Apamin-sensitive Currents Research Articles

Preferential formation of human heteromeric SK2:SK3 channels limits homomeric SK channel assembly and function

Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels in vivo. Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)] produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.

The Inhibition of the Small-Conductance Ca2+-Activated Potassium Channels Decreases the Sinus Node Pacemaking during Beta-Adrenergic Activation.

Sinus pacemaking is based on tight cooperation of intracellular Ca2+ handling and surface membrane ion channels. An important player of this synergistic crosstalk could be the small-conductance Ca2+-activated K+-channel (ISK) that could contribute to the sinoatrial node (SAN) pacemaking driven by the intracellular Ca2+ changes under normal conditions and beta-adrenergic activation, however, the exact role is not fully clarified. SK2 channel expression was verified by immunoblot technique in rabbit SAN cells. Ionic currents and action potentials were measured by patch-clamp technique. The ECG R-R intervals were obtained by Langendorff-perfusion method on a rabbit heart. Apamin, a selective inhibitor of SK channels, was used during the experiments. Patch-clamp experiments revealed an apamin-sensitive current. When 100 nM apamin was applied, we found no change in the action potential nor in the ECG R-R interval. In experiments where isoproterenol was employed, apamin increased the cycle length of the SAN action potentials and enhanced the ECG R-R interval. Apamin did not amplify the cycle length variability or ECG R-R interval variability. Our data indicate that ISK has no role under normal condition, however, it moderately contributes to the SAN automaticity under beta-adrenergic activation.

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.

Abstract 17319: Distinct Effects of Human Calmodulinopathy on the Function of Small Conductance Ca 2+ -Activated K + (SK) Channels

Background: Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca 2+ -dependent ion channels. Mutations in CALM1, CALM2, and CALM3 have recently been linked to cardiac arrhythmias, such as Long QT Syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and familial idiopathic ventricular fibrillation (IVF). Small-conductance Ca 2+ - activated K + channels (SK) are voltage-independent channels that are regulated solely from beat-to-beat changes in intracellular calcium. CaM regulates the function of multiple ion channels, including SK channels, although the effect of CaM mutations on these channels is not yet understood. We hypothesize that human CaM mutations linked to sudden cardiac death disrupt SK channel function by distinct mechanisms. Methods and Results: We tested the effects of LQTS (CaM D96V , CaM D130G ), CPVT (CaM N54I , CaM N98S ), and IVF (CaM F90L ) CaM mutants compared to CaM WT on SK channel function. Using whole-cell voltage-clamp recordings, we found that CaM D96V and CaM D130G mutants significantly inhibited apamin-sensitive currents. Similarly, action potential studies in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) also revealed significant knockdown of apamin-sensitive currents. Immunofluorescent confocal microscopy confirmed that this effect was not due to changes in SK channel trafficking. Rather, co-immunoprecipitation studies showed a significant decrease in the association of these CaM mutants with the SK channel. Rosetta molecular modeling was used to identify a conformational change in CaM F90L structure compared to that of CaM WT . Conclusions: We found that CaM D96V and CaM D130G mutants significantly reduced apamin-sensitive currents, through a dominant negative effect on SK channel function. Consistent with our hypothesis, CaM F90L resulted in the least inhibitory effects. The data suggests that specific mutations with phenylalanine to leucine (CaM F90L ) may disrupt the interaction between apo-CaM with CaMBD on the SK2 channel.

Apamin-Sensitive Small Conductance Calcium-Activated Potassium Channels were Negatively Regulated by Captopril in Volume-Overload Heart Failure Rats.

