Calcitonin gene-related peptide activates the K+ channels of vascular smooth muscle cells via adenylate cyclase

The aim of the present study was to examine the effects of calcitonin gene-related peptide (CGRP) on the K⁺ channels of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 nM CGRP activated two types of K⁺ channels the Ca²⁺-activated K⁺ channel (K_Ca channel) and the ATP-sensitive K⁺ channel (K_ATP channel) in cell-attached patch configurations. In cells pretreated with Rp-cAMPS, a membrane-permeable inhibitor of cAMP-dependent protein kinase (PKA), extracellular application of 100 nM CGRP could not activate the K_Ca or K_ATP channel, indicating that the activation of the K⁺ channels by CGRP occurs in connection with PKA. In the cell-attached patch configurations, extracellular application of 1 mM dibutyryl cAMP, a membrane permeable cAMP, activated K_Ca and K_ATP channels. In inside-out patch configurations, application of PKA to the cytosolic side activated both the K_Ca and K_ATP channels. These results indicate that CGRP modulates the K⁺ channels of vascular smooth muscle cells via adenylate cyclase, i.e., cAMP-PKA pathway, and contributes to control of vascular tone.     

HMR 1098 is not an SUR isotype specific inhibitor of heterologous or sarcolemmal K ATP channels

Murine ventricular and atrial ATP-sensitive potassium (K_ATP) channels contain different sulfonylurea receptors (ventricular K_ATP channels are Kir6.2/SUR2A complexes, while atrial K_ATP channels are Kir6.2/SUR1 complexes). HMR 1098, the sodium salt of HMR 1883 {1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea}, has been considered as a selective sarcolemmal (i.e. SUR2A-dependent) K_ATP channel inhibitor. However, it is not clear whether HMR 1098 would preferentially inhibit ventricular K_ATP channels over atrial K_ATP channels. To test this, we used whole-cell patch clamp techniques on mouse atrial and ventricular myocytes as well as ⁸⁶Rb⁺ efflux assays and excised inside-out patch clamp techniques on Kir6.2/SUR1 and Kir6.2/SUR2A channels heterologously expressed in COSm6 cells. In mouse atrial myocytes, both spontaneously activated and diazoxide-activated K_ATP currents were effectively inhibited by 10 μM HMR 1098. By contrast, in ventricular myocytes, pinacidil-activated K_ATP currents were inhibited by HMR 1098 at a high concentration (100 μM) but not at a low concentration (10 μM). Consistent with this finding, HMR 1098 inhibits ⁸⁶Rb⁺ effluxes through Kir6.2/SUR1 more effectively than Kir6.2/SUR2A channels in COSm6 cells. In excised inside-out patches, HMR 1098 inhibited Kir6.2/SUR1 channels more effectively, particularly in the presence of MgADP and MgATP (mimicking physiological stimulation). Finally, dose-dependent enhancement of insulin secretion from pancreatic islets and decrease of blood glucose level confirm that HMR 1098 is an inhibitor of Kir6.2/SUR1-composed K_ATP channels.     

NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1

GABA_B receptors are the G-protein–coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GABA_B receptors are abundant on dendritic spines, where they dampen postsynaptic excitability and inhibit Ca²⁺ influx through NMDA receptors when activated by spillover of GABA from neighboring GABAergic terminals. Here, we show that an excitatory signaling cascade enables spines to counteract this GABA_B-mediated inhibition. We found that NMDA application to cultured hippocampal neurons promotes dynamin-dependent endocytosis of GABA_B receptors. NMDA-dependent internalization of GABA_B receptors requires activation of Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII), which associates with GABA_B receptors in vivo and phosphorylates serine 867 (S867) in the intracellular C terminus of the GABA_B1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABA_B receptors refractory to NMDA-mediated internalization. Time-lapse two-photon imaging of organotypic hippocampal slices reveals that activation of NMDA receptors removes GABA_B receptors within minutes from the surface of dendritic spines and shafts. NMDA-dependent S867 phosphorylation and internalization is predominantly detectable with the GABA_B1b subunit isoform, which is the isoform that clusters with inhibitory effector K⁺ channels in the spines. Consistent with this, NMDA receptor activation in neurons impairs the ability of GABA_B receptors to activate K⁺ channels. Thus, our data support that NMDA receptor activity endocytoses postsynaptic GABA_B receptors through CaMKII-mediated phosphorylation of S867. This provides a means to spare NMDA receptors at individual glutamatergic synapses from reciprocal inhibition through GABA_B receptors.     

