Apamin-sensitive Ca2 Research Articles

Sex-specific IKAS activation in rabbit ventricles with drug-induced QT prolongation

Female sex is a known risk factor for drug-induced long QT syndrome (diLQTS). We recently demonstrated a sex difference in apamin-sensitive small-conductance Ca2+-activated K+ current (IKAS) activation during β-adrenergic stimulation. The purpose of this study was to test the hypothesis that there is a sex difference in IKAS in the rabbit models of diLQTS. We evaluated the sex difference in ventricular repolarization in 15 male and 22 female Langendorff-perfused rabbit hearts with optical mapping techniques during atrial pacing. HMR1556 (slowly activating delayed rectifier K+ current [IKs] blocker), E4031 (rapidly activating delayed rectifier K+ current [IKr] blocker) and sea anemone toxin (ATX-II, late Na+ current [INaL] activator) were used to simulate types 1-3 long QT syndrome, respectively. Apamin, an IKAS blocker, was then added to determine the magnitude of further QT prolongation. HMR1556, E4031, and ATX-II led to the prolongation of action potential duration at 80% repolarization (APD80) in both male and female ventricles at pacing cycle lengths of 300-400 ms. Apamin further prolonged APD80 (pacing cycle length 350 ms) from 187.8±4.3 to 206.9±7.1 (P=.014) in HMR1556-treated, from 209.9±7.8 to 224.9±7.8 (P=.003) in E4031-treated, and from 174.3±3.3 to 188.1±3.0 (P=.0002) in ATX-II-treated female hearts. Apamin did not further prolong the APD80 in male hearts. The Cai transient duration (CaiTD) was significantly longer in diLQTS than baseline but without sex differences. Apamin did not change CaiTD. We conclude that IKAS is abundantly increased in female but not in male ventricles with diLQTS. Increased IKAS helps preserve the repolarization reserve in female ventricles treated with IKs and IKr blockers or INaL activators.

Impact of ISK Voltage and Ca2+/Mg2+-Dependent Rectification on Cardiac Repolarization

Cardiac small conductance Ca2+-activated K+ (SK) channels are activated solely by Ca2+, but the SK current (ISK) is inwardly rectified. However, the impact of inward rectification in shaping action potentials (APs) in ventricular cardiomyocytes under β-adrenergic stimulation or in disease states remains undefined. Two processes underlie this inward rectification: an intrinsic rectification caused by an electrostatic energy barrier from positively charged amino acids at the inner pore and a voltage-dependent Ca2+/Mg2+ block. Thus, Ca2+ has a biphasic effect on ISK, activating at low [Ca2+] yet inhibiting ISK at high [Ca2+]. We examined the effect of ISK rectification on APs in rat cardiomyocytes by simultaneously recording whole-cell apamin-sensitive currents and Ca2+ transients during an AP waveform and developed a computer model of SK channels with rectification features. The typical profile of ISK during AP clamp included an initial peak (mean 1.6 pA/pF) followed by decay to the point that submembrane [Ca2+] reached ∼10 μM. During the rest of the AP stimulus, ISK either plateaued or gradually increased as the cell repolarized and submembrane [Ca2+] decreased further. We used a six-state gating model combined with intrinsic and Ca2+/Mg2+-dependent rectification to simulate ISK and investigated the relative contributions of each type of rectification to AP shape. This SK channel model replicates key features of ISK recording during AP clamp showing that intrinsic rectification limits ISK at high Vm during the early and plateau phase of APs. Furthermore, the initial rise of Ca2+ transients activates, but higher [Ca2+] blocks SK channels, yielding a transient outward-like ISK trajectory. During the decay phase of Ca2+, the Ca2+-dependent block is released, causing ISK to rise again and contribute to repolarization. Therefore, ISK is an important repolarizing current, and the rectification characteristics of an SK channel determine its impact on early, plateau, and repolarization phases of APs.

