Increased BK Ca Channel Activity Research Articles

MicroRNA-mediated downregulation of K+ channels in pulmonary arterial hypertension.

Downregulated expression of K+ channels and decreased K+ currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K+ channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3'-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K+ channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K+ channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K+ currents and downregulated voltage-gated K+ channel 1.5 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel β1 subunit (BKCaβ1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaβ1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.

Ability of naringenin, a bioflavonoid, to activate M-type potassium current in motor neuron-like cells and to increase BKCa-channel activity in HEK293T cells transfected with α-hSlo subunit.

BackgroundNaringenin (NGEN) is a citrus bioflavonoid known to have beneficial health properties; however, the ionic mechanism of its actions remains largely unclear. In this study, we attempted to evaluate the possible effects of NGEN on K+ currents in NSC-34 neuronal cells and in HEK293T cells expressing α-hSlo.ResultsNGEN increased M-type K+ current (IK(M)) in a concentration-dependent manner with an EC50 value of 9.8 μM in NSC-34 cells. NGEN shifted the activation curve of IK(M) conductance to the more negative potentials. In cell-attached recordings, NGEN or flupirtine enhanced the activity of M-type K+ (KM) channels with no changes in single-channel amplitude. NGEN (10 μM) had minimal effect on erg-mediated K+ currents. Under cell-attached voltage-clamp recordings, NGEN decreased the frequency of spontaneous action currents and further application of linopirdine can reverse NGEN-induced inhibition of firing. In HEK293T cells expressing α-hSlo, this compound increased the amplitude of Ca2+-activated K+ current (IK(Ca)). Under inside-out recordings, NGEN applied to the intracellular side of the detached patch enhanced the activity of large-conductance Ca2+-activated K+ (BKCa) channels. Moreover, from the study of a modeled neuron, burst firing of simulated action potentials (APs) was reduced in the presence of the increased conductances of both KM and KCa channels. Fast-slow analysis of AP bursting from this model also revealed that as the conductances of both KM and BKCa channels were increased by two-fold, the voltage nullcline was shifted in an upward direction accompanied by the compression of burst trajectory.ConclusionsThe present results demonstrate that activation of both KM and BKCa channels caused by NGEN might combine to influence neuronal activity if similar channels were functionally co-expressed in central neurons in vivo.

Role of KCNMA1gene in breast cancer invasion and metastasis to brain

BackgroundThe prognosis for patients with breast tumor metastases to brain is extremely poor. Identification of prognostic molecular markers of the metastatic process is critical for designing therapeutic modalities for reducing the occurrence of metastasis. Although ubiquitously present in most human organs, large-conductance calcium- and voltage-activated potassium channel (BKCa) channels are significantly upregulated in breast cancer cells. In this study we investigated the role of KCNMA1 gene that encodes for the pore-forming α-subunit of BKCa channels in breast cancer metastasis and invasion.MethodsWe performed Global exon array to study the expression of KCNMA1 in metastatic breast cancer to brain, compared its expression in primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immunohistochemistry was performed to study the expression and localization of BKCa channel protein in primary and metastatic breast cancer tissues and breast cancer cell lines. We performed matrigel invasion, transendothelial migration and membrane potential assays in established lines of normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether BKCa channel inhibition attenuates breast tumor invasion and metastasis using KCNMA1 knockdown with siRNA and biochemical inhibition with Iberiotoxin (IBTX).ResultsThe Global exon array and RT-PCR showed higher KCNMA1 expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased BKCa channel activity, leading to greater invasiveness and transendothelial migration, both of which could be attenuated by blocking KCNMA1.ConclusionDetermining the relative abundance of BKCa channel expression in breast cancer metastatic to brain and the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy.