Potassium secretion in rat distal colon during dietary potassium loading: role of pH regulated apical potassium channels

Background—Chronicdietary K⁺ loading increases the abundance of largeconductance (210 pS) apical K⁺ channels in surface cells ofrat distal colon, resulting in enhanced K⁺ secretion inthis epithelium. However, the factors involved in the regulation ofthese K⁺ channels are at present unclear.
Aims—To evaluate theeffect of dietary K⁺ loading on intracellular pH and itsrelation to large conductance apical K⁺ channel activity insurface cells of rat distal colon.
Methods/Results—Asassessed by fluorescent imaging, intracellular pH was higher inK⁺ loaded animals (7.48 (0.09)) than in controls (7.07 (0.04); p<0.01) when surface cells were bathed in NaCl solution, and asimilar difference in intracellular pH was observed when cells werebathed in Na₂SO₄ solution (7.67 (0.09) and 6.92 (0.05) respectively; p<0.001). Ethylisopropylamiloride (EIPA; aninhibitor of Na⁺-H⁺ exchange; 1 µM) decreasedintracellular pH when surface cells from K⁺ loaded animalswere bathed in either solution, although the decrease was greater whenthe solution contained NaCl (ΔpH 0.50 (0.03)) rather thanNa₂SO₄ (ΔpH 0.18 (0.02); p<0.05). Incontrast, EIPA had no effect in cells from control animals. As assessedby patch clamp recording techniques, the activity of large conductance K⁺ channels in excised inside-out membrane patches fromdistal colonic surface cells of K⁺ loaded animals increasedtwofold when the bath pH was raised from 7.40 to 7.60. As assessed bycell attached patches in distal colonic surface cells fromK⁺ loaded animals, the addition of 1 µM EIPA decreasedK⁺ channel activity by 50%, consistent with reversal ofNa⁺-H⁺ exchange mediated intracellular alkalinisation.
Conclusion—Intracellularalkalinisation stimulates pH sensitive large conductance apicalK⁺ channels in rat distal colonic surface cells as part ofthe K⁺ secretory response to chronic dietary K⁺loading. Intracellular alkalinisation seems to reflect an increase inEIPA sensitive Na⁺-H⁺ exchange, which may be amanifestation of the secondary hyperaldosteronism associated with thismodel of colonic K⁺ adaptation.

     

Collateral response to activation of potassium channels in vivo

Activation of ATP-sensitive K⁺ channels is involved in the coronary vascular response to decreases in perfusion pressure and ischemia. Since activation of ATP-sensitive K⁺ channels in collateral vessels may be important in determining flow to collateral-dependent myocardium, the ability of collaterals to respond to activation of the channel was tested. In the beating heart of dogs, we compared responses of non-collaterals less than 100 μm in diameter to collaterals of similar size using computer-controlled stroboscopic epi-illumination of the left ventricle coupled to a microscope-video system. Aprikalim, a selective activator of ATP-sensitive K⁺ channels (0.1–10 μM) produced similar dose-dependent dilation of non-collaterals and collaterals. Relaxation was decreased by inhibition of ATP-sensitive K⁺ channels with glibenclamide, but not by inhibition of nitric oxide synthase with nitro-L-arginine. Bradykinin (10–100 μM) produced similar dilation of non-collaterals and collaterals which was decreased by nitro-L-arginine but not glibenclamide. Thus, in microvascular collaterals, relaxation to both nitric oxide and activation of ATP-sensitive K⁺ channels is similar to non-collaterals. Received: 7 April 1997, Returned for revision: 5 May 1997, Revision received: 23 December 1997, Accepted: 13 January 1998     

Effects of extracellular potassium and 4-aminopyridine on corticosteroid secretion