In heart failure (HF), the malignant arrhythmias occur frequently; a study demonstrated that upregulation of I KAS resulted in recurrent spontaneous ventricular fibrillation in HF. However, the regulation of SK channels was poorly understood. The activation of SK channels depended on [Ca(2+)]i and PP2A; studies suggested that angiotensin II can regulate them. So, we hypothesized that in HF, the excess of angiotensin may regulate the SK channels and result in the remodeling of SK channels. To test the hypothesis, we used volume-overload-induced HF rat model, treated with captopril, performed whole-cell patch clamp to record apamin-sensitive currents (I KAS), and I-V curve was studied. The sensitivity of I KAS to [Ca(2+)]i was also explored by setting various [Ca(2+)]i (10, 100, 500, 900, 1000, and 10,000nM), and the steady-state Ca(2+) response of I KAS was attained and performed Hill fitting with the equation (y=1/[1+(EC50/x) (n) ]). Immunofluorescent staining, real-time PCR, Western blot were also carried out to furtherly investigate the underlying molecular mechanisms of the regulation. Captopril significantly decreased the mean density of I KAS when [Ca(2+)]i was 500, 900, 1000, and 10000nM. The Hill fitting showed significantly different EC50 values and the Hill coefficients and showed captopril significantly shifted rightward the steady-state Ca(2+) response of I KAS. The results of real-time PCR and Western blot demonstrated captopril decreased the mRNA and protein expression of SK3 channels. Captopril significantly downregulated the sensitivity of SK channels to [Ca(2+)]i and the SK3 channels expression in HF, and reversed the SK channels remodeling.

Abstract 16089: Functional Coupling of SK Channels and L-type Calcium Channels in Cardiomyocytes

Background. Small conductance Ca 2+ -activated K + (SK) channels play significant roles in regulating the excitability of cardiomyocytes (CMs). SK channels are unique in that they are gated solely by intracellular Ca 2+ and hence, function to integrate intracellular Ca 2+ and membrane potentials on a beat-to-beat basis in the heart. Our previous studies revealed that cardiac SK2 channels coupled with L-type Ca 2+ channels (LTCCs) through a physical bridge, α-actinin2, suggesting that LTCCs may be functionally coupled with SK2 channels by providing local Ca 2+ domain to activate the SK channels. However, a recent study suggested that sarcoplasmic reticulum (SR) Ca 2+ release is necessary and sufficient for the activation of cardiac SK channels. The objective of the study is to examine the mechanisms of SK channel activation in native CMs. Methods and Results. By using a voltage-clamp protocol in rabbit CMs to activate LTCCs followed immediately by a test voltage to monitor the SK currents, we recorded apamin-sensitive SK currents in response to the Ca 2+ influx. By altering the duration of activation of LTCCs, the activation of SK currents as a function of the increasing Ca 2+ influx was quantified. EGTA or BAPTA were then used to test the intracellular Ca 2+ domain necessary for SK channel activation. BAPTA but not EGTA completely disengaged the coupling between LTCCs and SK channels. SK currents coupled with Ca 2+ influx via LTCCs continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca 2+ store, suggesting that LTCCs provide the immediate Ca 2+ microdomain for the activation of SK channels in CMs. Super-resolution imaging of SK2, Ca v 1.2, and ryanodine receptor 2 (RyR2) using stimulated emission depletion (STED) microscopy was performed to directly quantify the distance among SK, LTCC and RyR2 in CMs. Conclusion. Ca 2+ influx through LTCCs provides the immediate and efficient Ca 2+ microdomain for the activation of SK channels. Furthermore, changes in SK channel activation kinetics following SR Ca 2+ depletion suggest both LTCC-mediated Ca 2+ influx and SR Ca 2+ release are necessary for the activation of cardiac SK channels. Funded by NIH (R01 HL085727 and R01 HL085844 to NC), VA (I01 BX000576 to NC) and AHA (14BGIA18870087 to XZ).

ASSA13-03-46 The Effects of Verapamil On SK2 Channel In Myocardium Cells From Human Chronic Atrial Fibrillation

<h3>Objective</h3> Small conductance calcium-activated potassium channel (SK channel) plays an important role in heart. Four subtype of SK channel are classified according to their sensitive to apamin. Electrophysiological properties of SK2 channel are voltage-independent as well as solely activated by changes in intracellular calcium concentration. This study investigated the alteration of SK2 channel currents in cAF patients. Moreover, L-type calcium channel inhibitor verapamil were adopted to observe the change of SK2 channel. We plan to explore the functional relationship between L-type calcium channel and SK2 channels. <h3>Methods</h3> Single human cardiac myocyte was isolated by two-step enzyme digestion. The whole-cell patch clamp technique was used to record the SK2 current in isolated cardiomyocytes from patients with siuns rhythm (SR) or chronic atrial fibrillation (cAF). Verapamil (10 μmol/L) and apamin (100 nmol/L) were added into bath solution respectively to observe the change of current. <h3>Results</h3> The current density (pA/pF) of SK2 channel as well as the ration in the integrated inward currents were up-regulated significantly in cAF groups. At wide range of test potentials, L-type calcium channel inhibitor verapamil reduced SK2 channel currents in cardiomyocytes from patients with SR or cAF. Compared with control group, the apamin-sensitive currents density of the verapamil group showed a significant decrease in human atrial myocytes. It indicates that part of SK2 current can be inhibited by verapamil. <h3>Conclusions</h3> It suggested that the up-regulation of SK2 currents underlies the occurrence and maintenance of cAF. There may be functionally coupling between SK2 channels and L-type calcium channels in human myocardium cells. The regulation of L-type calcium channel involves the remodelling of SK2 channels in the atrial myocytes from SR and cAF patients. This work was supported by the National Natural Science Foundation of China (No. 30870903) and the Natural Science Foundation of Luzhou.

