SK Channel Blocker Research Articles

Inhibitory neuromuscular transmission in the mouse distal colon is mediated by SK and calcium activated chloride channels

Neurogenic colonic smooth muscle relaxation is caused by inhibitory junction potentials (IJPs). We attempted to identify neurotransmitter, receptor and ion channel mechanisms that mediate IJPs in the mouse distal colon.MethodsIntracellular recordings were obtained from circular smooth muscle cells in vitro. Electrical field stimulation was used to evoke IJPs. Drugs were applied in the organ bath solution.ResultsThe P2Y1 receptor antagonist MRS‐2179 (10 μM) reduced IJP amplitude (66 ± 4%) and area (24 ± 11%). The nitric oxide synthase (NOS) inhibitor L‐NNA (100 μM) reduced IJP area (33 ± 8%) but not amplitude. Co‐application of MRS‐2179 and L‐NNA blocked IJPs. The SK channel blocker, apamin (0.1 μM) reduced IJP amplitude (36 ± 5%) and area 41 ± 12%. The Ca2+‐activated Cl‐channel blocker, niflumic acid (50 μM) reduced IJP amplitude (52 ± 6%) and area (44 ± 6%). Co‐application of niflumic acid and apamin blocked IJPs. ATP (5 mM) produced a larger hyperpolarization (20 ± 2 mV) compared to β‐NAD (10 ± 3 mV). MRS‐2179 and apamin reduced the ATP response amplitude by 34 ± 8% and 31 ± 1% respectively. MRS‐2179 and apamin reduced the β‐NAD (5 mM) response by 71 ± 14% and 61 ± 5% respectively.ConclusionIJPs are mediated via P2Y1 and NOS mediated pathways, which involve SK and Ca2+‐activated Cl‐channels. Responses caused by β‐NAD more closely mimic the IJP compared to ATP. Supported by NIH (DK094932).

Lisinopril augments the combined effect of nitric oxide and potassium channels on endothelium‐dependent relaxation in spontaneously hypertensive rats

Endothelium‐dependent relaxation is reported to be impaired in hypertension. However, there is equivocal evidence on the exact nature of the underlying dysfunction and whether it could be affected by antihypertensive drugs specifically angiotensin‐converting enzyme inhibitors. This study was conducted to evaluate the contribution of NO and potassium channels to endothelial function after lisinopril treatment in spontaneously hypertensive rats. Male animals (10–12 weeks old) were treated with lisinopril (20mg/Kg/day) for 10 weeks. ACh (0.1 nM–10μM)‐induced relaxation of third order mesenteric arteries was measured on a wire myograph with isometric recording in the presence and absence of different blockers. In the presence of inhibitor of nitric oxide synthase (LNMMA 100uM), the maximum relaxation of arteries from untreated (UT) and treated (T) rats to ACh was reduced by 19±3.5% and 5.0±01.6% respectively, while in the presence of SK and IK channel blockers (apamin 100nM and TRAM‐34 10μM) it was reduced by 35±3.5% and 34%±6.25%; respectively (n=10–22). With combined blockade, ACh‐induced relaxation was inhibited by 29±6.2% UT and 65±3.2 % T. Our results suggest that chronic treatment with lisinopril, although it reduced the relative contribution of nitric oxide alone it enhanced the combined effect of nitric oxide and potassium channels towards more endothelium‐dependent relaxation.

ASSA13-06-2 Characterization of Small-Conductance Ca2+-Activated K+Channels in Patients with Chronic Atrial Fibrillation