Transient alteration in [K⁺]₀ significantly influenced basal and stimulated corticosteroid secretory activity of superfused rat adrenal cortical tissue slices. A transient rise in [K⁺]₀ resulted in an increase in basal steroid secretion. Removal of extracellular K⁺ from the superfusing medium significantly reduced basal as well as ACTH and cAMP-stimulated steroid production. Elevated [K⁺]₀ significantly potentiated cAMP's and to a much lesser extent increased ACTH's steroid stimulatory activity. The K⁺ flux inhibitor, 4-aminopyridine (4AP) (10 mM) elicited an initial decline in basal steroid secretory activity. A single 10-ml dose of 10 mM but not 1 mM 4AP significantly diminished, for many hours, ACTH's and cAMP's ability to stimulate adrenal steroid secretion. The combination of elevated [K⁺]₀ and 4AP administration resulted in additional reduction of the ability of ACTH to stimulate steroid production. This combination of 4AP with [K⁺]₀ elevations also blocked the potentiating effect of K⁺ on cAMP-stimulated steroid secretion. Together these findings demonstrated that some procedures which can alter the distribution of K⁺ across the adrenal cell membrane are capable of profoundly influencing the secretion of the predominant glucocorticoid from the rat adrenal gland under in vitro superfusion conditions. The data support the suggestion that a slight rise in intracellular K⁺ concentration would be stimulatory and that further increase of, or a decline in, this ion's intracellular level would be inhibitory to basal and stimulated steroid secretory activity.     

Effects of ion channels on proliferation in cultured human cardiac fibroblasts

Our previous study demonstrated that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts, including a large-conductance Ca²⁺-activated K⁺ current (BKCa), a volume-sensitive chloride current (I_Cl.vol), and voltage-gated sodium currents (I_Na). The present study was designed to examine the possible involvement of these ion channels in proliferation of cultured human cardiac fibroblasts using approaches of cell proliferation assay, whole-cell patch voltage-clamp, siRNA and Western blot analysis. It was found that the blockade of BKCa with paxilline (1-3 μM) or I_Cl.vol with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium (DIDS, 100-200 μM), but not I_Na with tetrodotoxin (0.1-10 μM), remarkably suppressed proliferation in human cardiac fibroblasts. Knockdown of KCa1.1 or Clcn3 with specific siRNAs significantly reduced BKCa or I_Cl.vol current, mRNA and channel protein levels, and inhibited growth of human cardiac fibroblasts. Flow cytometry analysis showed accumulation of cardiac fibroblasts at G0/G1 phase and reduced cell number in S phase after inhibition of BKCa or I_Cl.vol with channel blockers or knock down of the corresponding channels with specific siRNAs; these effects were accompanied by a decreased expression of cyclin D1 and cyclin E. The present results demonstrate the novel information that BKCa and I_Cl.vol channels, but not I_Na channels, are involved in the regulation of proliferation in cultured human cardiac fibroblasts by promoting cell cycle progression via modulating cyclin D1 and cyclin E expression.     

Na+-mediated coupling between AMPA receptors and KNa channels shapes synaptic transmission

Na⁺-activated K⁺ (K_Na) channels are expressed in neurons and are activated by Na⁺ influx through voltage-dependent channels or ionotropic receptors, yet their function remains unclear. Here we show that K_Na channels are associated with AMPA receptors and that their activation depresses synaptic responses. Synaptic activation of K_Na channels by Na⁺ transients via AMPA receptors shapes the decay of AMPA-mediated current as well as the amplitude of the synaptic potential. Thus, the coupling between K_Na channels and AMPA receptors by synaptically induced Na⁺ transients represents an inherent negative feedback mechanism that scales down the magnitude of excitatory synaptic responses.     

Disopyramide and its metabolite enhance insulin release from clonal pancreatic beta-cells by blocking K(ATP) channels