Evidence for Functional SK Channels in Isolated Atrial Myocytes

Atrial fibrillation (AF) is the most common sustained arrhythmia and contributes to cardiac morbidity and mortality. Extension of the atrial effective refractory period through the block of cardiac potassium channels is a therapeutic strategy for the prevention of re-entrant arrhythmias and AF. However, many conventional potassium channel blockers carry an increased risk of potentially lethal ventricular arrhythmias such as torsades de pointes. Atrial selective compounds may offer attractive therapeutic alternatives. Small-conductance calcium-activated potassium (SK) channels have recently been suggested as a promising atrial selective target for the treatment of AF. Although there is growing evidence for SK channel expression in the mammalian heart, there is little consensus concerning SK function and it is possible that species differences may contribute to the controversy. We have investigated the expression and function of SK channels in atrial myocytes from mouse and rabbit. Immunocytochemistry using confocal microscopy showed the presence of SK2 protein in mouse atrial myocytes, with clear localization of staining along the z-lines. Whole-cell voltage clamp recordings from mouse and rabbit atrial myocytes, using a pipette solution with limited Ca2+ buffering capacity, revealed the presence of apamin-sensitive currents positive to −30 mV. In mouse cells, the apamin-sensitive difference currents slowly decayed during the sustained depolarization. In contrast, a rapidly decaying apamin-sensitive current that inactivated within 50 ms was observed in a proportion of rabbit atrial myocytes . These data suggest that both mouse and rabbit atrial myocytes exhibit functional SK channels, with the presence of SK2 protein confirmed in mouse cells. The apparent kinetic differences of apamin-sensitive current between species may underlie different physiological roles.

Ca2+-Activated K+ Channels in Gonadotropin-Releasing Hormone-Stimulated Mouse Gonadotrophs

GnRH receptor activation elicits release of intracellular Ca(2+), which leads to secretion and also activates Ca(2+)-activated ion channels underlying membrane voltage changes. The predominant Ca(2+)-activated ion channels in rat and mouse gonadotrophs are Ca(2+)-activated K(+) channels. To establish the temporal relationship between GnRH-induced changes in intracellular [Ca(2+)] ([Ca(2+)](i)) and membrane current (I(m)), and to identify specific Ca(2+)-activated K(+) channels linking GnRH-induced increase in [Ca(2+)](i) to changes in plasma membrane electrical activity, we used single female mouse gonadotrophs in the perforated patch configuration of the patch-clamp technique, which preserves signaling pathways. Simultaneous measurement of [Ca(2+)](i) and I(m) in voltage-clamped gonadotrophs revealed that GnRH stimulates an increase in [Ca(2+)](i) that precedes outward I(m), and that activates two kinetically distinct currents identified, using specific toxin inhibitors, as small conductance Ca(2+)-activated K(+) (SK) current (I(SK)) and large (big) conductance voltage- and Ca(2+)-activated K(+) (BK) current (I(BK)). We show that the apamin-sensitive current has an IC(50) of 69 pM, consistent with the SK2 channel subtype and confirmed by immunocytochemistry. The magnitude of the SK current response to GnRH was attenuated by 17beta-estradiol (E(2)) pretreatment. Iberiotoxin, an inhibitor of BK channels, completely blocked the residual apamin-insensitive outward I(m), substantiating that I(BK) is a component of the GnRH-induced outward I(m). In contrast to its suppression of I(SK), E(2) pretreatment augmented peak I(BK). SK or BK channel inhibition modulated GnRH-stimulated LH secretion, implicating a role for these channels in gonadotroph function. In summary, in mouse gonadotrophs the GnRH-stimulated increase in [Ca(2+)](i) activates I(SK) and I(BK), which are differentially regulated by E(2) and which may be targets for E(2) positive feedback in LH secretion.