Role of SK Current Rectification in Shaping Action Potential of Ventricular Cardiomyocytes

The role of cardiac small conductance Ca2+-activated K+ (SK) channels in shaping the action potential (AP) of ventricular cardiomyocytes remains undefined. Two processes underlie inward rectification of SK current (ISK), an intrinsic voltage-dependent rectification caused by positively charged amino acids at the inner pore, and a divalent ion block. The latter process gives Ca2+ a biphasic effect on ISK, activating at low [Ca2+] yet inhibiting ISK at high [Ca2+]. We examined the role of ISK rectification on AP in rat cardiomyocytes by simultaneously recording whole-cell apamin-sensitive currents and Ca2+ transients during an AP waveform, and developed a computer model of SK channel with rectification features. The typical profile of ISK during AP-clamp included an initial peak (mean 1.6 pA/pF) followed by decay to the point where submembrane [Ca2+] reached ∼10 μM. During the rest of the AP stimulus, ISK either plateaued or gradually increased as the cell repolarized and submembrane [Ca2+] continued to decrease. We used a six state gating model combined with intrinsic rectification and divalent block to simulate ISK and investigated the relative contributions of the two types of rectification to the AP shape. This SK channel model replicates key features of ISK recording during AP-clamp. The simulation shows that the rise of Ca2+ during Ca2+ transients activates SK channel opening, but higher [Ca2+] blocks channels, resulting in transient outward-like trajectory of ISK. During the decay phase of Ca2+, Ca2+-dependent block is released causing ISK to contribute to repolarization. The intrinsic rectification characteristics limit ISK during the early and plateau phase of APs to prevent excessive APD shortening. In conclusion, ISK is an important repolarizing current and the rectification characteristics of SK channel determines its impact on early, plateau and repolarization phases of APs.

Sex-specific activation of SK current by isoproterenol facilitates action potential triangulation and arrhythmogenesis in rabbit ventricles.

It is unknown if a sex difference exists in cardiac apamin-sensitive small conductance Ca2+ -activated K+ (SK) current (IKAS ). There is no sex difference in IKAS in the basal condition. However, there is larger IKAS in female rabbit ventricles than in male during isoproterenol infusion. IKAS activation by isoproterenol leads to action potential triangulation in females, indicating its abundant activation at early phases of repolarization. IKAS activation in females induces negative Ca2+ -voltage coupling and promotes electromechanically discordant phase 2 repolarization alternans. IKAS is important in the mechanisms of ventricular fibrillation in females during sympathetic stimulation. Sex has a large influence on cardiac electrophysiological properties. Whether sex differences exist in apamin-sensitive small conductance Ca2+ -activated K+ (SK) current (IKAS ) remains unknown. We performed optical mapping, transmembrane potential, patch clamp, western blot and immunostaining in 62 normal rabbit ventricles, including 32 females and 30 males. IKAS blockade by apamin only minimally prolonged action potential (AP) duration (APD) in the basal condition for both sexes, but significantly prolonged APD in the presence of isoproterenol in females. Apamin prolonged APD at the level of 25% repolarization (APD25 ) more prominently than APD at the level of 80% repolarization (APD80 ), consequently reversing isoproterenol-induced AP triangulation in females. In comparison, apamin prolonged APD to a significantly lesser extent in males and failed to restore the AP plateau during isoproterenol infusion. IKAS in males did not respond to the L-type calcium current agonist BayK8644, but was amplified by the casein kinase 2 (CK2) inhibitor 4,5,6,7-tetrabromobenzotriazole. In addition, whole-cell outward IKAS densities in ventricular cardiomyocytes were significantly larger in females than in males. SK channel subtype 2 (SK2) protein expression was higher and the CK2/SK2 ratio was lower in females than in males. IKAS activation in females induced negative intracellular Ca2+ -voltage coupling, promoted electromechanically discordant phase 2 repolarization alternans and facilitated ventricular fibrillation (VF). Apamin eliminated the negative Ca2+ -voltage coupling, attenuated alternans and reduced VF inducibility, phase singularities and dominant frequencies in females, but not in males. We conclude that β-adrenergic stimulation activates ventricular IKAS in females to a much greater extent than in males. IKAS activation plays an important role in ventricular arrhythmogenesis in females during sympathetic stimulation.

