of the muscarinic K+ current was successfully antagonized by atropine in SAN cells where inhibition of IKs persisted. Therefore, the lack of antagonist effects on inhibition of IKs cannot be attributed to either an absence of muscarinic cholinoceptors on SAN cells or a loss of antagonist activity under our experimental conditions. These data demonstrate that cholinergic agonists, including the endogenous neurotransmitter acetylcholine, decrease the amplitude of IKs in guinea pig SAN cells via a non-muscarinic, non-nicotinic, cAMP-independent mechanism. Although the precise nature of this signal transduction pathway has not been elucidated, it is clearly different from those described for regulation of other nodal currents. Differential regulation of IKs in guinea pig SAN and ventricle cannot be attributed to higher basal adenylate cyclase activity in SAN cells. The inhibitory effect of carbachol on IKs was not additive with that of verapamil, a drug that is both an allosteric muscarinic antagonist and a potassium channel-blocking agent. Cholinergic agonists may inhibit IKs in SAN cells via a direct interaction with the SAN IKs channel.

Allosteric Regulation↗
PubMed2005

Isolation and characterization of I(Kr) in cardiac myocytes by Cs+ permeation.

Isolation of the rapidly activating delayed rectifier potassium current (I(Kr)) from other cardiac currents has been a difficult task for quantitative study of this current. The present study was designed to separate I(Kr) using Cs+ in cardiac myocytes. Cs+ have been known to block a variety of K+ channels, including many of those involved in the cardiac action potential such as inward rectifier potassium current I(K1) and the transient outward potassium current I(to). However, under isotonic Cs+ conditions (135 mM Cs+), a significant membrane current was recorded in isolated rabbit ventricular myocytes. This current displayed the voltage-dependent onset of and recovery from inactivation that are characteristic to I(Kr). Consistently, the current was selectively inhibited by the specific I(Kr) blockers. The biophysical and pharmacological properties of the Cs+-carried human ether-a-go-go-related gene (hERG) current were very similar to those of the Cs+-carried I(Kr) in ventricular myocytes. The primary sequence of the selectivity filter in hERG was in part responsible for the Cs+ permeability, which was lost when the sequence was changed from GFG to GYG, characteristic of other, Cs+-impermeable K+ channels. Thus the unique high Cs+ permeability in I(Kr) channels provides an effective way to isolate I(Kr) current. Although the biophysical and pharmacological properties of the Cs+-carried I(Kr) are different from those of the K+-carried I(Kr), such an assay enables I(Kr) current to be recorded at a level that is large enough and sufficiently robust to evaluate any I(Kr) alterations in native tissues in response to physiological or pathological changes. It is particularly useful for exploring the role of reduction of I(Kr) in arrhythmias associated with heart failure and long QT syndrome due to the reduced hERG channel membrane expression.

Animals↗
PubMedDate not supplied

The [3H]dofetilide binding assay is a predictive screening tool for hERG blockade and proarrhythmia: Comparison of intact cell and membrane preparations and effects of altering [K+]o.

INTRODUCTION: The human ether-a-go-go-related gene (hERG) encodes a potassium channel responsible for the cardiac delayed rectifier current (IKr) involved in ventricular repolarization. Drugs that block hERG have been associated with QT interval prolongation and serious, sometimes fatal, cardiac arrhythmias (including torsade de pointes). While displacement of [3H]dofetilide, a potent methanesulfonanilide hERG blocker, from cells heterologously expressing hERG has been suggested as a screening assay, questions have been raised about its predictive value. METHODS: To validate the utility of this assay as a screening tool, we performed a series of saturation and competition binding studies using [3H]dofetilide as ligand and either intact cells or membrane preparations from HEK 293 cells stably transfected with hERG K+ channels. The object of these experiments was to (1) compare binding Ki values for 22 hERG blockers using intact cells or membrane homogenates to determine whether maintaining cell integrity enhanced assay reliability; (2) evaluate the ability of different K+ concentrations (2, 5, 10, 20, and 60 mM) to modulate hERG binding; and (3) to establish the predictive value of the assay by comparing Ki values from binding studies at 5 and 60 mM [K+]o to functional IC50 values for hERG current block using 56 structurally diverse drugs. RESULTS: We found (a) comparable Ki values in the intact cell and isolated membrane binding assays, although there were some differences in rank order; (b) increasing [K+]o lowered the Kd and increased the Bmax for [3H]dofetilide, particularly in the membrane assay; and (c) good correlation between binding Ki values and functional IC50 values for hERG current block. DISCUSSION: In conclusion, increasing K+ concentrations results in an increase in both [3H]dofetilide affinity for hERG and available binding sites, particularly when using membrane homogenates. There are no meaningful differences between Ki values when comparing intact cell versus membrane assay, neither are there meaningful trends with increasing [K+]o within assays. There is good correlation between binding Ki values and functional (whole-cell patch clamp) IC50 values at both 5 and 60 mM K+ concentrations (R2 values of .824 and .863, respectively). The simplicity, predictability, and adaptability to high-throughput platforms make the [3H]dofetilide membrane binding assay a useful tool for screening and ranking compounds for their potential to block the hERG K+ channel.