Potentiation of Potassium Currents by Beta-Adrenoceptor Agonists in Human Urinary Bladder Smooth Muscle Cells: A Possible Electrical Mechanism of Relaxation

We examined the effects of β-adrenoceptor agonists on the membrane currents of smooth muscle cells from the human urinary bladder using a whole-cell patch clamp to investigate the involvement of Ca²⁺-activated K⁺ (K_Ca) channels in relaxation by β-adrenergic agonists. With 0.05 mmol/l EGTA in the patch pipette, depolarizing pulses evoked outward rectifying currents. Isoproterenol (1 µmol/l) significantly increased the membrane currents by 75% at +80 mV with 0.05 mmol/l EGTA pipette solution. BRL 37344 (1 µmol/l) significantly increased the membrane currents by 44% at +80 mV. Iberiotoxin (100 nmol/l) significantly decreased the membrane currents by 60% at +80 mV. In the presence of iberiotoxin, the potentiation of the outward currents by isoproterenol was greatly suppressed and, in the presence of iberiotoxin and apamin (1 µmol/l), the potentiation by isoproterenol was totally abolished. On the other hand, with 5 mmol/l EGTA pipette solution, depolarizing pulses evoked smaller outward currents. Isoproterenol (1 µmol/l) did not change the membrane currents with 5 mmol/l EGTA pipette solution. The real-time PCR analysis revealed the expression of β₂-adrenoceptors in the cells. These results suggest that Ca²⁺-activated and iberiotoxin- and apamin-sensitive currents via both large-conductance and small-conductance K_Ca channels could be increased by stimulation of β₂-adrenoceptors.

Apical SK potassium channels and Ca2+‐dependent anion secretion in endometrial epithelial cells

Apical uridine triphosphate (UTP) stimulation was shown to increase short circuit current (I(sc)) in immortalized porcine endometrial gland epithelial monolayers. Pretreatment with the bee venom toxin apamin enhanced this response. Voltage-clamp experiments using amphotericin B-permeablized monolayers revealed that the apamin-sensitive current increased immediately after UTP stimulation and was K(+) dependent. The current-voltage relationship was slightly inwardly rectifying with a reversal potential of -52 +/- 2 mV, and the P(K)/P(Na) ratio was 14, indicating high selectivity for K(+). Concentration-response relationships for apamin and dequalinium had IC(50) values of 0.5 nm and 1.8 microm, respectively, consistent with data previously reported for SK3 channels in excitable cells and hepatocytes. Treatment of monolayers with 50 microm BAPTA-AM completely blocked the effects of UTP on K(+) channel activation, indicating that the apamin-sensitive current was also Ca(2+) dependent. Moreover, channel activation was blocked by calmidazolium (IC(50) = 5 microm), suggesting a role for calmodulin in Ca(2+)-dependent regulation of channel activity. RT-PCR experiments demonstrated expression of mRNA for the SK1 and SK3 channels, but not SK2 channels. Treatment of monolayers with 20 nm oestradiol-17beta produced a 2-fold increase in SK3 mRNA, a 2-fold decrease in SK1 mRNA, but no change in GAPDH mRNA expression. This result correlated with a 2.5-fold increase in apamin-sensitive K(+) channel activity in the apical membrane. We speculate that SK channels provide a mechanism for rapidly sensing changes in intracellular Ca(2+) near the apical membrane, evoking immediate hyperpolarization necessary for increasing the driving force for anion efflux following P2Y receptor activation.