Ondansetron blocks wild-type and p.F503L variant small-conductance Ca2+-activated K+ channels.

Apamin-sensitive small-conductance Ca2+-activated K+ (SK) current ( IKAS) is encoded by Ca2+-activated K+ channel subfamily N ( KCNN) genes. IKAS importantly contributes to cardiac repolarization in conditions associated with reduced repolarization reserve. To test the hypothesis that IKAS inhibition contributes to drug-induced long QT syndrome (diLQTS), we screened for KCNN variants among patients with diLQTS, determined the properties of heterologously expressed wild-type (WT) and variant KCNN channels, and determined if the 5-HT3 receptor antagonist ondansetron blocks IKAS. We searched 2,306,335 records in the Indiana Network for Patient Care and found 11 patients with diLQTS who had DNA available in the Indiana Biobank. DNA sequencing discovered a heterozygous KCNN2 variant (p.F503L) in a 52-yr-old woman presenting with corrected QT interval prolongation at baseline (473 ms) and further corrected QT interval lengthening (601 ms) after oral administration of ondansetron. That patient was also heterozygous for the p.S38G and p.P2835S variants of the QT-controlling genes KCNE1 and ankyrin 2, respectively. Patch-clamp experiments revealed that the p.F503L KCNN2 variant heterologously expressed in human embryonic kidney (HEK)-293 cells augmented Ca2+ sensitivity, increasing IKAS density. The fraction of total F503L-KCNN2 protein retained in the membrane was higher than that of WT KCNN2 protein. Ondansetron at nanomolar concentrations inhibited WT and p.F503L SK2 channels expressed in HEK-293 cells as well as native SK channels in ventricular cardiomyocytes. Ondansetron-induced IKAS inhibition was also demonstrated in Langendorff-perfused murine hearts. In conclusion, the heterozygous p.F503L KCNN2 variant increases Ca2+ sensitivity and IKAS density in transfected HEK-293 cells. Ondansetron at therapeutic (i.e., nanomolar) concentrations is a potent IKAS blocker. NEW & NOTEWORTHY We showed that ondansetron, a 5-HT3 receptor antagonist, blocks small-conductance Ca2+-activated K+ (SK) current. Ondansetron may be useful in controlling arrhythmias in which increased SK current is a likely contributor. However, its SK-blocking effects may also facilitate the development of drug-induced long QT syndrome.

Dependence of accumbens shell dialysate dopamine on the activity of apamin-sensitive slow-conducting Ca2+-activated K+ channels

Dependence of accumbens shell dialysate dopamine on the activity of apamin-sensitive slow-conducting Ca2+-activated K+ channels

Small conductance calcium-activated potassium current is important in transmural repolarization of failing human ventricles.

The transmural distribution of apamin-sensitive small conductance Ca(2+)-activated K(+) (SK) current (IKAS) in failing human ventricles remains unclear. We optically mapped left ventricular wedge preparations from 12 failing native hearts and 2 rejected cardiac allografts explanted during transplant surgery. We determined transmural action potential duration (APD) before and after 100 nmol/L apamin administration in all wedges and after sequential administration of apamin, chromanol, and E4031 in 4 wedges. Apamin prolonged APD from 363 ms (95% confidence interval [CI], 341-385) to 409 (95% CI, 385-434; P<0.001) in all hearts, and reduced the transmural conduction velocity from 36 cm/s (95% CI, 30-42) to 32 cm/s (95% CI, 27-37; P=0.001) in 12 native failing hearts at 1000 ms pacing cycle length (PCL). The percent APD prolongation is negatively correlated with baseline APD and positively correlated with PCL. Only 1 wedge had M-cell islands. The percentages of APD prolongation in the last 4 hearts at 2000 ms PCL after apamin, chromanol, and E4031 were 9.1% (95% CI, 3.9-14.2), 17.3% (95% CI, 3.1-31.5), and 35.9% (95% CI, 15.7-56.1), respectively. Immunohistochemical staining of subtype 2 of SK protein showed increased expression in intercalated discs of myocytes. SK current is important in the transmural repolarization in failing human ventricles. The magnitude of IKAS is positively correlated with the PCL, but negatively correlated with APD when PCL is fixed. There is abundant subtype 2 of SK protein in the intercalated discs of myocytes.

