BK Activation Research Articles

POI09 Trends in BK and JC polyomavirus and relationship to CD4+/CD8+ ratio in patients with relapsing remitting multiple sclerosis on natalizumab therapy

Natalizumab reduces clinical relapses and the risk of sustained progression of disability in patients with relapsing remitting multiple sclerosis (RR MS). It has been associated with the development of progressive...

The role of the large-conductance voltage-dependent and calcium-activated potassium (BKCa) channels in the regulation of rat ductus arteriosus tone

The role of large-conductance voltage-dependent and calcium-activated potassium (BK(Ca)) channels in the regulation of ductus arteriosus (DA) tone is not clear. This study aimed to examine whether BK(Ca) α and β subunits and BK(Ca) currents are present in the rat DA, as well as whether the BK(Ca) channels are involved in O₂-induced ductal constriction. BK(Ca) α and β subunit transcripts (mRNAs) were detected in the DA from premature (19D) and mature (21D) rat fetuses and full-term neonates (NB) by quantitative real-time PCR. The amount of BK(Ca) α mRNAs decreased with advancing development. β1 was the dominant β subunit in the DA, and the amount of β1 mRNAs was greatest in the mature DA. Immunofluorescence staining showed that the majority of BK(Ca) α and β1 proteins were colocated with alpha smooth muscle actin (α-SMA) in the tunica media of the DA in all age groups. The protein expression of the α subunit was greatest in the mature DA, while the expression of the β1 subunit did not differ among all three groups. The 19D and 21D ductus tensions were recorded under various conditions by myograph. The 19D ductus rings exhibited poor O₂ sensitivity and no response to BK(Ca) inhibitor (paxilline) or activator (NS1619). The 21D ductus rings developed significant constriction induced by O₂. Paxilline did not increase the 21D DA tension under either hypoxic or oxygenated conditions. NS1619 dilated the 21D DA only under oxygenated conditions. The recorded BK(Ca) currents were greatest in the 21D DA smooth muscle cells (SMCs) upon using a whole-cell patch clamp. Our study indicated that BK(Ca) channels exist in the DA but are not involved in O₂-induced ductal constriction. Activation of BK(Ca) channels led to vasodilatation in the preconstricted DA induced by O₂, possibly suggesting a way to maintain the patency of DA after birth.

Adaptation to Short-Term Stress Exposures Prevents Poststress Dysfunction of Calcium-Activated Potassium Channels in Coronary Vessels

Adaptation to short-term stress exposures prevents immobilization stress-induced decrease in functional activity of BK(Ca)channels in smooth muscle cells of coronary vessels developing against the background of NO overproduction and increase in the relative content of oxidized glutathione (change in redox state of the glutathione system). The protective antistress mechanisms of adaptation probably include prevention of NO overproduction by inducible NO synthase and maintenance of dependent glutathione redox state functional activity of BK(Ca)channels in smooth muscle cells of coronary vessels.

N‐arachidonoyl glycine, an endogenous lipid that acts as a vasorelaxant via nitric oxide and large conductance calcium‐activated potassium channels

N-arachidonoyl glycine (NAGly) is an endogenous lipid that is structurally similar to the endocannabinoid, N-arachidonoyl ethanolamide (anandamide). While NAGly does not activate cannabinoid receptors, it exerts cannabimimetic effects in pain regulation. Here, we have determined if NAGly, like anandamide, modulates vascular tone. In rat isolated small mesenteric arteries, the relaxant responses to NAGly were characterized. Effects of N-arachidonoyl serine and N-arachidonoyl gamma-aminobutyric acid were also examined. In endothelium-intact arteries, NAGly-induced relaxation (pEC(50%)= 5.7 +/- 0.2; relaxation at 30 microM = 98 +/- 1%) was attenuated by l-NAME (a nitric oxide synthase inhibitor) or iberiotoxin [selective blocker of large conductance Ca(2+)-activated K(+) channels (BK(Ca))], and abolished by high extracellular K(+) concentration. Endothelial removal reduced the potency of NAGly, and the resultant relaxation was inhibited by iberiotoxin, but not l-NAME. NAGly responses were sensitive to the novel cannabinoid receptor antagonist O-1918 independently of endothelial integrity, whereas pertussis toxin, which uncouples G(i/o) proteins, attenuated NAGly relaxation only in endothelium-intact arteries. Treatments with antagonists for CB(1), CB(2) and TRPV1 receptors, or inhibitors of fatty acid amide hydrolase and COX had no effect. The two other arachidonoyl amino acids also induced iberiotoxin- and L-NAME-sensitive relaxations. NAGly acts as a vasorelaxant predominantly via activation of BK(Ca) in rat small mesenteric arteries. We suggest that NAGly activates an unknown G(i/o)-coupled receptor, stimulating endothelial release of nitric oxide which in turn activates BK(Ca) in the smooth muscle. In addition, NAGly might also activate BK(Ca) through G(i/o)- and nitric oxide-independent mechanisms.