<h3>Introduction</h3> In 2003, Small-Conductance of Ca²⁺-activated K⁺ channels (SK) are first reported to present in Cardiac myocytes, which is expressed more abundant in the atria compared with the ventricles. Genetic knockout of the SK channels results in the delay in cardiac repolarization and showed inducible atrial fibrillation (AF). The purpose of this study was to confirm whether SK channels are involved in electrical remodelling of human chronic atrial fibrillation (CAF). <h3>Methods</h3> The ionic currents were recorded using whole-cell Conventional patch clamp techniques (35 appendages from patients with SR and 18 appendages from patients with CAF). SK2 channel protein expressions were assayed by western blot technique (12 appendages from patients with SR and 24 preparations from patients with CAF). <h3>Results</h3> Our result indicate that SK channels present in patients atrial with SR Group and CAF Group, identified it with SK channel blocker apamin (100 nmol/L). SK channels are voltage-independent and inwardly rectifying. The SK channel current densities were significantly increased in CAF (n = 24,[Ca²⁺]_i = 500nmol/L, p &lt; 0.05) compare to SR (n = 49, [Ca²⁺]_i = 500nmol/L). The increased proportion of SK channel current densities in CAF (n = 8, [Ca²⁺]_i = 1000 nmol/L, p &lt; 0.05) were larger than in SR (n = 10, [Ca²⁺]_i = 1000 nmol/L). it manifested that SK is more sensitive to intracellular Ca²⁺ in CAF than in SR. Western bloting result demonstrated that SK2 protein level is significant decrease in CAF than in SR. <h3>Conclusion</h3> Our results showed SK channels present in patients atrial with SR and CAF; SK channel density in CAF were significantly increase than in SR. In patients with CAF, the SK channels are more sensitive to [Ca²⁺]_i. Compared with SR, the protein level of SK2 was down-regulation in Patients atrial with CAF.these results indicated the increase densities of SK channel in CAF maybe depend on the more sensitive to [Ca²⁺]_i and higher intracellular Calcium level, which lead to increase channels activity, not by the numbers of channels. It might be one of the treatment targets of patients with Atrial Fibrillation, involving in human atrial electric remodelling in CAF.

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) express SK channels with low Ca2+ affinity

Chemosensitive RTN neurons regulate breathing in response to changes in tissue CO2/H+. despite the importance of these cells, little is known regarding molecular determinants of their intrinsic excitability. We have recently determined that RTN chemoreceptors express Ca2+‐activated small conductance K+ (SK) channels, however, the role of these channels in chemoreceptor function is unknown. Here, we use electrophysiological and computational approaches to explore SK channel function in RTN neurons. In brain slices (300 μm thick) from rat pups (P7–12), cell‐attached recordings of membrane potential show that bath application of apamin (selective SK channel blocker) decreased amplitude of the after hyperpolarization (AHP) evoked by depolarizing current injection but did not affect baseline activity. Based on computer simulations, we predict that under baseline conditions and high Ca2+ pump activity, [Ca2+]i should be sufficient to activate SK channels with a typical Ca2+affinity of &lt; 1μM. Therefore, we predict that RTN neurons express SK channels with low Ca2+ affinity possibly due to high casein kinase‐2 (CK2) activity. Consistent with this possibility, we found that TBB (a selective CK2 inhibitor) eliminated spontaneous activity of RTN neurons as expected for activation of a K+ conductance. These results suggest that the contribution of SK channels to activity of RTN neurons is regulated by CK2 activity.

Sympathoexcitation Induced by SK Channel Blockade in PVN Requires Activation of NMDA Receptors

Small conductance calcium activated potassium channels (SK) are abundantly expressed in the paraventricular nucleus (PVN). SK channel blockade in the PVN augments sympathetic nerve activity (SNA), yet the mechanism remains unclear. We hypothesized that the increased SNA evoked by SK channel blockade in PVN requires local activation of glutamate receptors. In anesthetized rats, bilateral PVN microinjection of SK channel blocker Apamin (25 pmol, 50 nl) significantly (n=5; p&lt;0.01) increased splanchnic SNA (SSNA) (304±60%) and mean arterial pressure (MAP) (33±6mmHg). Pre‐treatment with PVN injection of KYN (7.2 nmol), a non‐selective glutamate receptor blocker, significantly attenuated (n=4) the elevated SSNA (137±31%; p&lt;0.05 vs vehicle control) response to Apamin. Similarly, pre‐treatment with AP5 (6.0 nmol), a NMDA receptor blocker, also attenuated (n=4) the SSNA (155±17%; p&lt;0.05 vs vehicle control) response to Apamin. Pre‐treatment with KYN (n=4) and AP5 (n=4) obviously attenuated MAP responses to Apamin (17±6mmHg, p=0.08 for KYN; 14±11mmHg, p=0.06 for AP5) but didn't reach statistical significance. Immunohistochemistry studies demonstrated SK channel expression in PVN neurons with projections to the RVLM. This data indicates that the sympathoexcitatory response elicited by SK channel blockade in PVN, at least partly, requires local activation of NMDA receptors. Support: AHA2640130 (QHC).