Differential regulation of synaptic transmission by pre- and postsynaptic SK channels in the spinal locomotor network

The generation of activity in the central nervous system requires precise tuning of cellular properties and synaptic transmission. Neural networks in the spinal cord produce coordinated locomotor movements. Synapses in these networks need to be equipped with multiple mechanisms that regulate their operation over varying regimes to produce locomotor activity at different frequencies. Using the in vitro lamprey spinal cord, we explored whether Ca(2+) influx via different routes in postsynaptic soma and dendrites and in presynaptic terminals can activate apamin-sensitive Ca(2+)-activated K(+) (SK) channels and thereby shape synaptic transmission. We show that postsynaptic SK channels are tightly coupled to Ca(2+) influx via NMDA receptors. Activation of these channels by synaptically induced NMDA-dependent Ca(2+) transients restrains the time course of the synaptic current and the amplitude of the synaptic potential. In addition, presynaptic SK channels are activated by Ca(2+) influx via voltage-gated channels and control the waveform of the action potential and the resulting Ca(2+) dynamics in the axon terminals. The coupling of SK channels to different Ca(2+) sources, pre- and postsynaptically, acts as a negative feedback mechanism to shape synaptic transmission. Thus SK channels can play a pivotal role in setting the dynamic range of synapses and enabling short-term plasticity in the spinal locomotor network.

SK2 and SK3 expression differentially affect firing frequency and precision in dopamine neurons

The firing properties of dopamine (DA) neurons in the substantia nigra (SN) pars compacta are strongly influenced by the activity of apamin-sensitive small conductance Ca2+-activated K+ (SK) channels. Of the three SK channel genes expressed in central neurons, only SK3 expression has been identified in DA neurons. The present findings show that SK2 was also expressed in DA neurons. Immuno-electron microscopy (iEM) showed that SK2 was primarily expressed in the distal dendrites, while SK3 was heavily expressed in the soma and, to a lesser extent, throughout the dendritic arbor. Electrophysiological recordings of the effects of the SK channel blocker apamin on DA neurons from wild type and SK−/− mice show that SK2-containing channels contributed to the precision of action potential (AP) timing, while SK3-containing channels influenced AP frequency. The expression of SK2 in DA neurons may endow distinct signaling and subcellular localization to SK2-containing channels.

A Functional Role for the 'Fibroblast-Like Cells' in Gastrointestinal Smooth Muscles (J Physiol 2011;589[Pt 3]:697-710)

The physiologic role of fibroblast-like cells (FLCs) has not been well established. Recently Kurahashi et al1 suggested the involvement of FLCs in neurotransmission by Ca2+-activated K+ (SK3) channels. They demonstrated that a network of more extensively branching cells with platelet-derived growth factor receptor alpha (PDGFRα) existed between the circular and longitudinal muscles in the plane of the myenteric plexus. FLCs (PDGFRα+) were located in the same anatomical spaces and distributed in similar density as interstitial cells of Cajal (ICC), but PDGFRα+ cells were distinct from c-Kit+ ICC, as shown by double labeling. Other double labeling experiments with anti-SK3 antibody revealed that PDGFRα+ cells in both muscle layers and the region of the myenteric plexus expressed SK3. Some other experiments showed that PDGFRα+ cells labeled with enhanced green fluorescent protein (eGFP) produced robust expression of P2ry1 (P2Y1 receptors) and Kcnn3 (SK3 channels). Whole-cell patch clamp results suggested that PDGFRα+ cells express a high density of apamin-sensitive Ca2+-activated K+ channels, which is consistent with the expression of SK3 channels: small amplitude and time-dependent outward currents without voltage-dependent inward currents in step depolarization, and significant reduction of this currents by apamin 300 nM and charybdotoxin (block both BK and IK conductance Ca2+-activated K+ channels) caused a small but significant reduction in outward currents. Single channel patch recording (excised inside-out patch) to change Ca2+ at the intracellular surface produced the results consistent with previous reported data of Ca2+-activated K+ channels. Pharmacological data using purines (ATP, β-NAD and ADP) evoked significant outward currents in PDGFRα+ cells. Apamin and MRS2500 (P2Y1 antagonist) attenuated current amplitudes. The authors concluded that PDGFRα+ cells are a novel class of excitable cells with large current densities attributable to SK channels that are the molecular and ionic apparatus to mediate enteric inhibitory responses to purines in gastrointestinal (GI) muscles.