The Na +/Ca 2+ exchanger inhibitor KB-R7943 activates large-conductance Ca 2+-activated K + channels in endothelial and vascular smooth muscle cells

The effect of the selective inhibitor of Na +/Ca 2+ exchanger (NCX), KB-R7943, on large-conductance Ca 2+-activated K + (BK Ca) channels was examined in cultured human umbilical vein endothelial cells (HUVECs) and freshly isolated mouse aortic smooth muscle cells (MASMCs). In voltage-clamped cells, KB-R7943 reversibly activated BK Ca currents in HUVECs and MASMCs. The EC 50 of KB-R7943 for BK Ca current activation in HUVECs was determined to be 6.78 ± 0.7 μM. In inside-out and outside-out patches, KB-R7943 markedly increased BK Ca channel activity and slightly decreased single channel current amplitudes. In inside-out patches, KB-R7943 shifted the relationship between [Ca 2+] i and open probability ( P o) to the left; the [Ca 2+] i required to evoke half-maximal activation changed from 1220 ± 68 nM (in the absence of KB-R7943) to 620 ± 199 nM (in the presence of 10 μM KB-R7943). In addition, KB-R7943 shifted the relationship between membrane potential and P o to the left; the membrane potential to evoke half-maximal activation changed from 76.86 ± 1.09 mV (in the absence of KB-R7943) to 49.62 ± 2.55 mV (in the presence of 10 μM KB-R7943). In conclusion, KB-R7943 was found to act as a potent BK Ca channel activator, which increases the sensitivity of BK Ca channels to cytosolic free Ca 2+ and membrane potential, and thereby BK Ca channel activity. These results should be considered when KB-R7943 is used as NCX blocker.

Riluzole-induced block of voltage-gated Na + current and activation of BK Ca channels in cultured differentiated human skeletal muscle cells

Riluzole is known to be of therapeutic use in the management of amyotrophic lateral sclerosis. In this study, we investigated the effects of riluzole on ion currents in cultured differentiated human skeletal muscle cells (dHSkMCs). Western blotting revealed the protein expression of α-subunits for both large-conductance Ca 2+-activated K + (BK Ca) channel and Na + channel (Na v1.5) in these cells. Riluzole could reduce the frequency of spontaneous beating in dHSkMCs. In whole-cell configuration, riluzole suppressed voltage-gated Na + current ( I Na) in a concentration-dependent manner with an IC 50 value of 2.3 μM. Riluzole (10 μM) also effectively increased Ca 2+-activated K + current ( I K(Ca)) which could be reversed by iberiotoxin (200 nM) and paxilline (1 μM), but not by apamin (200 nM). In inside-out patches, when applied to the inside of the cell membrane, riluzole (10 μM) increased BK Ca-channel activity with a decrease in mean closed time. Simulation studies also unraveled that both decreased conductance of I Na and increased conductance of I K(Ca) utilized to mimic riluzole actions in skeletal muscle cells could combine to decrease the amplitude of action potentials and increase the repolarization of action potentials. Taken together, inhibition of I Na and stimulation of BK Ca-channel activity caused by this drug are partly, if not entirely, responsible for its muscle relaxant actions in clinical setting.

Evidence for direct stimulation by estrogen receptor agonists (diarylpropionitrile and propylpyrazole‐triol) of the large‐conductance Ca 2+ ‐activated K + channel in human cardiac fibroblasts

The agonists selective for estrogen receptor (ER)- α propylpyrazole-triol, PPT) and ER-β diarylpropionitrile, DPN) can bind to the ER-α and ER-β receptors which may be localized to the plasma membrane. It is unclear whether DPN or PPT influence ion channels linked to non-genomic mechanism. We tested the hypothesis that DPN or PPT directly stimulates the large-conductance Ca2+-activated K+ (BKCa) channels in human cardiac fibroblasts (HCFs). In whole-cell configuration, depolarizing pulses evoked K+ outward currents in an outward rectification in HCFs, the amplitude of which was increased by DPN or PPT. Applied to the intracellular surface of excised patches, PPT or DPN enhanced BKCa-channel activity with no change in single-channel conductance. DPN and PPT increased BKCa-channel activity with an EC50 value of 2.3 and 2.6 μM, respectively. The mean closed time of these channels measured during the exposure to DPN or PPT was reduced with no change in the gating charge of the channels. Intracellular Ca2+ was not altered in the presence of these two compounds. RT-PCR study also revealed that no change in the transcriptional level of the BKCa channel α-subunit was observed in treatment with DPN or PPT. Our data suggest that PPT- and DPN-mediated activation of BKCa channels reveal novel pharmacological properties attributable to the activity of these channels.