Effects of SK Channel Blockers on Atrial Myocytes Suggest SK Channel Heterogeneity

Atrial fibrillation (AF) is the most common sustained arrhythmia and contributes to cardiac morbidity and mortality. Extension of the atrial effective refractory period through block of potassium channels is a therapeutic strategy for AF, with an atrial-selective drug being desired to avoid potentially lethal ventricular arrhythmias. Small-conductance calcium-activated potassium (SK) channels have been recently suggested as a promising atrial selective target, but disagreement exists concerning expression and function of these channels in atrial myocytes. Visualization of antibody labelling using confocal microscopy showed the presence of SK2 protein in mouse atrial myocytes with localization of staining along the z-lines. Whole-cell recordings revealed outward currents positive to −20 mV that were sensitive to two SK inhibitors, apamin and UCL1684. Current was blocked by apamin with an IC50 of 118 pM, close to reported values for homomeric SK2 current in mammalian cell lines. Action potential duration (APD) was prolonged more by application of UCL1684 than apamin, at a firing frequency of 0.2 Hz. The effect of UCL1684 was greater with a firing frequency of 2 Hz, producing a decrease of the stability of APD and increasing beat-to-beat variability (BVR) in APD. These data suggest that functional SK channels are present in the mouse atrium. However, the effects of apamin were different under voltage- or current-clamp conditions, while the effects of UCL1684 were similar. This difference might arise from atrial myocytes expressing more than one population of SK channels, with one being apamin-insensitive and contributing to action potential repolarization. The effect of UCL1684 on APD is consistent with the recruitment of more SK channel activity at higher firing frequencies. Block of these SK channels increases BVR, a marker of drug induced repolarization-related proarrhythmias, raising the possibility that SK inhibition could be proarrhythmic.

Small-conductance Ca2+-activated K+ channels modulate action potential-induced Ca2+ transients in hippocampal neurons.

In hippocampal pyramidal neurons, voltage-gated Ca(2+) channels open in response to action potentials. This results in elevations in the intracellular concentration of Ca(2+) that are maximal in the proximal apical dendrites and decrease rapidly with distance from the soma. The control of these action potential-evoked Ca(2+) elevations is critical for the regulation of hippocampal neuronal activity. As part of Ca(2+) signaling microdomains, small-conductance Ca(2+)-activated K(+) (SK) channels have been shown to modulate the amplitude and duration of intracellular Ca(2+) signals by feedback regulation of synaptically activated Ca(2+) sources in small distal dendrites and dendritic spines, thus affecting synaptic plasticity in the hippocampus. In this study, we investigated the effect of the activation of SK channels on Ca(2+) transients specifically induced by action potentials in the proximal processes of hippocampal pyramidal neurons. Our results, obtained by using selective SK channel blockers and enhancers, show that SK channels act in a feedback loop, in which their activation by Ca(2+) entering mainly through L-type voltage-gated Ca(2+) channels leads to a reduction in the subsequent dendritic influx of Ca(2+). This underscores a new role of SK channels in the proximal apical dendrite of hippocampal pyramidal neurons.