Exercise training attenuates ageing‐induced BKCa channel downregulation in rat coronary arteries

Physical inactivity and ageing are widely recognized as risk factors for development of coronary artery disease. One of the characteristic changes that occurs in aged coronary artery is downregulation of their large-conductance voltage- and calcium-activated K(+) (BK(Ca)) channels. In this study, we investigated the effects of moderate exercise training (ET) on the activity of BK(Ca) channels in coronary arteries of aged rats. Old Fischer 344 rats (23-26 months old) were randomly assigned to sedentary (O-SED, n = 24) or exercise-trained groups (O-ET, n = 28). The O-ET rats underwent a progressive treadmill exercise-training programme for 60 min day(1), 5 days week(1) for 12 weeks. Young animals were used for comparison. Coronary arteries were mounted on a wire myograph, and contractions in response to 1, 10, 30, 50 and 100 nmoll(1) iberiotoxin were compared. Iberiotoxin (100 nmol l(1)) contracted coronary arteries of young, O-SED and O-ET rats by 115 +/- 14, 36 +/- 5.6 and 61 +/- 5% of 5-hydroxytryptamine-induced contractions, respectively. Patch-clamp studies revealed a larger magnitude of BK(Ca) current in young (104 +/- 15.6 pA pF(1)) compared with O-ET (44 +/- 9 pA pF(1)) and least in O-SED coronary smooth muscle cells (8.6 +/- 2 pA pF(1)). Western immunoblotting was performed to study expression levels of BK(Ca) channel proteins. The alpha and beta1 subunits of the BK(Ca) channel were reduced by 40 +/- 3.5 and 30 +/- 2.6%, respectively, in coronary arteries of old compared with young rats, and ET attenuated this reduction in expression level to 28 +/- 2 and 12 +/- 4%, respectively. Our results showed that ageing was associated with a reduction in BK(Ca) channels, and ET partly reversed this reduction. We conclude that low-intensity ET may be beneficial in restoring age-related decline in coronary vasodilatory properties mediated by BK(Ca) channels.

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase alpha-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).

Activation of BKCa channel is associated with increased apoptosis of cerebrovascular smooth muscle cells in simulated microgravity rats

Cerebral arterial remodeling is one of the critical factors in the occurrence of postspaceflight orthostatic intolerance. We hypothesize that large-conductance calcium-activated K(+) (BK(Ca)) channels in vascular smooth muscle cells (VSMCs) may play an important role in regulating cerebrovascular adaptation during microgravity exposure. The aim of this work was to investigate whether activation of BK(Ca) channels is involved in regulation of apoptotic remodeling of cerebral arteries in simulated microgravity rats. In animal studies, Sprague-Dawley rats were subjected to 1-wk hindlimb unweighting to simulate microgravity. Alterations of BK(Ca) channels in cerebral VSMCs were investigated by patch clamp and Western blotting; apoptosis was assessed by electron microscopy and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL). To evaluate the correlation of BK(Ca) channel and apoptosis, channel protein and cell nucleus were double-stained. In cell studies, hSloalpha+beta1 channel was coexpressed into human embryonic kidney 293 (HEK293) cells to observe the effects of BK(Ca) channels on apoptosis. In rats, enhanced activities and expression of BK(Ca) channels were found to be correlated with increased apoptosis in cerebral VSMCs after simulated microgravity. In transfected HEK293 cells, activation of cloned BK(Ca) channel induced apoptosis, whereas inhibition of cloned BK(Ca) channel decreased apoptosis. In conclusion, activation of BK(Ca) channels is associated with increased apoptosis in cerebral VSMCs of simulated microgravity rats.