Accumulation of cytoplasmic calcium, but not apamin‐sensitive afterhyperpolarization current, during high frequency firing in rat subthalamic nucleus cells

The autonomous firing pattern of neurons in the rat subthalamic nucleus (STN) is shaped by action potential afterhyperpolarization currents. One of these is an apamin-sensitive calcium-dependent potassium current (SK). The duration of SK current is usually considered to be limited by the clearance of calcium from the vicinity of the channel. When the cell is driven to fire faster, calcium is expected to accumulate, and this is expected to result in accumulation of calcium-dependent AHP current. We measured the time course of calcium transients in the soma and proximal dendrites of STN neurons during spontaneous firing and their accumulation during driven firing. We compared these to the time course and accumulation of AHP currents using whole-cell and perforated patch recordings. During spontaneous firing, a rise in free cytoplasmic calcium was seen after each action potential, and decayed with a time constant of about 200 ms in the soma, and 80 ms in the dendrites. At rates higher than 10 Hz, calcium transients accumulated as predicted. In addition, there was a slow calcium transient not predicted by summation of action potentials that became more pronounced at high firing frequency. Spike AHP currents were measured in voltage clamp as tail currents after 2 ms voltage pulses that triggered action currents. Apamin-sensitive AHP (SK) current was measured by subtraction of tail currents obtained before and after treatment with apamin. SK current peaked between 10 and 15 ms after an action potential, had a decay time constant of about 30 ms, and showed no accumulation. At frequencies between 5 and 200 spikes s(-1), the maximal SK current remained the same as that evoked by a single action potential. AHP current did not have time to decay between action potentials, so at frequencies above 50 spikes s(-1) the apamin-sensitive current was effectively constant. These results are inconsistent with the view that the decay of SK current is governed by calcium dynamics. They suggest that the calcium is present at the SK channel for a very short time after each action potential, and the current decays at a rate set by the deactivation kinetics of the SK channel. At high rates, repetitive firing was governed by a fast apamin-insensitive AHP current that did not accumulate, but rather showed depression with increases in activation frequency. A slowly accumulating AHP current, also insensitive to apamin, was extremely small at low rates but became significant with higher firing rates.

Ca2+‐dependent K+ currents and spike‐frequency adaptation in medial entorhinal cortex layer II stellate cells

The entorhinal cortex (EC), located in the medial temporal lobe (MTL) of the brain, plays an important functional role in the MTL memory circuit. Medial EC (MEC) Layer II stellate cells (SCs) serve as one of the most prominent target cell types within the EC for inputs arising from higher cortical areas, and these same cells provide most of the output from the EC to the hippocampal region. We used the whole-cell patch clamp technique in a rat in vitro slice preparation to test whether SCs express afterhyperpolarization (AHP) currents and if these currents can be modulated. Our results revealed that SCs contain medium (mI(K(Ca))) and slow (sI(AHP)) Ca(2+)-dependent K(+) currents. Furthermore, we determined that an apamin-sensitive current does not underlie the mAHP in SCs. Our studies also showed that a cAMP-dependent modulation process significantly reduces mI(K(Ca)), sI(AHP), and spike-frequency adaptation in MEC Layer II SCs. Modulation of the firing pattern of SCs resulting from this effect may play an important role in the encoding of information related to memory processes.

Early electrical remodeling in rabbit pulmonary vein results from trafficking of intracellular SK2 channels to membrane sites

Atrial fibrillation is often initiated by bursts of ectopic activity arising in the pulmonary veins. We have previously shown that a 3-h intermittent burst pacing protocol (BPP), mimicking ectopic pulmonary vein foci, shortens action potential duration (APD) locally at the pulmonary vein-atrial interface (PV) while having no effect elsewhere in rabbit atrium. This shortening is Ca(2+) dependent and is prevented by apamin, which blocks small conductance Ca(2+)-activated K(+) channels (SK(Ca)). The present study investigates the ionic and molecular mechanisms whereby two apamin-sensitive SK(Ca) channels, SK2 and SK3, might contribute to the regional APD changes. Microelectrode and patch clamp techniques were used to record APDs and apamin-sensitive currents in isolated rabbit left atria and cells dispersed from PV and Bachmann's bundle (BB) regions. SK2 and SK3 mRNA and protein levels were quantified, and immunofluorescence was used to observe channel protein distribution. There was a direct relationship between APD shortening and apamin-sensitive current in burst-paced but not sham-paced PV. Moreover, apamin-sensitive current density increased in PV but not BB after BPP. SK2 mRNA, protein, and current were increased in PV after BPP, while SK2 immunostaining shifted from a perinuclear pattern in sham atria to predominance at sites near or at the PV membrane. BPP-induced acceleration of repolarization in PV results from SK2 channel trafficking to the membrane, leading to increased apamin-sensitive outward current. This is the first indication of involvement of Ca(2+)-activated K(+) currents in atrial remodeling and provides a possible basis for evolution of an arrhythmogenic substrate.