SK channel-selective opening by SKA-31 induces hyperpolarization and decreases contractility in human urinary bladder smooth muscle

Overactive bladder (OAB) is often associated with increased involuntary detrusor smooth muscle (DSM) contractions during the bladder-filling phase. To develop novel therapies for OAB, it is critical to better understand the mechanisms that control DSM excitability and contractility. Recent studies showed that small-conductance Ca(2+)-activated K(+) (SK) channels, SK3 channels, in particular, regulate human DSM function. However, the concept that SK channel-selective pharmacological activation can decrease the excitability and contractility directly in human DSM needs further exploration. Here, we studied the effect of the novel and potent SK channel activator, SKA-31 (or naphtho [1,2-d]thiazol-2-ylamine), on human DSM excitability and contractility at the cellular and tissue level. We used isometric tension recordings on human DSM-isolated strips and the perforated patch-clamp technique on freshly isolated native human DSM cells. SKA-31 significantly decreased spontaneous phasic contractions of DSM-isolated strips. In the presence of the SK channel blocker, apamin, the inhibitory effects of SKA-31 on the DSM spontaneous phasic contractions were significantly reduced. SKA-31 decreased the carbachol- and KCl-induced contractions in human DSM strips. Electrical field stimulation-induced contractions were significantly attenuated in the presence of SKA-31 at all stimulation frequencies (0.5-50 Hz). SKA-31 hyperpolarized the resting membrane potential of human DSM cells. Apamin abolished the hyperpolarizing effect of SKA-31, indicating the involvement of SK channel activation. These results support the concept that pharmacological activation of SK channels with selective openers may represent an attractive new pharmacological approach for decreasing DSM excitability and contractility, thus controlling OAB.

Ventricular tachyarrhythmias in rats with acute myocardial infarction involves activation of small-conductance Ca2+-activated K+channels

In vitro experiments have shown that the upregulation of small-conductance Ca(2+)-activated K(+) (SK) channels in ventricular epicardial myocytes is responsible for spontaneous ventricular fibrillation (VF) in failing ventricles. However, the role of SK channels in regulating VF has not yet been described in in vivo acute myocardial infarction (AMI) animals. The present study determined the role of SK channels in regulating spontaneous sustained ventricular tachycardia (SVT) and VF, the inducibility of ventricular tachyarrhythmias, and the effect of inhibition of SK channels on spontaneous SVT/VF and electrical ventricular instability in AMI rats. AMI was induced by ligation of the left anterior descending coronary artery in anesthetized rats. Spontaneous SVT/VF was analyzed, and programmed electrical stimulation was performed to evaluate the inducibility of ventricular tachyarrhythmias, ventricular effective refractory period (VERP), and VF threshold (VFT). In AMI, the duration and episodes of spontaneous SVT/VF were increased, and the inducibility of ventricular tachyarrhythmias was elevated. Pretreatment in the AMI group with the SK channel blocker apamin or UCL-1684 significantly reduced SVT/VF and inducibility of ventricular tachyarrhythmias (P < 0.05). Various doses of apamin (7.5, 22.5, 37.5, and 75.0 μg/kg iv) inhibited SVT/VF and the inducibility of ventricular tachyarrhythmias in a dose-dependent manner. Notably, no effects were observed in sham-operated controls. Additionally, VERP was shortened in AMI animals. Pretreatment in AMI animals with the SK channel blocker significantly prolonged VERP (P < 0.05). No effects were observed in sham-operated controls. Furthermore, VFT was reduced in AMI animals, and block of SK channels increased VFT in AMI animals, but, again, this was without effect in sham-operated controls. Finally, the monophasic action potential duration at 90% repolarization (MAPD(90)) was examined in the myocardial infarcted (MI) and nonmyocardial infarcted areas (NMI) of the left ventricular epicardium. Electrophysiology recordings showed that MAPD(90) in the MI area was shortened in AMI animals, and pretreatment with SK channel blocker apamin or UCL-1684 significantly prolonged MAPD(90) (P < 0.05) in the MI area but was without effect in the NMI area or in sham-operated controls. We conclude that the activation of SK channels may underlie the mechanisms of spontaneous SVT/VF and susceptibility to ventricular tachyarrhythmias in AMI. Inhibition of SK channels normalized the shortening of MAPD(90) in the MI area, which may contribute to the inhibitory effect on spontaneous SVT/VF and inducibility of ventricular tachyarrhythmias in AMI.