Mechanisms involved in the regulation of bovine pulmonary vascular tone by the 5‐HT1B receptor

5-HT(1B) receptors may have a role in pulmonary hypertension. Their relationship with the activity of BK(Ca,) a T-type voltage-operated calcium channel (VOCC) and cyclic nucleotide-mediated relaxation was examined. Ring segments of bovine pulmonary arteries were mounted in organ baths in modified Krebs-Henseleit buffer (37 degrees C) under a tension of 20 mN and gassed with 95% O(2)/5% CO(2). Isometric recordings were made using Chart 5 software. Contractile responses to 5-HT (10 nM-300 microM) were inhibited similarly by the 5-HT(1B) receptor antagonist SB216641 (100 nM) and the T-type VOCC blockers mibefradil (10 microM) and NNC550396 (10 microM) with no additive effect between SB216641 and mibefradil. Inhibition by SB216641 was prevented by the potassium channel blocker, charybdotoxin (100 nM). 5-HT(1B) receptor activation and charybdotoxin produced a mibefradil-sensitive potentiation of responses to U46619. Bradykinin (0.1 nM-30 microM), sodium nitroprusside (0.01 nM-3 microM), zaprinast (1 nM-3 microM), isoprenaline (0.1 nM-10 microM) and rolipram (1 nM-3 microM) produced 50% relaxation of arteries constricted with 5-HT (1-3 microM) or U46619 (30-50 nM) in the presence of 5-HT(1B) receptor activation, but full relaxation of arteries constricted with U46619, the 5-HT(2A) receptor agonist 2,5 dimethoxy-4 iodoamphetamine (1 microM) or 5-HT in the presence of 5-HT(1B) receptor antagonism. Enhanced relaxation of 5-HT-constricted arteries by cGMP-dependent pathways, seen in the presence of the 5-HT(1B) receptor antagonist, was reversed by charybdotoxin whereas cAMP-dependent relaxation was only partly reversed by charybdotoxin. 5-HT(1B) receptors couple to inhibition of BK(Ca), thus increasing tissue sensitivity to contractile agonists by activating a T-type VOCC and impairing cGMP-mediated relaxation. Impaired cAMP-mediated relaxation was only partly mediated by inhibition of BK(Ca).

Angiotensin II Effects on BK Channel in Mesenteric Arterial Smooth Muscle Cells of SS, BN, and Congenic Renin+ and Renin- Rats

Cardiovascular sensitivity to anesthetics has been linked to the renin-angiotensin system (RAS), and it is thought to be related to the differences in cardiovascular collapse observed between SS and BN rats. In a previous study we found that congenics carrying the BN renin allele (renin+) in the SS background had the same cardiovascular sensitivity and low BK channel activity as BN rats. On the other hand, congenics carrying the SS renin allele (renin-) displayed high BK activity and behaved like SS rats, suggesting that RSA could be involved in differential responses. To test this hypothesis, the inhibition of BK channel by angiotensin II (AngII, 100 nM) was evaluated in four strains. Activity of BK was monitored from isolated mesenteric arterial smooth muscle cells of SS and BN rats, and renin+ and renin- congenic rats in the cell-attached mode at +80 mV Em and in symmetrical 150 mM K+. Blockade by paxilline (1 μM) confirmed identity of BK channel. Similar to findings from our previous study, in the cell-attached mode the probability of BK channel opening (Po) was different between SS (high Po) and BN (lower Po). The BK activity of renin2- resembled that of SS, whereas BK activity of renin+ matched BN. AngII had a greater inhibitory effect on channel Po in BN (−55+7%) and renin+ (−94+2%) strains than in SS (−9+6%) and renin- (−7+2%) strains. Impaired renin expression and impaired RAS are associated with lower sensitivity of BK to inhibition by AngII in SS than in BN rats. The fact that renin+ and renin- strains follow a similar pattern appears to support this conclusion.