Norepinephrine Causes a Biphasic Change in Mammalian Pinealocye Membrane Potential: Role of α1B-Adrenoreceptors, Phospholipase C, and Ca2+

Perforated patch clamp recording was used to study the control of membrane potential (V(m)) and spontaneous electrical activity in the rat pinealocyte by norepinephrine. Norepinephrine did not alter spiking frequency. However, it was found to act through α(1B)-adrenoreceptors in a concentration-dependent manner (0.1-10 μM) to produce a biphasic change in V(m). The initial response was a hyperpolarization (∼13 mV from a resting potential of -46 mV) due to a transient (∼5 sec) outward K(+) current (∼50 pA). This current appears to be triggered by Ca(2+) released from intracellular stores, based on the observation that it was also seen in cells bathed in Ca(2+)-deficient medium. In addition, pharmacological studies indicate that this current was dependent on phospholipase C (PLC) activation and was in part mediated by bicuculline methiodide and apamin-sensitive Ca(2+)-controlled K(+) channels. The initial transient hyperpolarization was followed by a sustained depolarization (∼4 mV) due to an inward current (∼10 pA). This response was dependent on PLC-dependent activation of Na(+)/Ca(2+) influx but did not involve nifedipine-sensitive voltage-gated Ca(2+) channels. Together, these results indicate for the first time that activation of α(1B)-adrenoreceptors initiates a PLC-dependent biphasic change in pinealocyte V(m) characterized by an initial transient hyperpolarization mediated by a mixture of Ca(2+)-activated K(+) channels followed by a sustained depolarization mediated by a Ca(2+)-conducting nonselective cation channel. These observations indicate that both continuous elevation of intracellular Ca(2+) and sustained depolarization at approximately -40 mV are associated with and are likely to be required for activation of the pinealocyte.

Small-Conductance Calcium-Activated Potassium Channel and Recurrent Ventricular Fibrillation in Failing Rabbit Ventricles

Fibrillation/defibrillation episodes in failing ventricles may be followed by action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (SVF). We hypothesized that activation of apamin-sensitive small-conductance Ca(2+)-activated K(+) (SK) channels is responsible for the postshock APD shortening in failing ventricles. A rabbit model of tachycardia-induced heart failure was used. Simultaneous optical mapping of intracellular Ca(2+) and membrane potential (V(m)) was performed in failing and nonfailing ventricles. Three failing ventricles developed SVF (SVF group); 9 did not (no-SVF group). None of the 10 nonfailing ventricles developed SVF. Increased pacing rate and duration augmented the magnitude of APD shortening. Apamin (1 μmol/L) eliminated recurrent SVF and increased postshock APD(80) in the SVF group from 126±5 to 153±4 ms (P<0.05) and from 147±2 to 162±3 ms (P<0.05) in the no-SVF group but did not change APD(80) in nonfailing group. Whole cell patch-clamp studies at 36°C showed that the apamin-sensitive K(+) current (I(KAS)) density was significantly larger in the failing than in the normal ventricular epicardial myocytes, and epicardial I(KAS) density was significantly higher than midmyocardial and endocardial myocytes. Steady-state Ca(2+) response of I(KAS) was leftward-shifted in the failing cells compared with the normal control cells, indicating increased Ca(2+) sensitivity of I(KAS) in failing ventricles. The K(d) was 232±5 nmol/L for failing myocytes and 553±78 nmol/L for normal myocytes (P=0.002). Heart failure heterogeneously increases the sensitivity of I(KAS) to intracellular Ca(2+), leading to upregulation of I(KAS), postshock APD shortening, and recurrent SVF.