Anti-Arrhythmia Agents↗
PubMed1997

Impulse encoding mechanisms of ganglion cells in the tiger salamander retina.

A study of nerve impulse generation in ganglion cells of the tiger salamander retina is carried out through a combination of experimental and analytic approaches, including computer simulations based on a single-compartment model. Whole cell recordings from ganglion cells were obtained using a superfused retina-eyecup preparation and studied with pharmacological and electrophysiological techniques, including phase plot analysis. Experimental efforts were guided by computer simulation studies of an excitability model consisting of five voltage- or ion-gated channels, which were identified from earlier voltage-clamp data. The ion channels include sodium, calcium, and three types of potassium channels, namely the A type (IK,A), Ca-activated potassium (IK,Ca), and the delayed rectifier (IK). A leakage channel was included to preserve input resistance continuity between model and experiment. Ion channel densities of Na and Ca currents (INa and ICa) for the single-compartment model were independently determined from phase plot analysis. The IK and IK,A current densities were determined from the measured width of impulses. The IK,Ca was modeled to respond to Ca influx, and a variable-rate Ca-sequestering mechanism was implemented to remove cytoplasmic calcium. Impulse frequency increases when either ICa or IK,Ca is eliminated from the model or blocked pharmacologically in whole cell recording experiments. Faithful simulations of experimental data show that the ionic currents may be grouped into small (IK,Ca, leakage, and stimulus), and large (INa, IK, IA, ICa) on the basis of their peak magnitudes throughout the impulse train. This division of the currents is reflected in their function of controlling the interspike interval (small currents) and impulse generation (large currents). Although the single-compartmental model is qualitatively successful in simulating impulse frequency behavior and its controlling mechanisms, limitations were found that specifically suggest the need to include morphological details. The spike train analysis points to a role for electrotonic currents in the control of the duration of the interspike intervals, which can be compensated by prolonged activation of gK,Ca in the single-compartment model. A detailed, multicompartmental model of the ganglion cell is presented in the companion paper.

Animals↗
PubMed1996

A patch-clamp study of delayed rectifier currents in skeletal muscle of control and mdx mice.

1. Potassium currents were measured in the extensor digitorum longus muscle of normal and mdx mice, which lack the protein dystrophin, using the cell-attached and inside-out patch clamp techniques, in the presence of asymmetrical K+ concentrations (3 mM in the pipette, 160 mM in the bath). 2. In cell-attached patches, the delayed rectifier was the most commonly found potassium channel, with a density of roughly 8 channels microns-2. Outward macroscopic currents were activated in macropatches depolarized to potentials positive to -60 mV. The probability of opening reached half-maximal values around -40 mV for control patches and -31 mV for patches from mdx mice. 3. Tail currents were linear in the range between -60 and +20 mV, reversing close to -100 mV. The single channel current at 0 mV, estimated from non-stationary analysis of variance, was used in conjunction with the slope of the linear part of the tail current to calculate the single channel conductance, yielding a value of 19 +/- 1 pS. 4. At 0 mV, the delayed rectifier inactivated with two time constants, of 70 +/- 20 ms and 600 +/- 200 ms. Prepulses of 500 ms duration to different potentials produced incomplete inactivation with inactivation reaching 50% of its maximum at -50 mV. 5. Single channel activity was recorded using small pipettes. Both single channel conductance and kinetic behaviour were in agreement with the macroscopic current data. 6. In excised patches, the delayed rectifier current ran down, unmasking other K+ channels. A Ca(2+)-dependent K+ channel of 186 pS (BK-like channel) was found frequently in patches bathed in solutions containing appropriate concentrations of calcium, especially at stronger depolarizations. A K+ channel of 63 pS was unmasked in control excised patches bathed in solutions devoid of ATP. This channel was not observed in patches excised from mdx fibers.