Small-Conductance Ca2+-Activated K+Channel Type 2 (SK2) Modulates Hippocampal Learning, Memory, and Synaptic Plasticity

Apamin-sensitive, small-conductance, Ca2+-activated K+ channels (SK channels) modulate neuronal excitability in CA1 neurons. Blocking all SK channel subtypes with apamin facilitates the induction of hippocampal synaptic plasticity and enhances hippocampal learning. In CA1 dendrites, SK channels are activated by Ca2+ through NMDA receptors and restrict glutamate-mediated EPSPs. Studies of SK channel knock-out mice reveal that of the three apamin-sensitive SK channel subunits (SK1-SK3), only SK2 subunits are necessary for the apamin-sensitive currents in CA1 hippocampal neurons. To determine the specific influence of SK2 channels on hippocampal synaptic plasticity, learning, and memory, we used gene targeting through homologous recombination in embryonic stem cells to generate transgenic mice that overexpress SK2 subunits by 10-fold (SK2+/T). In these mice, the apamin-sensitive current in CA1 neurons was increased by approximately fourfold, relative to wild-type (WT) littermates. In addition, the amplitude of synaptically evoked EPSPs recorded from SK2+/T CA1 neurons increased twice as much in response to SK channel blockade relative to EPSPs recorded from WT CA1 neurons. Consistent with this, SK2 overexpression reduced long-term potentiation after high-frequency stimulation compared with WT littermates and severely impaired learning in both hippocampus- and amygdala-dependent tasks. We conclude that SK2 channels regulate hippocampal synaptic plasticity and play a critical role in modulating mechanisms of learning and memory.

SK Channel Regulation of Dendritic Excitability and Dendrodendritic Inhibition in the Olfactory Bulb

Small-conductance calcium-activated potassium channels (SK) regulate dendritic excitability in many neurons. In the olfactory bulb, regulation of backpropagating action potentials and dendrodendritic inhibition depend on the dendritic excitability of mitral cells. We report here that SK channel currents are present in mitral cells but are not detectable in granule cells in the olfactory bulb. In brain slices from PND 14-21 mice, long step depolarizations (100 ms) in the mitral cell soma evoked a cadmium- and apamin-sensitive outward SK current lasting several hundred milliseconds. Block of the SK current unmasked an inward N-methyl-D-aspartate (NMDA) autoreceptor current due to glutamate released from mitral cell dendrites. In low extracellular Mg(2+) (100 microM), brief step depolarizations (2 ms) evoked an apamin-sensitive current that was reduced by D,L-2-amino-5-phosphonopentanoic acid. In current- clamp, block of SK channels increased action potential firing in mitral cells as well as dendrodendritic inhibition. Our results indicate that SK channels can be activated either by calcium channels or NMDA channels in mitral cell dendrites, providing a mechanism for local control of dendritic excitability.

Endothelial potassium channels, endothelium-dependent hyperpolarization and the regulation of vascular tone in health and disease.