The D5Dopamine Receptor Mediates Large-Conductance, Calcium- and Voltage-Activated Potassium Channel Activation in Human Coronary Artery Smooth Muscle Cells

Large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channels hyperpolarize coronary artery smooth muscle cells, causing vasorelaxation. Dopamine activates BK(Ca) channels by stimulating D(1)-like receptor-mediated increases in cAMP in porcine coronary artery myocytes. There are two D(1)-like receptors (R), D(1)R and D(5)R. We hypothesize that the specific D(1)-like receptor involved in BK(Ca) channel activation in human coronary artery smooth muscle cells (HCASMCs) is the D(5)R and that activation occurs via cAMP cross-activation of cGMP-dependent protein kinase (PKG), rather than cAMP-dependent protein kinase (PKA). The effects of D(1)-like receptor agonists and antagonists on BK(Ca) channel opening in HCASMCs were examined in the presence and absence of PKG/PKA inhibition by cell-attached patch clamp. In the absence of commercially available ligands specific for D(1)R or D(5)R, D(1)R or D(5)R protein was down-regulated by transfecting HCASMCs with human D(1)R or D(5)R antisense oligonucleotides, respectively: cells transfected with scrambled oligonucleotides and nontransfected HCASMCs served as controls. The predominant ion channel conducting outward currents in nontransfected HCASMCs was identified as the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel, which was activated by D(1)-like receptor agonists despite PKA inhibition with (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid (KT 5720) (300 nM), but was abolished by inhibiting PKG with 9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b-11a-triazadibenzo(a,g) cycloocta(cde)-trinden-1-one (KT 5823) (300 nM). D(1)-like receptor agonists activated BK(Ca) channels in all transfected cells except those transfected with D(5)R antisense oligonucleotides. Thus, the dopamine (D(1)-like) receptor mediates activation of BK(Ca) channels in HCASMCs by D(5)R, not D(1)R, and via PKG, not PKA. This is the first report of differential D(1)-like receptor regulation of vascular smooth muscle function in human cells.

Role of Large-conductance Calcium-activated Potassium Channels of Coronary Arteries in Heart Preservation

Large-conductance calcium-activated potassium channels (BK(Ca)) of vascular smooth muscle cells (VSMCs) play an important role in the regulation of vascular tone. We tested the hypotheses that the alteration of arterial relaxation after exposure to ischemia or hyperkalemia is attributable to the alteration in the activities of BK(Ca) by using the patch-clamp technique and force measurement. VSMCs were enzymatically isolated from porcine coronary arteries, with the primary cultures used for patch-clamp study. BK(Ca) currents were recorded in a whole-cell configuration (n = 5 in each group). The BK(Ca) activator NS1619 (-7 to -4.5 logM)-induced relaxation was studied myographically in small porcine coronary arteries (n = 6 per group). The effects of global ischemia for 1 hour and hyperkalemia (20 to 120 mmol/liter) were tested. Ischemia for 1 hour markedly reduced the BK(Ca) current from 119.8 +/- 11.4 pA/pF to 86.0 +/- 3.7 pA/pF (p < 0.05). The rise of extracellular K(+) resulted in increased BK(Ca) currents in a concentration-dependent manner (5.4 mmol/liter: 31.6 +/- 3.4 pA/pF; 20 mmol/liter: 73.6 +/- 11.4 pA/pF; 60 mmol/liter: 108.6 +/- 20.6 pA/pF; 120 mmol/liter: 135.2 +/- 20.5 pA/pF; p < 0.05). NS1619-induced relaxation was suppressed by ischemia (71.4 +/- 2.2% vs 95.3 +/- 1.6%; p < 0.01) and was inferior in K(+) pre-contraction, as compared with U(46619) (a thromboxane A(2) mimetic) pre-contraction (p < 0.05). In coronary circulation: (1) the BK(Ca) current density and related vasorelaxation are reduced after ischemia; and (2) hyperkalemia induces dual effects; the depolarization obscures the compensatory increase of the BK(Ca) activity. These findings imply that BK(Ca) activators may be used in cardioplegia or heart preservation solutions to protect the function of this ion channel.