In an insulin-secreting pancreatic -cell line (MIN6), insulin release was caused by disopyramide, an antiarrhythmic drug with Na-channel blocking action, and its main metabolite mono-isopropyl disopyramide (MIP). Insulin secretion, measured as immunoreactive insulin (IRI), was accelerated to 265.7% of the control by disopyramide and to 184.4% by MIP, with half-effective concentrations (EC₅₀) of 30.9 ± 1.5 M and 92.4 ± 2.2 M. We tested the possibility that these drugs induce insulin release by inhibiting ATP-sensitive K⁺ (K_ATP) channels of MIN6 cells. In the cell-attached or ATP-free inside-out mode with patch membranes on MIN6 cells, K-selective channels were recorded with unitary conductance of 70.5 ± 3.5 pS (150 mM external K⁺ ions at room temperature). The channels were concluded to be MIN6-K_ATP channels because they were closed by extracellular high glucose (11.0 mM) or glibenclamide (200 nM) and were reversibly activated by diazoxide (50 M). In the inside-out patch mode, they were inhibited by micromolar ATP. In both cell-attached and insideout mode, disopyramide and MIP inhibited single MIN6-K_ATP channels. In the inside-out mode, they produced a dose-dependent inhibition of channel activity: the half-blocking concentrations (IC⁵⁰) were 4.8 ± 0.2 M for disopyramide and 40.4 ± 3.1 M for MIP. It was therefore concluded that both agents exert insulinotrphic effect through the inhibition of membrane K_ATP channels in MIN6 cells.     

Dependence of prolactin release on coupling between Ca(2+) mobilization and voltage-gated Ca(2+) influx pathways in rat lactotrophs

Two Ca²⁺-mobilizing receptors expressed in lactotrophs, endothelin-A (ET_A) and thyrotropin-releasing hormone (TRH), induce a rapid Ca²⁺ release from intracellular stores and prolactin (PRL) secretion but differ in their actions during the sustained stimulation; TRH facilitates and ET-1 inhibits voltage-gated calcium influx (VGCI) and PRL secretion. In pertussis toxin (PTX)-treated cells, ET-1-induced inhibition of VGCI was abolished and the pattern of Ca²⁺ signaling was highly comparable with that observed in TRH-stimulated cells. The addition of Cs⁺, a relatively specific blocker of inward rectifier K⁺ channels, mimicked the effect of PTX on the pattern of ET-1-induced sustained Ca²⁺ signaling, but only in about 50% of cells, and did not affect agonist-induced inhibition of PRL secretion. Extracellular Cs⁺ was also ineffective in altering the TRH-induced facilitation of VGCI and PRL secretion. Furthermore, apamin and paxilline, specific blockers of Ca²⁺-activated SK-and BK-type K⁺ channels, respectively; E-4031, a blocker of ether a-go-go K⁺ channel; and linopirdine, a blocker of M-type K⁺ channel, did not affect the agonist-specific patterns of calcium signaling and PRL secretion. These results suggest that ET-1 inhibits VGCI through activation of Cs⁺-sensitive channels, presumably the G_i/o-controlled inward rectifier K⁺ channels, and that this agonist also inhibits PRL release, but downstream of Ca²⁺ influx. Further studies are required to identify the mechanism of sustained TRH-induced facilitation of VGCI and PRL secretion.     

Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange between plants and the atmosphere in response to light, CO₂, and the plant hormone abscisic acid. Light-induced stomatal opening is mediated by at least three key components: the blue light receptor phototropin (phot1 and phot2), plasma membrane H⁺-ATPase, and plasma membrane inward-rectifying K⁺ channels. Very few attempts have been made to enhance stomatal opening with the goal of increasing photosynthesis and plant growth, even though stomatal resistance is thought to be the major limiting factor for CO₂ uptake by plants. Here, we show that transgenic Arabidopsis plants overexpressing H⁺-ATPase using the strong guard cell promoter GC1 showed enhanced light-induced stomatal opening, photosynthesis, and plant growth. The transgenic plants produced larger and increased numbers of rosette leaves, with ∼42–63% greater fresh and dry weights than the wild type in the first 25 d of growth. The dry weights of total flowering stems of 45-d-old transgenic plants, including seeds, siliques, and flowers, were ∼36–41% greater than those of the wild type. In addition, stomata in the transgenic plants closed normally in response to darkness and abscisic acid. In contrast, the overexpression of phototropin or inward-rectifying K⁺ channels in guard cells had no effect on these phenotypes. These results demonstrate that stomatal aperture is a limiting factor in photosynthesis and plant growth, and that manipulation of stomatal opening by overexpressing H⁺-ATPase in guard cells is useful for the promotion of plant growth.In the present era of global climate changes and the threat of food insufficiency, finding ways to improve the uptake of CO₂ by terrestrial plants is an increasingly important problem. Stomata, key organs in the uptake of CO₂, are microscopic pores surrounded by two specialized cells in the epidermis (named guard cells) and are mainly found on the leaf surface in terrestrial plants. Because the leaf surface is nearly impermeable to air and water, stomata provide the major pathway for the diffusion of CO₂, O₂, and water vapor between the ambient atmosphere and the interior of the leaf. This facilitation of gas exchange by stomatal opening is one of the most essential processes in plant photosynthesis and transpiration (1, 2). A recent study indicated that stomatal transpiration limited photosynthesis in rice (3). Therefore, increased stomatal opening/transpiration is expected to promote photosynthesis and thereby increase plant growth. Condon et al. (4) examined diverse wheat genotypes and showed that increased stomatal conductance, especially abaxial stomatal conductance, may have a positive effect on crop biomass. However, to our knowledge, no previous studies have determined stomatal effects on plant growth by manipulating stomatal aperture via gene regulation in guard cells, perhaps because of the difficulty in balancing the counteracting effects of taking up CO₂ while losing water vapor through the stomata (5).Light is one of the principal factors that stimulates stomatal opening, and various mechanisms underlie stomatal opening in response to different light wavelengths (6–8). Red light is thought to induce stomatal opening via photosynthesis in the mesophyll and guard cell chloroplasts, as well as the reduction of the intercellular CO₂ concentration (Ci) (5, 9, 10). However, the detailed mechanisms of stomatal responses to red light are under debate (11, 12). In contrast, blue light acts as a signal and exerts the most pronounced effect on stomatal opening. The blue light receptors phototropins (phot1 and phot2) activate plasma membrane H⁺-ATPase through the phosphorylation of the penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated threonine (13–15). Blue light-activated H⁺-ATPase induces hyperpolarization of the plasma membrane, which allows K⁺ uptake through inward-rectifying K⁺ (K⁺_in) channels (16–21). Accumulation of K⁺ induces the swelling of guard cells and stomatal opening. Thus, these three components (phototropins, plasma membrane H⁺-ATPase, and K⁺_in channels) have important roles in blue light-induced stomatal opening. In addition to these components, FLOWERING LOCUS T (FT) is suggested to be a positive regulator for stomatal opening via its effect on the activation status of the plasma membrane H⁺-ATPase (22).In this study, we produced transgenic plants expressing key components active in stomatal opening under the control of the strong guard cell promoter GC1 to promote stomatal opening in Arabidopsis thaliana (23). We showed that transgenic Arabidopsis plants overexpressing H⁺-ATPase in guard cells exhibited enhanced light-induced stomatal opening, photosynthesis, and plant growth, and that stomatal aperture is a limiting factor in photosynthesis and plant growth.     

Genotype–phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor of Kv7.2 potassium channel subunits

Mutations in the K_V7.2 gene encoding for voltage-dependent K⁺ channel subunits cause neonatal epilepsies with wide phenotypic heterogeneity. Two mutations affecting the same positively charged residue in the S₄ domain of K_V7.2 have been found in children affected with benign familial neonatal seizures (R213W mutation) or with neonatal epileptic encephalopathy with severe pharmacoresistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct neuroradiological features (R213Q mutation). To examine the molecular basis for this strikingly different phenotype, we studied the functional characteristics of mutant channels by using electrophysiological techniques, computational modeling, and homology modeling. Functional studies revealed that, in homomeric or heteromeric configuration with K_V7.2 and/or K_V7.3 subunits, both mutations markedly destabilized the open state, causing a dramatic decrease in channel voltage sensitivity. These functional changes were (i) more pronounced for channels incorporating R213Q- than R213W-carrying K_V7.2 subunits; (ii) proportional to the number of mutant subunits incorporated; and (iii) fully restored by the neuronal K_v7 activator retigabine. Homology modeling confirmed a critical role for the R213 residue in stabilizing the activated voltage sensor configuration. Modeling experiments in CA1 hippocampal pyramidal cells revealed that both mutations increased cell firing frequency, with the R213Q mutation prompting more dramatic functional changes compared with the R213W mutation. These results suggest that the clinical disease severity may be related to the extent of the mutation-induced functional K⁺ channel impairment, and set the preclinical basis for the potential use of K_v7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates with K_V7.2 encephalopathy.     