The activation by estrogen receptor agonists of the BKCa-channel in human cardiac fibroblasts

The agonists selective for estrogen receptor (ER)-α (4,4′,4′′-(4-propyl-[1H]-pyrazole-1,3,5-triyl) tris-phenol, PPT) and ER-β (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN) are known to stimulate ER-α and ER-β receptors, respectively. It remains unknown whether these two agents regulate the activity of ion channels via a direct stimulation. In this study, we tested the hypothesis that DPN or PPT stimulates the large-conductance Ca2+-activated K+ (BKCa) channels in cultured human cardiac fibroblasts (HCFs). In whole-cell configuration, depolarizing pulses evoked K+ outward currents in an outward rectification in HCFs, the amplitude of which was increased in the presence of DPN or PPT. In inside-out patches, the activity of BKCa-channel with a conductance of 167±8pS was observed in these cells. PPT or DPN applied to the intracellular face of the membrane enhanced the activity of BKCa channels with no change in single-channel conductance. DPN and PPT increased BKCa-channel activity with an EC50 value of 2.3 and 2.6μM, respectively. The mean closed time of these channels during the exposure to these compounds was reduced with no change in the gating charge of the channels. Intracellular Ca2+ was not altered by these two compounds. RT-PCR analysis revealed that no change in the transcriptional level of the BKCa-channel α-subunit was observed in chronic treatment with these two compounds. PPT- and DPN-stimulated increase in BKCa channels reveal novel pharmacological properties attributable to the activity of these channels, and their increase in BKCa channels activity in HCFs may contribute to cell function.

Glucocorticoids stimulate the activity of large-conductance Ca 2+-activated K + channels in pituitary GH 3 and AtT-20 cells via a non-genomic mechanism

The effects of glucocorticoids on ion currents were investigated in pituitary GH 3 and AtT-20 cells. In whole-cell configuration, dexamethasone, a synthetic glucocorticoid, reversibly increased the density of Ca 2+-activated K + current ( I K(Ca)) with an EC 50 value of 21 ± 5 μM. Dexamethasone-induced increase in I K(Ca) density was suppressed by paxilline (1 μM), yet not by glibenclamide (10 μM), pandinotoxin −Kα (1 μM) or mifepristone (10 μM). Paxilline is a blocker of large-conductance Ca 2+-activated K + (BK Ca) channels, while glibenclamide and pandinotoxin-Kα are blockers of ATP-sensitive and A-type K + channels, respectively. Mifepristone can block cytosolic glucocorticoid receptors. In inside-out configuration, the application of dexamethasone (30 μM) into the intracellular surface caused no change in single-channel conductance; however, it did increase BK Ca-channel activity. Its effect was associated with a negative shift of the activation curve. However, no Ca 2+-sensitiviy of these channels was altered by dexamethasone. Dexamethasone-stimulated channel activity involves an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, dexamethasone decreased the firing frequency of action potentials. In pituitary AtT-20 cells, dexamethasone (30 μM) also increased BK Ca-channel activity. Dexamethasone-mediated stimulation of I K(Ca) presented here that is likely pharmacological, seems to be not linked to a genomic mechanism. The non-genomic, channel-stimulating properties of dexamethasone may partly contribute to the underlying mechanisms by which glucocorticoids affect neuroendocrine function.

Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochromeP-450 epoxygenase

Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large-conductance calcium-activated potassium (BK(Ca)) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BK(Ca) channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14(S),15(R)-EET. Adenovirus containing the cytomegalovirus promoter-Escherichia coli beta-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa immunoreactive protein was detected only in CYP BM-3-transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3-transduced cells were incubated with [14C]arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3-transduced cells, methacholine (10(-5) M) increased 14,15-EET release twofold and BK(Ca) channel activity fourfold in cell-attached patches. Methacholine-induced increases in BK(Ca) channel activity were blocked by the CYP inhibitor 17-octadecynoic acid (10(-5) M). 14(S),15(R)-EET was more potent than 14(R),15(S)-EET in relaxing bovine coronary arteries and activating BK(Ca) channels. Thus CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14(S),15(R)-EET from endogenous arachidonic acid to activate BK(Ca) channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BK(Ca) channels in coronary SMCs.