Nitric Oxide Suppresses Cerebral Vasomotion by sGC-Independent Effects on Ryanodine Receptors and Voltage-Gated Calcium Channels

Background/Aims: In cerebral arteries, nitric oxide (NO) release plays a key role in suppressing vasomotion. Our aim was to establish the pathways affected by NO in rat middle cerebral arteries. Methods: In isolated segments of artery, isometric tension and simultaneous measurements of either smooth muscle membrane potential or intracellular [Ca²⁺] ([Ca²⁺]_SMC) changes were recorded. Results: In the absence of L-NAME, asynchronous propagating Ca²⁺ waves were recorded that were sensitive to block with ryanodine, but not nifedipine. L-NAME stimulated pronounced vasomotion and synchronous Ca²⁺ oscillations with close temporal coupling between membrane potential, tone and [Ca²⁺]_SMC. If nifedipine was applied together with L-NAME, [Ca²⁺]_SMC decreased and synchronous Ca²⁺ oscillations were lost, but asynchronous propagating Ca²⁺ waves persisted. Vasomotion was similarly evoked by either iberiotoxin, or by ryanodine, and to a lesser extent by ODQ. Exogenous application of NONOate stimulated endothelium-independent hyperpolarization and relaxation of either L-NAME-induced or spontaneous arterial tone. NO-evoked hyperpolarization involved activation of BK_Ca channels via ryanodine receptors (RYRs), with little involvement of sGC. Further, in whole cell mode, NO inhibited current through L-type voltage-gated Ca²⁺ channels (VGCC), which was independent of both voltage and sGC. Conclusion: NO exerts sGC-independent actions at RYRs and at VGCC, both of which normally suppress cerebral artery myogenic tone.

Large Conductance Calcium-Activated Potassium Channels Contribute to the Reduced Myogenic Tone of Peripheral Microvasculature After Cardiopulmonary Bypass

We investigated the role of calcium-activated potassium (K(Ca)) channel activation in myogenic tone in human peripheral microvasculature after heart surgery. Human skeletal muscle arterioles (90-180microm diameter) were dissected from tissue harvested pre- and post-cardiopulmonary bypass (CPB) during cardiac surgery. Myogenic reactivity in response to stepwise increases in intraluminal pressure was studied between pressure steps. Microvessel tone was determined pre-CPB, post-CPB, and after blockade of K(Ca) channels. Expression and localization of large conductance (BK) K(Ca) channels in the coronary microvasculature was assessed by immunoblot and immunofluorescence photomicroscopy. Myogenic tone of skeletal muscle arterioles was significantly reduced post-CPB compared with pre-CPB. Decrease in myogenic tone after CPB was reflected by the increase in microvessel internal diameter. Myogenic tone of post-CPB microvessels was significantly increased after treatment with BK(Ca)-blocker iberiotoxin, but unchanged in the combined presence of the blockers of intermediate (IK(Ca)) and small conductance (SK(Ca)) K(Ca) channels, TRAM34/apamin. The increases in myogenic tone after iberiotoxin treatment were demonstrated as a decrease in microvessel internal diameter. No significant differences in BK(Ca) protein levels were noted comparing pre- and post-CPB conditions judged by immunoblot and by immunofluorescence staining of skeletal muscle microvessels. Prominent staining for BK(Ca)-alpha and BK(Ca)-beta(1) subunits localized to the microvascular smooth muscle. CPB-associated decrease in peripheral myogenic reactivity is likely due to activation of BK(Ca), but not IK(Ca) or SK(Ca). CPB may increase BK(Ca) activity without increasing BK polypeptide level.