Potassium channels in cardiac cells

K⁺ channels in heart can be subdivided into two groups, voltage-operated and ligand-operated channels. Only the voltage-operated channels—i_K, i_to, and i_K1—and one ligand-operated channel—i_K(ACh)—are activated under physiological conditions; the i_K1 only carries large currents at the resting potential. The delayed K⁺ current, i_K, and the transient outward current, i_to, are important for the plateau and repolarization phase of the action potential, and thus affect the refractory period in atrial and ventricular cells and also the frequency in the sinoatrial node. A high density of i_to and i_Ca is responsible for the spike-dome appearance of the plateau phase in adult atrial cells, epicardial myocytes, and Purkinje cells stimulated by catecholamines. The action-potential duration in these cells is more sensitive to K_e ⁺, frequency, and drugs. The i_K(ACh) can also be activated by adenosine and somatostatin. Its density is high in nodal and atrial tissue. Under pathological conditions, K⁺ channels dependent on ATP, Na_i ⁺, and fatty acids are activated and can carry large currents at depolarized levels. Local changes in the concentration of ATP or Na close to the membrane are probably important for the activation process. The study of pharmacological modulation of K⁺ currents should include frequency effects for the voltage-activated channels.     

Activation of ATP-Sensitive Potassium Channels Underlies Contractile Failure in Single Human Cardiac Myocytes During Complete Metabolic Inhibition

ATP-Sensitive Potassium Channels in Human Heart. Introduction: The purpose of this study was to examine the electrophysiologic derangements that underlie contractile failure in single human heart muscle cells exposed to metabolic inhibition. Methods and Results: Single myocytes were isolated from right atrial appendage specimens obtained intraoperatively from patients undergoing routine cardiac surgery. On exposure to lO-mM 2-deoxyglucose (to inhibit glycolysis) and 2-mM cyanide (to inhibit oxidative phosphorylation), twitch shortening decreased to undetectable levels over 5–6 minutes. The action potential duration declined in parallel with the contractile failure. Using voltage clamp depolarizations of a fixed duration the twitch was maintained in metabolic blockade until the development of maintained (rigor) contracture. At this time a large increase in K⁺ conductance, which can be attributed to the activation of ATP-sensitive K ⁺ channels (K _ATP channels), was measured. In isolated inside-out membrane patches, the ATP dependence of K_aTP channel activity was described by a sigmoid curve with K_i,_ATP (ATP concentration required for half-maximal inhibition of K _ATPchannel activity) = 8 μM and Hill coefficient (n_H) = 1.2. The single channel current-voltage relationship reversed close to the K ⁺ equilibrium potential and the conductance was approximately linear (g = 29 pS) over the voltage range included in the action potential (-60 m V to +20 mV). Conclusion: In human atrial cardiac myocytes subjected to complete metabolic inhibition, contractile failure is caused by action potential shortening resulting from an increase in K + conductance presumably through the activation of K_ATP channels. (J Cardiovasc Electrophysiol, Vol. 3, pp. 56–63, February 1992)     

The mode of action of tedisamil on voltage-dependent K+ channels

The mechanism of the interaction of tedisamil with voltage-dependent K⁺ channels was studied using whole-cell and single-channel recordings in a variety of species and cell types. In K⁺ channels with rapid activation kinetics (I_to of rat ventricular myocytes; I_A of mouse astroglial cells), tedisamil enhanced the kinetics of inactivation of the current without significantly suppressing the amplitude of the initial current. In K⁺ channels with slower activation/inactivation kinetics, tedisamil had a divergent effect. On I_K of the glial cells, which have slow activation and inactivation kinetics, the kinetics of inactivation were enhanced and the initial peak current was reduced. On the other hand, in I_K of guinea-pig ventricular myocytes, which have even slower activation kinetics with no inactivation, tedisamil slowed or completely suppressed the activation of the current. Finally, in K⁺ channels with rapid activation but slow inactivation kinetics (pedestal-type current of rat ventricular myocytes), tedisamil accelerated the inactivation without affecting the initial current. Thus, the prime determinant of the blocking mode of tedisamil appeared to be the kinetics of activation of the K⁺ channel; that is, the slower the kinetics of activation of the channel, the greater the initial block by the drug. Unitary I_to currents recorded in rat ventricular myocytes showed that tedisamil induced a rapid flicker block of the open channel and prolonged the time between the burst of openings without any effect on the unitary conductance. These effects were modeled by assuming that the drug bound to the open channel at a finite rate. Thus, tedisamil appears to decrease K⁺ currents by interacting uniformly with the open state of the channel.     