Properties of BKCa Channels in Oral Keratinocytes

Keratinocytes are important for epithelial antimicrobial barrier function. The activity of ion channels can affect the proliferation of keratinocytes. Little is known about Ca2+-activated K+ currents in these cells. Ion currents in normal human oral keratinocytes were characterized with a patch-clamp technique. In whole-cell configuration, depolarizing pulses evoked K+ outward currents (I(K)) in oral keratinocytes. Iberiotoxin (200 nM) and paxilline (1 microM) suppressed I(K); however, neither apamin (200 nM) nor 5-hydroxydecanoate (30 microM) had any effects on it. Caffeic acid phenethyl ester, a compound of honeybee propolis, increased I(K) with an EC50 value of 12.8 +/- 1.2 microM. In inside-out patches, a BK(Ca) channel was observed in keratinocytes, but not in oral squamous carcinoma (OCE-M1) cells. Caffeic acid phenethyl ester or cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate applied to the intracellular surface of a detached patch increased BK(Ca)-channel activity. The results demonstrate that the properties of BK(Ca) channels in normal human oral keratinocytes are similar to those described in other types of cells. Caffeic acid derivatives can also stimulate BK(Ca)-channel activity directly.

Assembly of a Ca2+-dependent BK channel signaling complex by binding to beta2 adrenergic receptor.

Large-conductance voltage and Ca2+-activated potassium channels (BKCa) play a critical role in modulating contractile tone of smooth muscle, and neuronal processes. In most mammalian tissues, activation of beta-adrenergic receptors and protein kinase A (PKAc) increases BKCa channel activity, contributing to sympathetic nervous system/hormonal regulation of membrane excitability. Here we report the requirement of an association of the beta2-adrenergic receptor (beta2AR) with the pore forming alpha subunit of BKCa and an A-kinase-anchoring protein (AKAP79/150) for beta2 agonist regulation. beta2AR can simultaneously interact with both BKCa and L-type Ca2+ channels (Cav1.2) in vivo, which enables the assembly of a unique, highly localized signal transduction complex to mediate Ca2+- and phosphorylation-dependent modulation of BKCa current. Our findings reveal a novel function for G protein-coupled receptors as a scaffold to couple two families of ion channels into a physical and functional signaling complex to modulate beta-adrenergic regulation of membrane excitability.

Cilostazol, an inhibitor of type 3 phosphodiesterase, stimulates large-conductance, calcium-activated potassium channels in pituitary GH3 cells and pheochromocytoma PC12 cells.

The effects of cilostazol, a dual inhibitor of type 3 phosphodiesterase and adenosine uptake, on ion currents were investigated in pituitary GH(3) cells and pheochromocytoma PC12 cells. In whole-cell configuration, cilostazol (10 microm) reversibly increased the amplitude of Ca(2+)-activated K(+) current [I(K(Ca))]. Cilostazol-induced increase in I(K(Ca)) was suppressed by paxilline (1 microM) but not glibenclamide (10 microm), dequalinium dichloride (10 microM), or beta-bungarotoxin (200 nM). Pretreatment of adenosine deaminase (1 U/ml) or alpha,beta-methylene-ADP (100 microM) for 5 h did not alter the magnitude of cilostazol-stimulated I(K(Ca)). Cilostazol (30 microM) slightly suppressed voltage-dependent l-type Ca(2+) current. In inside-out configuration, bath application of cilostazol (10 microM) into intracellular surface caused no change in single-channel conductance; however, it did increase the activity of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. Cilostazol enhanced the channel activity in a concentration-dependent manner with an EC(50) value of 3.5 microM. Cilostazol (10 microM) shifted the activation curve of BK(Ca) channels to less positive membrane potentials. Changes in the kinetic behavior of BK(Ca) channels caused by cilostazol were related to an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, cilostazol decreased the firing frequency of action potentials. In pheochromocytoma PC12 cells, cilostazol (10 microM) also increased BK(Ca) channel activity. Cilostazol-mediated stimulation of I(K(Ca)) appeared to be not linked to its inhibition of adenosine uptake or phosphodiesterase. The channel-stimulating properties of cilostazol may, at least in part, contribute to the underlying mechanisms by which it affects neuroendocrine function.