Effects of docosahexaenoic acid on large-conductance Ca2+-activated K+ channels and voltage-dependent K+ channels in rat coronary artery smooth muscle cells

To investigate the effects of docosahexaenoic acid (DHA) on large-conductance Ca(2+)-activated K(+)(BK(Ca)) channels and voltage-dependent K(+) (K(V)) channels in rat coronary artery smooth muscle cells (CASMCs). Rat CASMCs were isolated by an enzyme digestion method. BK(Ca) and K(V) currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BK(Ca) and K(V) channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 micromol/L. When DHA concentrations were greater than 10 micromol/L, BK(Ca) currents increased in a dose-dependent manner. At a testing potential of +80 mV, 6.1%+/-0.3%, 76.5%+/-3.8%, 120.6%+/-5.5%, 248.0%+/-12.3%, 348.7%+/-17.3%, 374.2%+/-18.7%, 432.2%+/-21.6%, and 443.1%+/-22.1% of BK(Ca) currents were increased at the above concentrations, respectively. The half-effective concentration (EC(50)) of DHA on BK(Ca) currents was 37.53+/-1.65 micromol/L. When DHA concentrations were greater than 20 micromol/L, K(V) currents were gradually blocked by increasing concentrations of DHA. At a testing potential of +50 mV, 0.40%+/-0.02%, 1.37%+/-0.06%, 11.80%+/-0.59%, 26.50%+/-1.75%, 56.50%+/-2.89%, 73.30%+/-3.66%, 79.70%+/-3.94%, and 78.1%+/-3.91% of K(V) currents were blocked at the different concentrations listed above, respectively. The EC(50) of DHA on K(V) currents was 44.20+/-0.63 micromol/L. DHA can activate BK(Ca) channels and block K(V) channels in rat CASMCs, and the EC(50) of DHA for BK(Ca) channels is lower than that for K(V) channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BK(Ca) channels.

Canonical Transient Receptor Potential Channel (TRPC) 3 and TRPC6 Associate with Large-Conductance Ca2+-Activated K+(BKCa) Channels: Role in BKCaTrafficking to the Surface of Cultured Podocytes

Large-conductance (BK(Ca) type) Ca(2+)-activated K(+) channels encoded by the Slo1 gene and various canonical transient receptor potential channels (TRPCs) are coexpressed in many cell types, including podocytes (visceral epithelial cells) of the renal glomerulus. In this study, we show by coimmunoprecipitation and GST pull-down assays that BK(Ca) channels can associate with endogenous TRPC3 and TRPC6 channels in differentiated cells of a podocyte cell line. Both types of TRPC channels colocalize with Slo1 in podocytes and in human embryonic kidney (HEK) 293T cells transiently coexpressing the TRPC channels with Slo1. In HEK293T cells, coexpression of TRPC6 increased surface expression of a Slo1 subunit splice variant (Slo1(VEDEC)) that is typically retained in intracellular compartments, as assessed by cell-surface biotinylation assays and confocal microscopy. Corresponding currents through BK(Ca) channels were also increased with TRPC6 coexpression, as assessed by whole-cell and excised inside-out patch recordings. By contrast, coexpression of TRPC3 had no effect on the surface expression of BK(Ca) channels in HEK293T cells or on the amplitudes of currents in whole cells or excised patches. In podocytes, small interfering RNA knockdown of endogenous TRPC6 reduced steady-state surface expression of endogenous Slo1 channels, but knockdown of TRPC3 had no effect. TRPC6, but not TRPC3 knockdown also reduced voltage-evoked outward current through podocyte BK(Ca) channels. These data indicate that TRPC6 and TRPC3 channels can bind to Slo1, and this colocalization may allow them to serve as a source of Ca(2+) for the activation of BK(Ca) channels. TRPC6 channels also play a role in the regulation of surface expression of a subset of podocyte BK(Ca) channels.

Resveratrol and Large-Conductance Calcium-Activated Potassium Channels in the Protection of Human Retinal Pigment Epithelial Cells