Abstract 506: Long-term High Salt Intake Involves Reduced Small Conductance Ca2+-activated K+ (SK) Current in Pre-sympathetic Paraventricular Nucleus Neurons and Increased Sympathetic Nerve Activity (SNA)

It is well-documented that elevated sympathetic outflow is an important contributor to salt-sensitive hypertension. Accumulating evidence indicates that high salt (HS) intake is involved in the activation of the sympathetic nervous system. However, the neural mechanisms responsible for HS-induced sympathetic activation are not fully understood. Recently, we reported that hypertension resulting from angiotensin II and HS intake is characterized by reduced small conductance Ca 2+ -activated K + (SK) current and increased excitability of RVLM projecting paraventricular nucleus (PVN-RVLM) neurons. Here, we hypothesized that HS intake may involve both reduced SK current in pre-sympathetic PVN neurons and increased neuronal excitability to contribute to the sympathetic activation. First, we determined whether 5 weeks of HS (2% NaCl) intake alters the SK current in PVN-RVLM neurons and whether this change affects the intrinsic excitability. In whole cell voltage-clamp recordings, the amplitude of SK current in neurons from the HS group (13 ± 5 pA, n=6) was significantly (P &lt; 0.05) less than that from normal salt (NS, 0.4% NaCl) controls (63 ± 11 pA, n=6). In whole cell current-clamp recordings, graded current injections evoked graded increases in discharge frequency among neurons from both groups. Maximum discharge evoked by +200 pA current injection in the HS group was 43 ± 4 Hz (n=8), which was significantly greater (P &lt; 0.01) than that of NS controls (22 ± 1 Hz, n=9). The SK channel blocker apamin (100 nM) significantly increased (P &lt; 0.05) discharge in NS controls (47 ± 4 Hz, n=6), but had no effect in HS rats (45 ± 2 Hz, n=5). Next, the contribution of SK channel activity in regulating SNA was assessed by bilateral PVN microinjection of apamin in anesthetized rats with and without HS intake. Respectively, maximum increases in splanchnic SNA and renal SNA by apamin (12.5 pmol) were 319 ± 41% and 277 ± 48% in rats on a NS diet (n=6), contrasted by a much smaller increase of 116 ± 41% (P &lt; 0.01) and 81 ± 28% (P &lt; 0.01) in rats on HS (n=6). Our data indicate that reduced SK current in PVN-RVLM neurons contributes to the increased excitability in neurons from rats on a HS diet, which may be a contributing factor in the sympathetic activation that appears to accompany elevated salt intake.

SK2 Channel Modulation Contributes to Compartment-Specific Dendritic Plasticity in Cerebellar Purkinje Cells

Small-conductance Ca(2+)-activated K(+) channels (SK channels) modulate excitability and curtail excitatory postsynaptic potentials (EPSPs) in neuronal dendrites. Here, we demonstrate long-lasting plasticity of intrinsic excitability (IE) in dendrites that results from changes in the gain of this regulatory mechanism. Using dendritic patch-clamp recordings from rat cerebellar Purkinje cells, we find that somatic depolarization or parallel fiber (PF) burst stimulation induce long-term amplification of synaptic responses to climbing fiber (CF) or PF stimulation and enhance the amplitude of passively propagated sodium spikes. Dendritic plasticity is mimicked and occluded by the SK channel blocker apamin and is absent in Purkinje cells from SK2 null mice. Triple-patch recordings from two dendritic sites and the soma and confocal calcium imaging studies show that local stimulation limits dendritic plasticity to the activated compartment of the dendrite. This plasticity mechanism allows Purkinje cells to adjust the SK2-mediated control of dendritic excitability in an activity-dependent manner.