Adenosine Triphosphate↗
PubMed2002

Imipramine, mianserine and maprotiline block delayed rectifier potassium current in ventricular myocytes.

Imipramine, mianserine and maprotiline are three widely used antidepressant drugs with different chemical structure. In the present work we have studied the effects of these drugs on the delayed rectifier potassium current (I(K)) in myocytes isolated from rat ventricle. The delayed rectifier potassium current, responsible for action potential termination, is blocked by all of the three drugs I(K)studied in a state-independent manner. Imipramine and mianserine block I(K)in a 1 : 1 drug-receptor interaction, whereas maprotiline shows a negative cooperativity in the interaction between the channel complex and drug molecules.

Action Potentials↗
PubMed2003

[Effects of AlCl3 on transient outward K+ current and delayed rectifier K(+) current in acutely isolated rat hippocampal CA1 neurons].

The effects of aluminum chloride (AlCl3) on the transient outward potassium and delayed rectifier K(+) current in hippocampal CA1 neurons of rats were studied by the whole-cell patch clamp technique. It was found that AlCl3 reduced the transient outward potassium current and delayed rectifier K(+) current in a dose-dependent manner. 1000 micromol/L AlCl3 resulted in change in voltage and slope of the half-activation and the half-inactivation of I(A) and I(K). These results imply that AlCl3 may damage potassium channel of the hippocampal CA1 neurons from rats and this may be related to the mechanism of the damage to the central nervous system by aluminum.

Aluminum Chloride↗
PubMed1991

Voltage-activated potassium channels in blowfly photoreceptors and their role in light adaptation.

1. The membrane properties of the photoreceptors of the blowfly (Calliphora vicina) were investigated in situ by making intracellular recordings in the intact retina, using discontinuous single-electrode current and voltage clamp techniques. Single channels were investigated using inside-out patches from dissociated photoreceptors. 2. Photoreceptors have a resting potential in darkness of -60.4 +/- 6.6 mV (mean +/- S.D.; n = 43), a resting input resistance of 32 +/- 3 M omega (n = 11) and membrane time constant of 4.1 +/- 1 ms (n = 9). These values give a total cell capacitance of 0.13 nF and an effective membrane area of 1.3 x 10(-4) cm2. 3. Single-electrode voltage clamp reveals a voltage-sensitive outward current with an activation threshold at approximately -75 mV. This conductance has two kinetic components, the slower component activating at more depolarized levels. On the basis of its kinetics, a reversal potential of -85 +/- 6 mV (n = 6), sensitivity to intracellularly injected tetraethylammonium chloride (TEA), and its slow and partial inactivation (approximately 25%) this mechanism is classified as a delayed rectifier potassium conductance. 4. Voltage-sensitive potassium channels showing similar properties were found in excised inside-out patches from dissociated photoreceptors. Single-channel conductances are ca 20 pS for both fast and slow kinetic components, indicating a channel density in the intact cell of ca 2 microns -2. The reversal potential follows the Nernst slope for potassium ions. 5. The voltage dependence of the conductance was determined in patches containing channels of predominantly one or the other kinetic component. The midpoint of the activation curve is -65 mV for the fast and -50 mV for the slow component. Activation time constants (measured from a holding potential of -100 mV) are voltage dependent, and in the range 1-10 ms for the fast and 5-40 ms for the slow component. Both kinetic components are blocked by TEA (greater than 2.5 mM). The slow component is more sensitive to quinidine (greater than 200 microM), and the fast component to 4-aminopyridine (4-AP; greater than 200 microM). 6. In the intact preparation the outward current shows no dependence on light stimulation in the studied ranges of voltage (up to -25 mV) and intensity (up to 5.5 x 10(4) effective photons). Ensemble averages of channel openings in perfused inside-out patches show no dependence on calcium concentration in the range 10 nM-1.8 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Ocular↗
PubMed2006

Long QT 1 mutation KCNQ1A344V increases local anesthetic sensitivity of the slowly activating delayed rectifier potassium current.