Contribution of Kir2 potassium channels to ATP-induced cell death in brain capillary endothelial cells and reconstructed HEK293 cell model

Cellular turnover of brain capillary endothelial cells (BCECs) by the balance of cell proliferation and death is essential for maintaining the homeostasis of the blood-brain barrier. Stimulation of metabotropic ATP receptors (P2Y) transiently increased intracellular Ca²(+) concentration ([Ca²(+)](i)) in t-BBEC 117, a cell line derived from bovine BCECs. The [Ca²(+)](i) rise induced membrane hyperpolarization via the activation of apamin-sensitive small-conductance Ca²(+)-activated K(+) channels (SK2) and enhanced cell proliferation in t-BBEC 117. Here, we found anomalous membrane hyperpolarization lasting for over 10 min in response to ATP in ∼15% of t-BBEC 117, in which inward rectifier K(+) channel (K(ir)2.1) was extensively expressed. Once anomalous hyperpolarization was triggered by ATP, it was removed by Ba²(+) but not by apamin. Prolonged exposure to ATPγS increased the relative population of t-BBEC 117, in which the expression of K(ir)2.1 mRNAs was significantly higher and Ba²(+)-sensitive anomalous hyperpolarization was observed. The cultivation of t-BBEC 117 in serum-free medium also increased this population and reduced the cell number. The reduction of cell number was enhanced by the addition of ATPγS and the enhancement was antagonized by Ba²(+). In the human embryonic kidney 293 cell model, where SK2 and K(ir)2.1 were heterologously coexpressed, [Ca²(+)](i) rise by P2Y stimulation triggered anomalous hyperpolarization and cell death. In conclusion, P2Y stimulation in BCECs enhances cell proliferation by SK2 activation in the majority of cells but also triggers cell death in a certain population showing a substantial expression of K(ir)2.1. This dual action of P2Y stimulation may effectively facilitate BCEC turnover.

Relationship between afterhyperpolarization profiles and the regularity of spontaneous firings in rat medial vestibular nucleus neurons

Our previous in vivo and in vitro whole-cell patch-clamp recording studies demonstrated that neurons in the medial vestibular nucleus (MVN) could be characterized on the basis of three electrophysiological properties: afterhyperpolarization (AHP) profile; firing pattern; and response pattern to hyperpolarizing current pulses. In the present study, to clarify which types of the classified MVN neurons correspond to neurons with regular or irregular firing, we investigated their spike discharge patterns using whole-cell patch-clamp recording in both in vivo and in vitro preparations. The discharge regularity was related to AHP profiles, and we found that: (i) the coefficient of variation (CV) of interspike intervals during spike discharges was smaller in neurons exhibiting AHP with a slow component [AHP(s+)] than in those without a slow component [AHP(s-)], or with a slow AHP component preceded by afterdepolarization (ADP) [AHP(s+) with ADP]; (ii) the blockade of Ca(2+)-dependent K(+) channels by 100 nm apamin abolished the slow component and increased the CV in neurons exhibiting AHP(s+); and (iii) the modulation of firing (firing gain) in response to ramp current was larger in neurons exhibiting AHP(s-) than in the other two neuronal types. These results suggest that neurons exhibiting AHP(s+) are regularly discharging neurons with small firing gains to stimulus, neurons exhibiting AHP(s+) with ADP are irregularly discharging neurons with small firing gains, and neurons exhibiting AHP(s-) are irregularly discharging neurons with large firing gains. The regular firing of neurons exhibiting AHP(s+) is attributed to the activation of apamin-sensitive Ca(2+)-dependent K(+) channels.