Role of small conductance calcium-activated potassium channels expressed in PVN in regulating sympathetic nerve activity and arterial blood pressure in rats

Small conductance Ca(2+)-activated K(+) (SK) channels regulate membrane properties of rostral ventrolateral medulla (RVLM) projecting hypothalamic paraventricular nucleus (PVN) neurons and inhibition of SK channels increases in vitro excitability. Here, we determined in vivo the role of PVN SK channels in regulating sympathetic nerve activity (SNA) and mean arterial pressure (MAP). In anesthetized rats, bilateral PVN microinjection of SK channel blocker with peptide apamin (0, 0.125, 1.25, 3.75, 12.5, and 25 pmol) increased splanchnic SNA (SSNA), renal SNA (RSNA), MAP, and heart rate (HR) in a dose-dependent manner. Maximum increases in SSNA, RSNA, MAP, and HR elicited by apamin (12.5 pmol, n = 7) were 330 ± 40% (P < 0.01), 271 ± 40% (P < 0.01), 29 ± 4 mmHg (P < 0.01), and 34 ± 9 beats/min (P < 0.01), respectively. PVN injection of the nonpeptide SK channel blocker UCL1684 (250 pmol, n = 7) significantly increased SSNA (P < 0.05), RSNA (P < 0.05), MAP (P < 0.05), and HR (P < 0.05). Neither apamin injected outside the PVN (12.5 pmol, n = 6) nor peripheral administration of the same dose of apamin (12.5 pmol, n = 5) evoked any significant changes in the recorded variables. PVN-injected SK channel enhancer 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO, 5 nmol, n = 4) or N-cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidin]amine (CyPPA, 5 nmol, n = 6) did not significantly alter the SSNA, RSNA, MAP, and HR. Western blot and RT-PCR analysis of punched PVN tissue showed abundant expression of SK1-3 channels. We conclude that SK channels expressed in the PVN play an important role in the regulation of sympathetic outflow and cardiovascular function.

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.

Inhibition of Small‐Conductance Ca2+‐activated K+ Channels Protects Against Ventricular Fibrillation (VF) In Rats With Acute Myocardial Infarction (AMI)

In vitro study has shown that up‐regulation of SK channels in the ventricular epicardial myocytes is responsible for the spontaneous VF in failing ventricle. The present study determined the role of SK channels in regulating VF in AMI rats. AMI was induced by coronary artery ligation and ventricular programmed stimulation was used to evaluate ventricular vulnerable period (VVP), VF threshold (VFT) and ventricular effective refractory period (VERP) in anesthetized rats. In AMI animals, VVP was prolonged and VERP was shortened (p&lt;0.05, n=6). Pre‐treatment in the AMI group with the SK channel blocker UCL1684 significantly shortened VVP and prolonged VERP (p&lt;0.05, n=6). No effects were observed in sham‐operated controls (n=9). Additionally, VT was reduced in the AMI group, and UCL1684 increased VFT in the AMI group (p&lt;0.05, n=6), but, again, was without effect in sham‐operated controls (n=9). Finally, monophasic action potential duration (MAPD90) was examined using an electrode placed on the left ventricular epicardium. The data showed that MAPD90 was shortened in AMI group (p&lt;0.05, n=7), and UCL1684 significantly prolonged the MAPD90 (p&lt;0.05, n=5), but was without effect in sham‐operated controls (n=9). We conclude that activation of SK channels may underlie the mechanisms of VF. Inhibition of SK channels reduced shorting of MAPD90, which may be responsible for the inhibitory effect on VF in AMI rats. Support: NNSF‐30971223 (JHZ); AHA 10SDG2640130 (QHC).

Calcium‐activated potassium channels expressed in retinal pigment epithelial cell