BACKGROUND: Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go-related gene) channel inhibition. The aim of this study was to investigate whether the LQT1 mutation A344V in the S6 region of KCNQ1, at a position corresponding to the local anesthetic binding site in HERG, may render drug insensitive KCNQ1 channels into a toxicologically relevant target of these pharmacologic agents. This may suggest that LQTS constitutes not only a nonspecific but also a specific pharmacogenetic risk factor for anesthesia. METHODS: The authors examined electrophysiologic and pharmacologic properties of wild-type and mutant KCNQ1 channels. The effects of bupivacaine, ropivacaine, and mepivacaine were investigated using two-electrode voltage clamp and whole cell patch clamp recordings. RESULTS: The mutation A344V induced voltage-dependent inactivation in homomeric KCNQ1 channels and shifted the voltage dependence of KCNQ1/KCNE1 channel activation by +30 mV. The mutation furthermore increased the sensitivity of KCNQ1/KCNE1 channels for bupivacaine 22-fold (KCNQ1wt/KCNE1: IC50 = 2,431 +/- 582 microM, n = 20; KCNQ1A344V/KCNE1: IC50 = 110 +/- 9 microM, n = 24). Pharmacologic effects of the mutant channels were dominant when mutant and wild-type channels were coexpressed. Simulation of cardiac action potentials with the Luo-Rudy model yielded a prolongation of the cardiac action potential duration and induction of early afterdepolarizations by the mutation A344V that were aggravated by local anesthetic intoxication. CONCLUSIONS: The results indicate that certain forms of the LQTS may constitute a specific pharmacogenetic risk factor for regional anesthesia.

Action Potentials↗
PubMed1996

Sodium and potassium currents of type I spiral ganglion cells from rat.

Ion channel activity of acutely dissociated type I spiral ganglion cells isolated from rats was investigated using the whole-cell variation of the patch clamp technique. Tetrodotoxin-sensitive sodium current and tetraethylammonium-sensitive potassium current were recorded. With a holding potential of -80 mV, peak sodium currents were generated by depolarizations to membrane potentials more positive than -50 mV. Potassium currents were elicited at membrane potentials more positive than -40 mV. Some cells which did not exhibit sodium current exhibited delayed rectifier potassium currents. Steady-state sodium channel inactivation curve yielded a slope of 12 mV and a half-inactivated potential of -83 mV. Recovery from inactivation of the sodium channel proceeded with a fast and slow time course; recovery began as early as 0.8 ms and proceeded with a time constant of 7.5 ms. It is concluded that type I spiral ganglion cells are endowed with sodium and potassium channels which are responsible for generation and propagation of auditory nerve action potentials.

Action Potentials↗
PubMed2002

Electrophysiological and antiarrhythmic effects of the novel I(Kur) channel blockers, S9947 and S20951, on left vs. right pig atrium in vivo in comparison with the I(Kr) blockers dofetilide, azimilide, d,l-sotalol and ibutilide.

Inhibition of the cardiac Kv1.5 channel, the molecular base for the human cardiac ultrarapid delayed rectifier potassium current (I(Kur)), is considered a new promising atrial selective antiarrhythmic concept since this channel is presumed to contribute to atrial but not ventricular repolarization in the human heart. In a previous study in pigs we found clear baseline differences in refractoriness between left and right atrium with shorter effective refractory periods (ERPs) of the left atrium associated with a high left atrial vulnerability for tachyarrhythmias. In this newly established model we compared atrial and ventricular effects of two novel I(Kur) blockers, S9947 and S20951, with the I(Kr) blockers dofetilide, azimilide, ibutilide and d,l-sotalol. In pentobarbital anesthetized pigs (n=45) we determined ERPs in the free walls of both atria with the S1-S2-stimulus method at three basic cycle lengths (BCL 240/300/400 ms) and QTc-intervals. The incidence of atrial tachyarrhythmias triggered by the S2-extrastimulus of the left atrium was evaluated (referred to as left atrial vulnerability). In contrast to I(Kr) blockade, I(Kur) blockade had no effect on the QT-interval, but prolonged the atrial ERP. The I(Kur) blockers were significantly stronger on left atrial ERP, I(Kr) blockers on right atrial ERP (P<0.05 for all compounds tested). At 240 ms BCL the I(Kur) blocker S20951, 3 mg/kg, prolonged left vs. right atrial ERP by 28+/-5 ms vs. 12+/-3 ms and S9947, 3 mg/kg, by 45+/-7 ms vs. 19+/-6 ms. By contrast the effect of dofetilide, 10 microg/kg, was stronger on the right than left atrium (47+/-6 ms vs. 25+/-2 ms), a profile also found with azimilide (5 mg/kg, 43+/-3 ms vs. 17+/-3 ms), ibutilide (15 microg/kg, 70+/-10 ms vs. 29+/-4 ms) and d,l-sotalol (1.5 mg/kg, 57+/-6 ms vs. 36+/-4 ms). The I(Kur) blockers, S20951and S9947, significantly decreased left atrial vulnerability (-82% and -100%, respectively, P<0.01) in contrast to the selective I(Kr) blocker dofetilide (-14%; n.s.). In conclusion, I(Kur) and I(Kr) blockers showed substantial differences in their atrial and ventricular actions in pigs. I(Kr) blockers were stronger on right atrial ERP, I(Kur) blockers on left atrial ERP, suggesting interatrial differences in the expression of potassium channels. In contrast to selective I(Kr) blockade, I(Kur) blockade inhibited left atrial vulnerability and had no effect on the QT-interval. Thus, blockade of I(Kur) seems to be a promising atrial selective antiarrhythmic concept.