1. The elusive nature of endothelium-derived hyperpolarizing factor (EDHF) has hampered detailed study of the ionic mechanisms that underlie the EDHF hyperpolarization and relaxation. Most studies have relied on a pharmacological approach in which interpretations of results can be confounded by limited specificity of action of the drugs used. Nevertheless, small-, intermediate- and large-conductance Ca2+-activated K+ channels (SKCa, IKCa and BKCa, respectively) have been implicated, with inward rectifier K+ channels (KIR) and Na+/K+-ATPase also suggested by some studies. 2. Endothelium-dependent membrane currents recorded using single-electrode voltage-clamp from electrically short lengths of arterioles in which the smooth muscle and endothelial cells remained in their normal functional relationship have provided useful insights into the mechanisms mediating EDHF. Charybdotoxin (ChTx) or apamin reduced, whereas apamin plus ChTx abolished, the EDHF current. The ChTx- and apamin-sensitive currents both reversed near the expected K+ equilibrium potential, were weakly outwardly rectifying and displayed little, if any, time- or voltage-dependent gating, thus having the biophysical and pharmacological characteristics of IKCa and SKCa channels, respectively. 3. The IKCa and SKCa channels occur in abundance in endothelial cells and their activation results in EDHF-like hyperpolarization of these cells. There is little evidence for a significant number of these channels in healthy, contractile vascular smooth muscle cells. 4. In a number of blood vessels in which EDHF occurs, the endothelial and smooth muscle cells are coupled electrically via myoendothelial gap junctions. In contrast, in the adult rat femoral artery, in which the smooth muscle and endothelial layers are not coupled electrically, EDHF does not occur, even though acetylcholine evokes hyperpolarization in the endothelial cells. 5. In vivo studies indicate that EDHF contributes little to basal conductance of the vasculature, but it contributes appreciably to evoked increases in conductance. 6. Endothelium-derived hyperpolarizing factor responses are diminished in some diseases, including hypertension, pre-eclampsia and some models of diabetes. 7. The most economical explanation for EDHF in vitro and in vivo in small vessels is that it arises from the activation of IKCa and SKCa channels in endothelial cells. The resulting endothelial hyperpolarization spreads via myoendothelial gap junctions to result in the EDHF-attributed hyperpolarization and relaxation of the smooth muscle.

Role of endothelial cell hyperpolarization in EDHF-mediated responses in the guinea-pig carotid artery.

1. Experiments were performed to identify the potassium channels involved in the acetylcholine-induced endothelium-dependent hyperpolarization of the guinea-pig internal carotid artery. Smooth muscle and endothelial cell membrane potentials were recorded in isolated arteries with intracellular microelectrodes. Potassium currents were recorded in freshly-dissociated smooth muscle cells using patch clamp techniques. 2. In single myocytes, iberiotoxin (0.1 microM)-, charybdotoxin (0.1 microM)-, apamin (0.5 microM)- and 4-aminopyridine (5 mM)-sensitive potassium currents were identified indicating the presence of large- and small-conductance calcium-sensitive potassium channels (BK(Ca) and SK(Ca)) as well as voltage-dependent potassium channels (K(V)). Charybdotoxin and iberiotoxin inhibited the same population of BK(Ca) but a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected. 4-aminopyridine (0. 1 - 25 mM) induced a concentration-dependent inhibition of K(V) without affecting the iberiotoxin- or the apamin-sensitive currents. 3. In isolated arteries, both the endothelium-dependent hyperpolarization of smooth muscle and the hyperpolarization of endothelial cells induced by acetylcholine or by substance P were inhibited by 5 mM 4-aminopyridine. 4. These results indicate that in the vascular smooth muscle cells of the guinea-pig carotid artery, a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected, comforting the hypothesis that the combination of these two toxins should act on the endothelial cells. Furthermore, the inhibition by 4-aminopyridine of both smooth muscle and endothelial hyperpolarizations, suggests that in order to observe an endothelium-dependent hyperpolarization of the vascular smooth muscle cells, the activation of endothelial potassium channels is likely to be required.

Spontaneous apamin-sensitive hyperpolarizations in dopaminergic neurons of neonatal rats.

Spontaneous apamin-sensitive hyperpolarizations in dopaminergic neurons of neonatal rats. J. Neurophysiol. 80: 3361-3364, 1998. Intracellular recordings from substantia nigra slices revealed the existence of spontaneous hyperpolarizations (amplitude 2-8 mV, duration 100-400 ms) at -60 mV in most dopaminergic neurons of neonatal (9-15 days) but not adult rats. These events were blocked by apamin (300 nM) and bicuculline methochloride (100-300 microM), which blocks apamin-sensitive currents. They were unaffected by the selective gamma-aminobutyric acid-A (GABAA) antagonists SR95531 (100 microM) and picrotoxin (30-50 microM), the GABAB antagonist CGP35348 (300 microM), the D2 antagonist haloperidol (1 microM), and the metabotropic antagonist MCPG (1 mM). The hyperpolarizations were strongly attenuated or abolished when recording electrodes contained 200 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. They were resistant to tetrodotoxin in the majority of the cells. They had some voltage dependency and were in some cases transiently potentiated when cells were briefly depolarized by current injection. We conclude that dopaminergic neurons have developmentally regulated physiological properties. These spontaneous hyperpolarizations might affect the firing rate of these cells, which was found to be lower in neonates than in adults.