This study was undertaken to examine the possible association of large-conductance calcium-activated potassium channels (BK(Ca) channels) and human retinal pigment epithelial (RPE) R-50 cell phagocytosis. The potential antioxidative effect of resveratrol in human RPE cells also was investigated. Cultured human RPE R-50cells were treated with hydrogen peroxide ( H(2)O(2), 10 microM, 20 min), meclofenamic acid (30 microM, 20 min), paxilline (100 nM, 20 min), or resveratrol (10 microM, 20 min), respectively. Meclofenamic acid (30 microM, 20 min) or resveratrol (10 microM, 20 min) was given after exposure to H(2)O(2) . Pretreatment with meclofenamic acid, resveratrol, or paxilline before H(2)O(2) exposure also was performed. Fluorescent latex beads then were fed for 4 h, and phagocytic function was assessed by flow cytometry. H(2)O(2) inhibited the phagocytic function of human RPE R-50 cells. The BK(Ca) channel inhibitor, paxilline, inhibited RPE phagocytosis, as did hyperoxide stress. The BK(Ca) channel opener, meclofenamic acid, prevented the damage caused by H(2)O(2) . Pretreatment with resveratrol also provided protection against damage caused by H(2)O(2) . However, further treatment with resveratrol or meclofenamic acid was not found to offer protection from H(2)O(2) exposure. In conclusion, the dietary antioxidant, resveratrol, significantly reduced oxidative damage on phagocytic function in human RPE R-50 cells. One of the underlying mechanisms might be linked to the activity of BK(Ca) channels in RPE cells.

Functional coupling of TRPV4 cationic channel and large conductance, calcium-dependent potassium channel in human bronchial epithelial cell lines

Calcium-dependent potassium channels are implicated in electrolyte transport, cell volume regulation and mechanical responses in epithelia, although the pathways for calcium entry and their coupling to the activation of potassium channels are not fully understood. We now show molecular evidence for the presence of TRPV4, a calcium permeable channel sensitive to osmotic and mechanical stress, and its functional coupling to the large conductance calcium-dependent potassium channel (BK(Ca)) in a human bronchial epithelial cell line (HBE). Reverse transcriptase polymerase chain reaction, intracellular calcium imaging and whole-cell patch-clamp experiments using HBE cells demonstrated the presence of TRPV4 messenger and Ca(2+) entry, and outwardly rectifying cationic currents elicited by the TRPV4 specific activator 4alpha-phorbol 12,13-didecanoate (4alphaPDD). Cell-attached and whole-cell patch-clamp of HBE cells exposed to 4alphaPDD, and hypotonic and high-viscosity solutions (related to mechanical stress) revealed the activation of BK(Ca) channels subsequent to extracellular Ca(2+) influx via TRPV4, an effect lost upon antisense-mediated knock-down of TRPV4. Further analysis of BK(Ca) modulation after TRPV4 activation showed that the Ca(2+) signal can be generated away from the BK(Ca) location at the plasma membrane, and it is not mediated by intracellular Ca(2+) release via ryanodine receptors. Finally, we have shown that, unlike the reported disengagement of TRPV4 and BK(Ca) in response to hypotonic solutions, cystic fibrosis bronchial epithelial cells (CFBE) preserve the functional coupling of TRPV4 and BK(Ca) in response to high-viscous solutions.

An Extracellular Cu2+ Binding Site in the Voltage Sensor of BK and Shaker Potassium Channels

Copper is an essential trace element that may serve as a signaling molecule in the nervous system. Here we show that extracellular Cu2+ is a potent inhibitor of BK and Shaker K+ channels. At low micromolar concentrations, Cu2+ rapidly and reversibly reduces macrosocopic K+ conductance (GK) evoked from mSlo1 BK channels by membrane depolarization. GK is reduced in a dose-dependent manner with an IC50 and Hill coefficient of ∼2 μM and 1.0, respectively. Saturating 100 μM Cu2+ shifts the GK-V relation by +74 mV and reduces GKmax by 27% without affecting single channel conductance. However, 100 μM Cu2+ fails to inhibit GK when applied during membrane depolarization, suggesting that Cu2+ interacts poorly with the activated channel. Of other transition metal ions tested, only Zn2+ and Cd2+ had significant effects at 100 μM with IC50s > 0.5 mM, suggesting the binding site is Cu2+ selective. Mutation of external Cys or His residues did not alter Cu2+ sensitivity. However, four putative Cu2+-coordinating residues were identified (D133, Q151, D153, and R207) in transmembrane segments S1, S2, and S4 of the mSlo1 voltage sensor, based on the ability of substitutions at these positions to alter Cu2+ and/or Cd2+ sensitivity. Consistent with the presence of acidic residues in the binding site, Cu2+ sensitivity was reduced at low extracellular pH. The three charged positions in S1, S2, and S4 are highly conserved among voltage-gated channels and could play a general role in metal sensitivity. We demonstrate that Shaker, like mSlo1, is much more sensitive to Cu2+ than Zn2+ and that sensitivity to these metals is altered by mutating the conserved positions in S1 or S4 or reducing pH. Our results suggest that the voltage sensor forms a state- and pH-dependent, metal-selective binding pocket that may be occupied by Cu2+ at physiologically relevant concentrations to inhibit activation of BK and other channels.