Bis-Quinolinium Cyclophanes: Highly Potent and Selective Non-Peptidic Blockers of the Apamin-Sensitive Ca2+-Activated K+ Channel

Small conductance Ca(2+)-activated K(+) (SK(Ca)) channels comprise an important subclass of K(+) channels. Selective blockade of SK(Ca) channels may find application in the therapy of myotonic muscular dystrophy, gastrointestinal dysmotilities, memory disorders, narcolepsy, and alcohol abuse. In the cyclophanes described herein the two 4-aminoquinolinium groups are joined at the ring N atoms (linker L) and at the exocyclic N atoms (linker A). When both the spacer A and L have only one benzene ring, the blocking potency changes dramatically with simple structural variations in the linkers. One of these smaller cyclophanes having A = benzene-1,4-diylbis(methylene) and L = benzene-1,3-diylbis(methylene) shows activity in the low nanomolar range. Furthermore, the results with the present series add significantly to the structure-activity knowledge in the field, since they incorporate the first example of molecules in which the activity depends critically on the nature of the linkers joining the two quinolinium (Q) groups. Later on, a novel series of bis-quinolinium bis-alkylene cyclophanes was described. The biological results of the present series add support to the suggestion that the linkers of the two Q groups do not form direct interactions with the channel protein but comprise a molecular support for the two Q groups. Two important structural features of the pharmacophore for SK(Ca) channel blockade have been identified. These are (1) an optimum distance of ca. 5.8 A between the centroids of the pyridinium rings of the two quinolinium groups, and (2) a preference for conformations having the Q groups in a synperiplanar orientation.

Contextual memory deficits observed in mice overexpressing small conductance Ca2+-activated K+ type 2 (KCa2.2, SK2) channels are caused by an encoding deficit

Hippocampal-dependent synaptic plasticity and memory are modulated by apamin-sensitive small conductance Ca2+-activated K+ (SK) channels. Transgenic mice overexpressing SK2 channels (SK2+/T mice) exhibit marked deficits in hippocampal memory and synaptic plasticity, as previously reported. Here, we examined whether SK2 overexpression affects the encoding or retention of contextual memory. Compared with wild-type littermates, SK2+/T mice exhibited significantly less context-dependent freezing 10 min and 24 h after conditioning. Interestingly, this contextual memory impairment was eliminated if SK2+/T mice were permitted longer pre-exposure to the conditioning chamber. These data support converging evidence that SK2 channels restrict the encoding of hippocampal memory.

Nitrergic and purinergic interplay in inhibitory transmission in rat gastric fundus

1 This study was undertaken to analyse the involvement of ATP in non-adrenergic non- cholinergic (NANC) relaxation and possible interplay between nitrergic and purinergic systems in rat gastric fundus. 2 Experiments were performed in vitro on strips of longitudinal muscle from rat gastric fundus, recording the mechanical activity as changes in isometric force. In addition, NO release induced by different experimental conditions was assayed. 3 Under NANC conditions in serotonin-precontracted strips, electrical field stimulation (EFS) elicited a tetrodotoxin (TTX)-sensitive relaxation accompanied by nitric oxide (NO) release. This effect was antagonized by pretreatment with the NO synthase antagonist Nomega-nitro-L-arginine (L-NA) or by desensitization of purinergic receptors. Purinergic desensitization was also able to further antagonize the residual EFS-induced relaxation remaining after L-NA treatment. Exogenously applied NO [delivered as sodium nitroprusside (SNP)] or ATP (and related purines) induced concentration-dependent, TTX-insensitive relaxant responses. ATP also induced the release of NO. A reduction in the responses to ATP was observed in the presence of L-NA. In contrast, SNP-induced relaxation remained unchanged after desensitization of purinergic receptors. Finally, apamin, a blocker of the small conductance Ca2+ -dependent K+ channels, reduced the amplitude of the muscular relaxation evoked by either EFS, ATP or SNP. 4 In conclusion, this study provides evidence that in rat gastric fundus, ATP is one of the inhibitory transmitters released from NANC intramural neurones acting directly on the muscle, through receptors coupled to apamin-sensitive Ca2+ -dependent K+ channels and, indirectly, through the stimulation of NO production.