Animals↗
PubMed1997

Action potential-generating cells in human glioblastomas.

We studied the electrophysiological properties of cells from human glioblastomas obtained after surgery. The membrane currents were compared in cells of acute tissue slices and primary cultures using the whole cell mode of the patch-clamp technique. Very strikingly, in about a third of the tumor cells in situ and in vitro, depolarizing voltage steps elicited large, tetrodotoxin-sensitive inward currents with a threshold of about -30 mV, indicating the presence of voltage-gated sodium channels. In addition, three types of potassium currents, a delayed rectifying, an A-type, and an inward rectifying, were observed. Such a set of voltage-gated channels is characteristic for neurons. Indeed, in these glioblastoma cells, depolarizing current pulses in the current clamp mode were able to generate action potentials with properties similar to those observed in neurons. We interpret this finding as the ability of glioblastoma cells to acquire neuronlike properties but retain some glial features, since they still express markers typical for astrocytes and their precursors. The role of sodium channels in glioblastoma cells is unclear at this moment and needs further investigation. Our findings, however, imply that the tumor tissue can be intrinsically excitable and that neoplastic glial cells themselves may be an etiologic factor for epileptic seizures.

Action Potentials↗
PubMed2000

Molecular evidence for a role of Shaw (Kv3) potassium channel subunits in potassium currents of dog atrium.

We previously described an ultrarapid delayed rectifier current in dog atrial myocytes (IKur,d) with properties resembling currents reported for Kv3.1 channels in neural tissue; however, there was no direct molecular evidence for Shaw subfamily (Kv3) subunit expression in the heart. To identify the molecular basis of IKur,d, we cloned a full-length cDNA (dKv3.1) from canine atrium with homology-based reverse transcription (RT)- polymerase chain reaction (PCR) cloning techniques. A 1755 bp full-length cDNA (dKv3.1) was obtained, with 94.2 % homology to rat brain Kv3.1 (rbKv3.1). The deduced amino acid sequence had 99.3 % homology with rbKv3.1. Heterologous expression of dKv3.1 in Xenopus oocytes produced currents with activation voltage dependence, rectification, and activation and deactivation kinetics that strongly resemble native IKur,d. Like IKur,d, dKv3.1 was found to be highly sensitive to extracellular 4-aminopyridine (4-AP) and tetraethylammonium (TEA). RNase protection assays, Western blots and immunohistochemical studies demonstrated the presence of dKv3.1 transcripts and proteins in dog atrial preparations and isolated canine atrial myocytes. Protein corresponding to the Kv1.5 subunit, which can also carry ultrarapid delayed rectifier current, was absent. Unlike neural tissues, which express two splice variants (Kv3.1a and Kv3.1b), canine atrium showed only Kv3.1b transcripts. Whole-cell patch-clamp studies showed that IKur,d is absent in canine ventricular myocytes, and immunohistochemical and Western blot analysis demonstrated the absence of dKv3.1 protein in canine ventricle. We conclude that the Shaw-type channel dKv3.1 is present in dog atrium, but not ventricle, and is the likely molecular basis of canine atrial IKur,d.

4-Aminopyridine↗
PubMed1995

Beta-adrenergic modulation of glial inwardly rectifying potassium channels.