Hyperosmotic perfusion of the beating rat heart and the role of the Na+/K+/2CI− co-transporter and the Na+/H+ exchanger

The aim of the present study was to investigate the role of the Na⁺/K⁺/2CI⁻ co-transporter and the Na⁺/H⁺ exchanger on contractile function and electrolyte regulation during hyperosmotic perfusion of the heart. Langendorff perfused rat hearts were subjected to hyperosmolal perfusion in 10-min intervals. Perfusates were made hyperosmotic by adding mannitol to the buffer (370, 450 and 600 mOsmol/kg H₂O). Cardiac contractile function was monitored with a balloon in the left ventricle (LV) coupled to a pressure transducer. Cardiac effluent was sampled repeatedly throughout and after hyperosmotic perfusion and analyzed for content of Na⁺, K⁺ and CI⁻. All three hyperosmotic perfusates initially reduced LV developed pressure (LVDP), but for 370 and 450 mOsmol/kg H₂, LVDP recovered to baseline within 4 min of perfusion. With 600 mOsmol/kg H₂, LVDP recovered slowly and was 50% below baseline after 10 min of hyperosmotic perfusion. Inhibition of the Na⁺/H⁺ exchanger with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and 3-methyl-sulfonyl-4-piperidinobenzoyl-guanidine methanesulfonate (HOE 694) abolished the recovery of LVDP to the 600 mOsmol/kg H₂ perfusate, whereas inhibition of the Na⁺/K⁺/2CI⁻ co-transporter had no impact on LVDP. Potassium was taken up by the heart during hyperosmotic perfusion and this uptake was significantly reduced with inhibition of the Na⁺/H⁺ exchanger. Intracellular pH was assessed with ³¹P magnetic resonance spectroscopy and hyperosmolality induced a significant alkalosis that was dependent upon the Na⁺/H⁺ exchanger. The rat heart responds to moderate elevations in osmolality with a transient reduction in contractile function, whereas an elevation of 300 mOsmol/kg H₂ persistently reduces contractile function. The Na⁺/H⁺ exchanger, but not the Na⁺/K⁺/2CI⁻ co-transporter, is of importance in contractile recovery and electrolyte regulation during hyperosmotic perfusion in the rat heart. Received: 10 December 1998, Returned for revision: 18 January 1999, Revision received: 1 July 1999, Accepted: 19 July 1999     

Excitation of rat cerebellar Golgi cells by ethanol: further characterization of the mechanism

Background: Studies with rodents suggest that acute ethanol exposure impairs information flow through the cerebellar cortex, in part, by increasing GABAergic input to granule cells. Experiments suggest that an increase in the excitability of specialized GABAergic interneurons that regulate granule cell activity (i.e., Golgi cells [GoCs]) contributes to this effect. In GoCs, ethanol increases spontaneous action potential firing frequency, decreases the afterhyperpolarization amplitude, and depolarizes the membrane potential. Studies suggest that these effects could be mediated by inhibition of the Na⁺/K⁺ ATPase. The purpose of this study was to characterize the potential role of other GoC conductances in the mechanism of action of ethanol. Methods: Computer modeling techniques and patch‐clamp electrophysiological recordings with acute slices from rat cerebella were used for these studies. Results: Computer modeling suggested that modulation of subthreshold Na⁺ channels, hyperpolarization‐activated currents, and several K⁺ conductances could explain some but not all actions of ethanol on GoCs. Electrophysiological studies did not find evidence consistent with a contribution of these conductances. Quinidine, a nonselective blocker of several types of channels (including several K⁺ channels) that also antagonizes the Na⁺/K⁺ ATPase, reduced the effect of ethanol on GoC firing. Conclusions: These findings further support that ethanol increases GoC excitability via modulation of the Na⁺/K⁺ ATPase and suggest that a quinidine‐sensitive K⁺ channel may also play a role in the mechanism of action of ethanol.     