Retinal pigment epithelium (RPE) cell serves very important functions to support vision. The aim of this study was to identify which the expression of KCa channels and ATP‐induced Ca2+ transient lead to the activation of SK channels as well as those of BK channels in the ARPE‐19 cells. We used reverse transcription polymerase chain reaction and electrophysiological patch clamp recordings to investigate the expression and characteristics of 3 types of the Kca channel subunits, BK, IK and SK.The presence of Ca2+ activated K+ channels were identified by expression profile of transcripts encoding BK and SK channel subunits (i.e., BK α1, BK β3, BK β4, SK1, SK3 and SK4/IK). 1‐ethyl‐2‐benzimidazolinone (EBIO), a specific SK channel activator, increased the whole‐cell current amplitude to 440.9% of control (at 50 mV, n = 8, p &lt; 0.01), whereas apamin, a specific SK channel blockers, attenuated the outward currents by to 72.5% of control when BK and IK channels were blocked with iberiotoxin (IBTX) and clotrimazole (CLT) (at 50 mV, n = 6, p &lt; 0.05). The single channel conductance of SK channel was 17.5 ± 0.8 pS in the symmetrical high K+ solutions (145/145 mM, n = 6). ATP (100μM) increased the NPo of the channels in cell‐attached patch mode (from 0.11 ± 0.26 to 0.40 ± 0.10 at 0 mV, n = 6, p &lt; 0.05).We suppose that SK channels related closely to these purinergic signals may play a role of important functions in the physiology of RPE cells.

Combined Contribution of Endothelial Relaxing Autacoides in the Rat Femoral Artery Response to CPCA: An Adenosine A2Receptor Agonist

We examined the contribution of endothelial relaxing factors and potassium channels in actions of CPCA, potent adenosine A2 receptor agonist, on isolated intact male rat femoral artery (FA). CPCA produced concentration-dependent relaxation of FA, which was notably, but not completely, reduced after endothelial denudation. DPCPX, A1 receptor antagonist, had no significant effect, while SCH 58261 (A2A receptor antagonist) notably reduced CPCA-evoked effect. Pharmacological inhibition of nitric oxide synthase or cyclooxygenase comparably reduced CPCA-evoked action, still in a lesser degree than after denudation. In the presence of buffer with high K+ (100 mM), CPCA-produced relaxations were almost abolished. TEA (nonselective KCa blocker), glibenclamide (KATP blocker), Ba++ (KIR blocker), or ouabain (Na+/K+-ATPase inhibitor) did not change CPCA-induced relaxation. Concentration-response curve for CPCA was significantly shifted to the right after the incubation of apamin (SK channel blocker). CPCA produced concentration-dependent relaxation of FA that was partly dependent on endothelial cells. Endothelium-related portion of CPCA-elicited effect was mediated by combined action of endothelial NO, prostacyclin, and EDHF after activation of endothelial A2A receptors. Small conductance KCa channels were involved in this action.

Relaxant effect of a novel calcium‐activated potassium channel modulator on human myometrial spontaneous contractility in vitro

To investigate the effect of 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), a novel SK/IK channels positive modulator, on human myometrial activity. Organ bath studies were performed on myometrial preparations obtained from women undergoing elective caesarean section at term (N = 11) or hysterectomy (N = 11). NS4591 was added cumulatively in the concentration range of 0.3-30 μm. In separate experiments, the effects of pre-incubation of muscle preparation with the SK or IK channel blockers apamin (1 μm) and TRAM34 (10 μm) on the outcomes of NS4591 were evaluated. Simultaneous vehicle controls were performed for all experiments. The effects of drugs were studied on spontaneous contractions. NS4591 exerted an inhibitory effect on myometrial contractions in muscle strips from non-pregnant and pregnant women. The contractility in non-pregnant and pregnant myometrium was reduced to the following values respectively: amplitude 20.65 ± 7.38% (P < 0.001) and 42.85 ± 11.04% (P < 0.05) and area under the curve 11.72 ± 7.39% (P < 0.001) and 34.84 ± 10.50% (P < 0.001) and are reflective of 30 μm NS4591 compared to vehicle control. In non-pregnant tissue, apamin partially reduced the inhibitory effects of NS4591, but we observed relaxation mediated by NS4591 despite pre-incubation with TRAM34. In contrast, in pregnant tissue, neither apamin nor TRAM34 could reverse the relaxatory effects of NS4591. Our findings imply that SK/IK channels are present and functional in myometrium from pregnant and non-pregnant women. The SK/IK channel-positive modulator NS4591 exerts relaxation of human myometrium in vitro, and this may have implications for the clinical management of preterm labour.