Cultured spinal cord astrocytes (2-13 days in vitro) express several different potassium current types, including delayed rectifier, transient A-type, and inward rectifier (Kir) K+ currents. Of these, Kir is believed to be of critical importance in the modulation of extracellular [K+] in the CNS. Using the whole-cell patch-clamp technique, we analyzed modulation of Kir currents by beta-adrenergic receptor activation. The selective beta-adrenergic agonist isoproterenol (1-100 microM) and epinephrine (1-100 microM) each reduced peak Kir current amplitudes to 52.7 +/- 12.5 and 63.6 +/- 7.0%, respectively, at 100 microM. Forskolin (KD of approximately 25 microM), an activator of adenylate cyclase (AC), and dibutyryl-cyclic AMP (1 mM), a membrane-permeable analogue of cyclic AMP (cAMP), were each used to increase [cAMP]i, the product of AC, and resulted in similar reductions of Kir currents. By contrast, 1,9-dideoxyforskolin (1-50 microM), a forskolin analogue that does not activate AC, did not affect Kir currents, indicating that AC activity is a required element for Kir modulation. Three inhibitors of PKA--Rp-adenosine 3',5'-cyclic monophosphothioate, H-7, and adenosine 3',5'-cyclic monophosphate-dependent protein kinase inhibitor--failed to inhibit Kir current reduction by beta-adrenergic agonists. These results indicate that beta-adrenergic receptor ligands can modulate Kir currents and suggest that this modulation involves activation of AC but not protein kinase A. Such modulation may provide a mechanism by which neurons can modulate glial Kir currents and thereby may affect glial K+ "spatial buffering" in the CNS.

Adenylyl Cyclases↗
PubMedDate not supplied

Unique interaction of scorpion toxins with the hERG channel.

ERG potassium channels specify one component of the delayed rectifier in the heart and are likely to play an important functional role in other excitable cells. Compared to other K+ channels, the human ERG (hERG) channel possesses an unusually long S5-P linker that presumably forms an alpha-helix important for channel function. hERG-specific toxins bind to the outer mouth of the hERG channel. Channel residues in the middle of the S5-P linker and at the pore entrance are critical for toxin binding. One of these scorpion toxins is BeKm-1. Residues critical for BeKm-1 binding to the hERG channel are located in the alpha-helix and the following loop, whereas the "traditional" interaction surface of other short scorpion toxins is formed by residues on the beta-sheet. This unique localization of BeKm-1's interaction surface and its specific action on the hERG channel suggest a unique outer mouth structure of the hERG channel. We used the mutant cycle analysis approach to define contacts in the toxin-channel complex. This information provides critical constraints and is important for molecular modeling of the hERG pore structure.

Animals↗
PubMed1990

K+ current diversity is produced by an extended gene family conserved in Drosophila and mouse.

The Drosophila Shaker gene on the X chromosome has three sister genes, Shal, Shab, and Shaw, which map to the second and third chromosomes. This extended gene family encodes voltage-gated potassium channels with widely varying kinetics (rate of macroscopic current activation and inactivation) and voltage sensitivity of steady-state inactivation. The differences in the currents of the various gene products are greater than the differences produced by alternative splicing of the Shaker gene. In Drosophila, the transient (A current) subtype of the potassium channel (Shaker and Shal) and the delayed-rectifier subtype (Shab and Shaw) are encoded by homologous genes, and there is more than one gene for each subtype of channel. Homologs of Shaker, Shal, Shab, and Shaw are present in mammals; each Drosophila potassium-channel gene may be represented as a multigene subfamily in mammals.

Amino Acid Sequence↗
PubMedDate not supplied

Voltage-gated K+ channels in human T lymphocytes: a role in mitogenesis?

Membrane receptors and ion transport mechanisms probably have an important role in lymphocyte activation leading to T-lymphocyte proliferation in the immune response. Here we have applied a gigaohm-seal patch clamp technique to reveal the identity and properties of ion channels in human T lymphocytes. A voltage-dependent potassium channel bearing a resemblance to the delayed rectifier of nerve and muscle cells was found to be the predominant ion channel in these cells. In the whole cell recording conformation, the channels open with sigmoid kinetics during depolarizing voltage steps, reaching a maximum K+ conductance of 3-5 nS. The current subsequently becomes almost completely inactivated during a long-lasting depolarization. Currents through single K+ channels recorded in whole cell and outside-out patch recording conformations reveal a unitary channel conductance of about 16 pS in normal Ringer solution. Thus, the peak current corresponds to approximately 200-300 conducting K+ channels per cell. Phytohaemagglutinin (PHA), at concentrations that produce mitogenesis, alters K+ channel gating within 1 min of addition to the bathing solution, causing channels to open more rapidly and at more negative membrane potentials. 3H-thymidine incorporation by T lymphocytes following PHA stimulation is inhibited by the 'classical' K+ channel blockers tetraethylammonium and 4-aminopyridine, and also by quinine, at doses found to block the K+ channel in voltage-clamped T lymphocytes, suggesting that K+ channels may play a part in mitogenesis.

Calcium↗