Synthesis and pharmacological testing of polyaminoquinolines as blockers of the apamin-sensitive Ca 2+-activated K + channel (SK Ca)

The synthesis and pharmacological testing of a series of non-peptidic blockers of the SK Ca (SK-3) channel is described. Target compounds were designed to mimic the spatial relationships of selected key residues in the energy-minimised structure of the octadecapeptide apamin, which are a highly potent blocker of this channel. Structures consist of a central unit, either a fumaric acid or an aromatic ring, to which are attached two alkylguanidine or two to four alkylaminoquinoline substituents. Potency was tested by the ability to inhibit the SK Ca channel-mediated after-hyperpolarization (AHP) in cultured rat sympathetic neurones. It was found that bis-aminoquinoline derivatives are significantly more potent as channel blockers than are the corresponding guanidines. This adds to the earlier evidence that delocalisation of positive charge through the more extensive aminoquinolinium ring system is important for effective channel binding. It was also found that an increase in activity can be gained by the addition of a third aminoquinoline residue to give non-quaternized amines which have submicromolar potencies (IC 50 = 0.13–0.36 μM). Extension to four aminoquinoline residues increased the potency to IC 50 = 93 nM.

Synthesis and Radioligand Binding Studies of Methoxylated 1,2,3,4-Tetrahydroisoquinolinium Derivatives as Ligands of the Apamin-Sensitive Ca2+-Activated K+Channels

Several methoxylated 1,2,3,4-tetrahydroisoquinoliniums derived from N-methyl-laudanosine and N-methyl-noscapine were synthesized and evaluated for their affinity for apamin-sensitive binding sites. The quaternary ammonium derivatives have a higher affinity with regard to the tertiary amines. 6,7-Dimethoxy analogues possess a higher affinity than the 6,8- and 7,8-dimethoxy isomers. A 3,4-dimethoxybenzyl or a 2-naphthylmethyl moiety in C-1 position are more favorable than a 3,4-dimethoxyphenethyl group. Smaller groups such as propyl or isobutyl are unfavorable. In 6,7-dimethoxy analogues, increasing the size and lipophilicity with a naphthyl group in the C-1 position leads to a slight increase of affinity, while the same group in the 6,7,8-trimethoxy series is less favorable. The 6,7,8-trimethoxy derivative 3f is the first tertiary amine in the series to possess an affinity close to that of N-methyl-laudanosine and N-methyl-noscapine. Moreover, electrophysiological studies show that the most effective compound 4f blocks the apamin-sensitive afterhyperpolarization in rat dopaminergic neurons.

Ionic mechanisms of autorhythmic firing in rat cerebellar Golgi cells

Although Golgi cells (GoCs), the main type of inhibitory interneuron in the cerebellar granular layer (GL), are thought to play a central role in cerebellar network function, their excitable properties have remained unexplored. GoCs fire rhythmically in vivo and in slices, but it was unclear whether this activity originated from pacemaker ionic mechanisms. We explored this issue in acute cerebellar slices from 3-week-old rats by combining loose cell-attached (LCA) and whole-cell (WC) recordings. GoCs displayed spontaneous firing at 1-10 Hz (room temperature) and 2-20 Hz (35-37 degrees C), which persisted in the presence of blockers of fast synaptic receptors and mGluR and GABAB receptors, thus behaving, in our conditions, as pacemaker neurons. ZD 7288 (20 microM), a potent hyperpolarization-activated current (Ih) blocker, slowed down pacemaker frequency. The role of subthreshold Na+ currents (INa,sub) could not be tested directly, but we observed a robust TTX-sensitive, non-inactivating Na+ current in the subthreshold voltage range. When studying repolarizing currents, we found that retigabine (5 microM), an activator of KCNQ K+ channels generating neuronal M-type K+ (IM) currents, reduced GoC excitability in the threshold region. The KCNQ channel antagonist XE991 (5 microM) did not modify firing, suggesting that GoC IM has low XE991 sensitivity. Spike repolarization was followed by an after-hyperpolarization (AHP) supported by apamin-sensitive Ca2+-dependent K+ currents (I(apa)). Block of I(apa) decreased pacemaker precision without altering average frequency. We propose that feed-forward depolarization is sustained by Ih and INa,sub, and that delayed repolarizing feedback involves an IM-like current whose properties remain to be characterized. The multiple ionic mechanisms shown here to contribute to GoC pacemaking should provide the substrate for fine regulation of firing frequency and precision, thus influencing the cyclic inhibition exerted by GoCs onto the cerebellar GL.