Altered Na+ transport after an intracellular α-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump

The Na/K pump actively exports 3 Na⁺ in exchange for 2 K⁺ across the plasmalemma of animal cells. As in other P-type ATPases, pump function is more effective when the relative affinity for transported ions is altered as the ion binding sites alternate between opposite sides of the membrane. Deletion of the five C-terminal residues from the α-subunit diminishes internal Na⁺ (Na_i⁺) affinity ≈25-fold [Morth et al. (2007) Nature 450:1043–1049]. Because external Na⁺ (Na_o⁺) binding is voltage-dependent, we studied the reactions involving this process by using two-electrode and inside-out patch voltage clamp in normal and truncated (ΔKESYY) Xenopus-α1 pumps expressed in oocytes. We observed that ΔKESYY (i) decreased both Na_o⁺ and Na_i⁺ apparent affinities in the absence of K_o⁺, and (ii) did not affect apparent Na_o⁺ affinity at high K_o⁺. These results support a model of strict sequential external release of Na⁺ ions, where the Na⁺-exclusive site releases Na⁺ before the sites shared with K⁺ and the ΔKESYY deletion only reduces Na_o⁺ affinity at the shared sites. Moreover, at nonsaturating K_o⁺, ΔKESYY induced an inward flow of Na⁺ through Na/K pumps at negative potentials. Guanidinium⁺ can also permeate truncated pumps, whereas N-methyl-D-glucamine cannot. Because guanidinium_o⁺ can also traverse normal Na/K pumps in the absence of both Na_o⁺ and K_o⁺ and can also inhibit Na/K pump currents in a Na⁺-like voltage-dependent manner, we conclude that the normal pathway transited by the first externally released Na⁺ is large enough to accommodate guanidinium⁺.     

Expression of voltage-gated K<Superscript>+</Superscript> channels in human atrium

Voltage-gated K⁺ channels underlie repolarisation of the cardiac action potential and represent a potential therapeutic target in the treatment of cardiac dysrhythmias. However, very little is known about the relative expression of K⁺ channel subunits in the human myocardium. We used a semi-quantitative RT-PCR technique to examine the relative expression of mRNAs for the voltage-gated K⁺ channel subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, KvLQT1, HERG and IsK in samples of human atrial appendage. Data were expressed as a percentage expression density relative to an 18S ribosomal RNA internal standard. The most abundant K⁺ channel mRNAs were Kv4.3 (80.7 ± 10.1 %), Kv1.5 (69.7 ± 11.2 %) and HERG (55.9 ± 21.5 %). Significant expression of KvLQT1 (33.5 ± 5.5 %,) and Kv1.4 (26.7 ± 9.6 %) was also detected. Levels of mRNAs for Kv1.2 and IsK were very low and neither Kv2.1 nor Kv4.2 mRNA were detected in any experiments. Whole-cell patch-clamp techniques were used to examine the outward currents of isolated human atrial myocytes at 37 °C. These recordings demonstrated the existence of transient (I_to1) and sustained (I_so) outward currents in isolated human atrial myocytes. I_to1, and not I_so, showed voltage-dependent inactivation during 100 ms pre-pulses. Both I_to1 and I_so were inhibited by high concentrations (2 mM) of the K⁺ channel blocker, 4-aminopyridine (4-AP). However, lower concentrations of 4-AP (10 μM) inhibited I_so selectively. I_to1 recovered from inactivation relatively rapidly (t ∼21 ms). These data, with published information regarding the properties of expressed K⁺ channels, suggest that Kv4.3 represents the predominant K⁺ channel subunit underlying I_to1 with little contribution of Kv1.4. The sensitivity of Iso to very low concentrations of 4-aminopyridine and the relatively low expression of mRNA for Kv1.2 and Kv2.1 is consistent with the major contribution of Kv1.5 to this current. The physiological significance of the expression of KvLQT1 and Kv1.4 mRNA in the human atrium warrants further investigation. Received: 30 August 2000, Returned for 1. revision: 21 September 2000, 1. Revision received: 21 June 2002, Returned for 2. revision: 15 July 2002, 2. Revision received: 30 July 2002, Accepted: 31 July 2002 Correspondence to: Dr. A. F. James