Developmental profile of SK2 channel expression and function in CA1 neurons

We investigated the temporal and spatial expression of SK2 in the developing mouse hippocampus using molecular and biochemical techniques, quantitative immunogold electron microscopy, and electrophysiology. The mRNA encoding SK2 was expressed in the developing and adult hippocampus. Western blotting and immunohistochemistry showed that SK2 protein increased with age. This was accompanied by a shift in subcellular localization. Early in development (P5), SK2 was predominantly localized to the endoplasmic reticulum in the pyramidal cell layer. But by P30 SK2 was almost exclusively expressed in the dendrites and spines. The level of SK2 at the postsynaptic density (PSD) also increased during development. In the adult, SK2 expression on the spine plasma membrane showed a proximal-to-distal gradient. Consistent with this redistribution and gradient of SK2, the selective SK channel blocker apamin increased evoked excitatory postsynaptic potentials (EPSPs) only in CA1 pyramidal neurons from mice older than P15. However, the effect of apamin on EPSPs was not different between synapses in proximal or distal stratum radiatum or stratum lacunosum-moleculare in adult. These results show a developmental increase and gradient in SK2-containing channel surface expression that underlie their influence on neurotransmission, and that may contribute to increased memory acquisition during early development.

Synthesis and radioligand binding studies of bis-(8-isopropyl-isoquinolinium) derivatives as ligands for apamin-sensitive sites on cloned SK2 and SK3 channels

A structure-activity relationship study of N-methyl-laudanosine, a SK channel blocker, has indicated that the 6,7-dimethoxy group could be successfully replaced by a hydrophobic moiety such as an isopropyl substituent in position 8 of the isoquinoline ring. In the present study, bis-(8-isopropyl-isoquinolinium) derivatives (2a-e) were synthesized and tested for their affinity for cloned SK2 and SK3 channels in comparison with their 6,7-dimethoxy analogues (4a-f). Several ligands were investigated, both in flexible (propyl, butyl and pentyl) and rigid (m- or p-xylyl) series, the m-xylyl derivative (2d) having the best profile in terms of affinity and selectivity for SK3/SK2 channels. Molecular studies showed that the optimal conformation of compound 2d fits well with our SK pharmacophore model.

Investigation of neurogenic excitatory and inhibitory motor responses and their control by 5-HT 4 receptors in circular smooth muscle of pig descending colon

The aim of this study was to investigate whether the pig colon descendens might be a good model for the responses mediated via the different locations of human colonic 5-HT 4 receptors. The intrinsic excitatory and inhibitory motor neurotransmission in pig colon descendens was therefore first characterized. In circular smooth muscle strips, electrical field stimulation (EFS) at basal tone induced only in the combined presence of the NO synthase inhibitor N ω-nitro- L-arginine methyl ester hydrochloride (L-NAME) and the SK channel blocker apamin voltage-dependent on-contractions. These on-contractions were largely reduced by the neuronal conductance blocker tetrodotoxin (TTX) and by the muscarinic receptor antagonist atropine, illustrating activation of cholinergic neurons. The 5-HT 4 receptor agonist prucalopride facilitated submaximal EFS-evoked cholinergic contractions and this effect was prevented by the 5-HT 4 receptor antagonist GR113808, supporting the presence of facilitating 5-HT 4 receptors on the cholinergic nerve endings innervating circular muscle in pig colon descendens. Relaxations were induced by EFS in strips pre-contracted with substance P in the presence of atropine. The responses at lower stimulation voltages were abolished by TTX. L-NAME or apamin alone did not influence or only moderately reduced the relaxations, but L-NAME plus apamin abolished the relaxations at lower stimulation voltages, suggesting that NO and ATP act as inhibitory neurotransmitters in a redundant way. Prucalopride did not influence the EFS-induced relaxations at lower stimulation voltage, nor did it per se relax contracted circular muscle strips. No evidence for relaxing 5-HT 4 receptors, either on inhibitory neurons or on the muscle cells was thus obtained in pig colon descendens circular muscle.