Effect of Testosterone on Potassium Channel Opening in Human Corporal Smooth Muscle Cells

In humans, the role of testosterone in sexual functions, including sexual desire, nocturnal penile erections, and ejaculatory volume, has been relatively well established. However, the effects of testosterone on intrapenile structure in humans remains controversial. We assessed the direct effects of testosterone on potassium channels in human corporal smooth muscle cells, in an effort to understand the mechanisms inherent to the testosterone-induced relaxation of corporal smooth muscle cells at the cellular and molecular levels. Methods. We conducted electrophysiologic studies using cultured human corporal smooth muscle cells. We evaluated the effects of testosterone on potassium channels-BK(Ca) and K(ATP) channels-by determining the whole-cell currents and single-channel activities. For the electrophysiologic recordings, whole-cell and cell-attached configuration patch-clamp techniques were utilized. Changes in whole-cell currents and channel activities of BK(Ca) and K(ATP) channels by testosterone. Results. Testosterone (200 nM) significantly increased the single-channel activity of calcium-activated potassium (BK(Ca)) channels and whole-cell K(+) currents by 443.4 +/- 83.4% (at +60 mV; N = 11, P < 0.05), and this effect was abolished by tetraethylammonium (TEA) (1 mM), a BK(Ca) channel blocker. The whole-cell inward K(+) currents of the K(ATP) channels were also increased by 226.5 +/- 49.3% (at -100 mV; N = 7, P < 0.05). In the presence of a combination of vardenafil (10 nM) and testosterone (200 nM), the BK(Ca) channel was activated to a significantly higher degree than was induced by testosterone alone. The results of patch-clamp studies provided direct molecular evidence that testosterone stimulates the activity of BK(Ca) channels and K(ATP) channels. An understanding of the signaling mechanisms that couple testosterone receptor activation to potassium channel stimulation will provide us with an insight into the cellular processes underlying the vasorelaxant effects of testosterone.

Upregulation of colonic ion channels in APC Min/+ mice

The adenomatous polyposis coli (APC) tumor suppressor gene is mutated in almost all human colonic cancers. Disturbances in Na(+) absorption have been observed in colonic cancer, and ion channels such as ether a go-go (Eag) or Ca(2+)-sensitive BK channels have been recognized for their oncogenic potential. APC ( Min/+ ) mice have reduced APC expression and develop multiple intestinal neoplasias (Min). Ion channels in the colonic epithelium were examined using electrophysiology and molecular techniques. APC ( Min/+ ) mice developed intestinal neoplasia and experienced a significant weight loss. Due to intestinal bleedings, the hematocrit was largely reduced and plasma aldosterone levels were enhanced. Rectal potential measurements in vivo indicated an increase in amiloride-sensitive Na(+) absorption in APC ( Min/+ ) mice. Quantitative Ussing chamber studies demonstrated enhanced Na(+) absorption via epithelial Na(+) channels (ENaC) and suggested enhanced activity of oncogenic BK and Eag-1 channels. Patch clamp and fluorescence measurements on isolated crypts suggested enhanced K(+) channel activity in the surface epithelium. ENaC-mRNA and membrane protein expression was enhanced in colonic surface epithelial cells. The data suggest that reduced expression of the APC gene with upregulation of the downstream proteins Akt and mTOR and subsequent hyperaldosteronism is paralleled by upregulation of oncogenic potassium channels and enhanced colonic Na(+) absorption.