ATP modulation of large conductance Ca 2+-activated K + channels via a functionally associated protein kinase A in CA1 pyramidal neurons from rat hippocampus

Using inside-out configuration of patch clamp techniques, ATP modulation of BK Ca channels was studied in hippocampal CA1 pyramidal neurons of adult rat. Intracellular ATP application markedly increased BK Ca channel activity, and this ATP-produced increase in BK Ca channel activity was characterized by a higher opening frequency with no changes in channel open times. In the presence of specific inhibitor against protein kinase A, H-89, ATP did not induce any increase in the channel activity. Furthermore, adding H-89 after addition of ATP reversed the modulation produced by ATP. In contrast, protein kinase C inhibitor chelerythrine exerted no apparent effects on ATP-induced channel activation. The present study suggests that BK Ca channels from hippocampal CA1 pyramidal neurons could be modulated by ATP via a functionally associated protein kinase A-like protein.

Natural Bile Acids and Synthetic Analogues Modulate Large Conductance Ca2+-activated K+ (BKCa) Channel Activity in Smooth Muscle Cells

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle.

Natural bile acids and synthetic analogues modulate large conductance Ca2+-activated K+ (BKCa) channel activity in smooth muscle cells.

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle.

Transient forebrain ischemia induces persistent hyperactivity of large conductance Ca2+-activated potassium channels via oxidation modulation in rat hippocampal CA1 pyramidal neurons.

The present study examined temporal changes in activity of large conductance, Ca2+-activated potassium (BKCa) channels in postischemic CA1 pyramidal neurons at 2, 6, 24 and 48 h after reperfusion. These changes in activity and possible cellular mechanisms were examined using the inside--out configuration of patch clamp. The unitary conductance of postischemic BKCa channels increased transiently to 119% of the control at 2 h after reperfusion, and recovered to the control level thereafter. A persistent increase in [Ca2+]i sensitivity of BKCa channels was observed in postischemic CA1 neurons with the maximal sensitivity to [Ca2+]i at 6 h after reperfusion while channel voltage- dependence showed no obvious changes. Kinetic analyses showed that the postischemic enhancement of BKCa channel activity was due to longer open times and shorter closed times as there was no significant changes in opening frequency after ischemia. Glutathione disulphide markedly increased BKCa channel activity in normal CA1 neurons, while reducing glutathione caused a decrease in BKCa channel activity by reducing the sensitivity of this channel to [Ca2+]i in postischemic CA1 neurons. Similar modulatory effects on postischemic BKCa channels were also observed with another redox couple, DTNB and DTT, suggesting an oxidation modulation of BKCa channel function after ischemia. The present results indicate that a persistent enhancement in activity of BKCa channels, probably via oxidation of channels, in postischemic CA1 pyramidal neurons may account for the decrease in neuronal excitability and increase in fAHP after ischemia. The ischemia-induced augmentation in BKCa channel activity may be also associated with the postischemic neuronal injury.

Involvement of K + channels in the relaxant effects of YC-1 in vascular smooth muscle

This study addresses the question whether K + channels are involved in the vasorelaxant effects of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl-indazole (YC-1 ). In rat aorta, guinea pig aorta, and guinea pig a. carotis, YC-1 inhibited contractions induced by phenylephrine (3 μM) more potently than those induced by K +(48 mM). In rat aorta, tetraethylammonium (10 mM), charybdotoxin (0.2 μM), and iberiotoxin (0.1 μM), but not glibenclamide (10 μM), attenuated the relaxant effects of YC-1. In guinea pig a. carotis, YC-1 (30 μM) induced a hyperpolarisation which was antagonised by 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ; 50 μM). In rat aorta, YC-1 (30 μM) increased the rate constant of 86Rb-efflux. The effect of YC-1 was potentiated by zaprinast (10 μM), but inhibited by ODQ (50 μM) or charybdotoxin (0.2 μM). In smooth muscle cells from rat aorta, YC-1 (10 μM) increased BK Ca channel activity. It is suggested that YC-1-induced vasorelaxation is partially mediated by the activation of K + channels.