scholarly journals Regulation of colonic apical potassium (BK) channels by cAMP and somatostatin

2009 ◽  
Vol 297 (1) ◽  
pp. G159-G167 ◽  
Author(s):  
M. D. Perry ◽  
G. I. Sandle
Keyword(s):  
Tyrosine Phosphatase ◽  
Distal Colon ◽  
Human Colon ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Secretory Diarrhea ◽  
Inside Out ◽  
Protein Phosphatase Type

High-conductance apical K+(BK) channels are present in surface colonocytes of mammalian (including human) colon. Their location makes them well fitted to contribute to the excessive intestinal K+losses often associated with infective diarrhea. Since many channel proteins are regulated by phosphorylation, we evaluated the roles of protein kinase A (PKA) and phosphatases in the modulation of apical BK channel activity in surface colonocytes from rat distal colon using patch-clamp techniques, having first increased channel abundance by chronic dietary K+enrichment. We found that PKA activation using 50 μmol/l forskolin and 5 mmol/l 3-isobutyl-1-methylxanthine stimulated BK channels in cell-attached patches and the catalytic subunit of PKA (200 U/ml) had a similar effect in excised inside-out patches. The antidiarrheal peptide somatostatin (SOM; 2 μmol/l) had a G protein-dependent inhibitory effect on BK channels in cell-attached patches, which was unaffected by pretreatment with 10 μmol/l okadaic acid (an inhibitor of protein phosphatase type 1 and type 2A) but completely prevented by pretreatment with 100 μmol/l Na+orthovanadate and 10 μmol/l BpV (inhibitors of phosphoprotein tyrosine phosphatase). SOM also inhibited apical BK channels in surface colonocytes in human distal colon. We conclude that cAMP-dependent PKA activates apical BK channels and may enhance colonic K+losses in some cases of secretory diarrhea. SOM inhibits apical BK channels through a phosphoprotein tyrosine phosphatase-dependent mechanism, which could form the basis of new antidiarrheal strategies.

scholarly journals Segmental differences in upregulated apical potassium channels in mammalian colon during potassium adaptation

2016 ◽  
Vol 311 (5) ◽  
pp. G785-G793 ◽  
Author(s):  
Matthew D. Perry ◽  
Vazhaikkurichi M. Rajendran ◽  
Kenneth A. MacLennan ◽  
Geoffrey I. Sandle
Keyword(s):  
Distal Colon ◽  
Proximal Colon ◽  
Bk Channels ◽  
Bk Channel ◽  
Patch Clamp Recording ◽  
Α Subunit ◽  
Subunit Protein ◽  
Clear Cut ◽  
Potassium Adaptation ◽  
Channel Expression

Rat proximal and distal colon are net K+secretory and net K+absorptive epithelia, respectively. Chronic dietary K+loading increases net K+secretion in the proximal colon and transforms net K+absorption to net K+secretion in the distal colon, but changes in apical K+channel expression are unclear. We evaluated expression/activity of apical K+(BK) channels in surface colonocytes in proximal and distal colon of control and K+-loaded animals using patch-clamp recording, immunohistochemistry, and Western blot analyses. In controls, BK channels were more abundant in surface colonocytes from K+secretory proximal colon (39% of patches) than in those from K+-absorptive distal colon (12% of patches). Immunostaining demonstrated more pronounced BK channel α-subunit protein expression in surface cells and cells in the upper 25% of crypts in proximal colon, compared with distal colon. Dietary K+loading had no clear-cut effects on the abundance, immunolocalization, or expression of BK channels in proximal colon. By contrast, in distal colon, K+loading 1) increased BK channel abundance in patches from 12 to 41%; 2) increased density of immunostaining in surface cells, which extended along the upper 50% of crypts; and 3) increased expression of BK channel α-subunit protein when assessed by Western blotting ( P < 0.001). Thus apical BK channels are normally more abundant in K+secretory proximal colon than in K+absorptive distal colon, and apical BK channel expression in distal (but not proximal) colon is greatly stimulated as part of the enhanced K+secretory response to dietary K+loading.

Peroxynitrite reversibly inhibits Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

2000 ◽  
Vol 278 (6) ◽  
pp. H1883-H1890 ◽  
Author(s):  
Anna K. Brzezinska ◽  
Debebe Gebremedhin ◽  
William M. Chilian ◽  
Balaraman Kalyanaraman ◽  
Stephen J. Elliott
Keyword(s):  
Single Channel ◽  
Cerebral Arteries ◽  
Ionic Current ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Cell Current

Peroxynitrite (ONOO−) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO−bioactivity, the present study examined the effect of ONOO− on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K+ current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca2+-activated K+ (BK) channels, and ONOO− (10–100 μM) were determined. At a pipette potential of +50 mV, ONOO− inhibited 39% of iberiotoxin-sensitive current. ONOO− was selective for iberiotoxin-sensitive current, whereas decomposed ONOO− had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K+solutions. Unitary currents were sensitive to increases in internal Ca2+ concentration, consistent with activity due to BK channels. Internal ONOO− dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO− could be overcome by reduced glutathione. Glutathione, added after ONOO−, restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO− on membrane K+ current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO− has the potential to alter cerebral vascular tone by inhibiting BK channel activity.

scholarly journals Regulation of endothelial BK channels by heme oxygenase-derived carbon monoxide and caveolin-1

2012 ◽  
Vol 303 (1) ◽  
pp. C92-C101 ◽  
Author(s):  
Melissa A. Riddle ◽  
Benjimen R. Walker
Keyword(s):  
Carbon Monoxide ◽  
Heme Oxygenase ◽  
Barometric Pressure ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Outward Currents ◽  
In Cells

A novel vasodilatory influence of endothelial cell (EC) large-conductance Ca2+-activated K+ (BK) channels is present after in vivo exposure to chronic hypoxia (CH) and may exist in other pathological states. However, the mechanism of channel activation that results in altered vasoreactivity is unknown. Previously, we demonstrated that inhibition of either BK channels or heme oxygenase (HO) restores vasoconstrictor reactivity after CH. Additionally, administration of the scaffolding domain of caveolin (Cav)-1 inhibits EC BK activity and restores vasoconstrictor reactivity in this setting. These results led us to hypothesize that CH exposure results in a loss in Cav-1 inhibition of EC BK channels, resulting in their activation by HO-derived carbon monoxide (CO). Experiments were conducted on freshly dispersed aortic ECs from control and CH-exposed (barometric pressure: 380 mmHg for 48 h) rats. In electrophysiology experiments, outward currents were greater in cells from CH rats as well as from cells from control rats treated with the cholesterol-depleting agent methyl-β-cyclodextrin. These enhanced currents were returned to control by HO inhibition. Channel activity could be restored by the CO donor CO-releasing molecule (CORM)-2 during HO inhibition. Administration of the Cav-1 scaffolding domain eliminated BK currents in cells from CH rats, and current was not restored by the addition of CORM-2. Colocalization experiments in ECs from control and CH rats demonstrated an association between HO-2, Cav-1, and BK. We conclude that EC BK channel activity is HO dependent in the absence of the inhibitory effect of the Cav-1 scaffolding domain.

scholarly journals Coronary arterial BK channel dysfunction exacerbates ischemia/reperfusion-induced myocardial injury in diabetic mice

2016 ◽  
Vol 41 (9) ◽  
pp. 992-1001 ◽  
Author(s):  
Tong Lu ◽  
Bin Jiang ◽  
Xiao-Li Wang ◽  
Hon-Chi Lee
Keyword(s):  
Myocardial Injury ◽  
Ischemia Reperfusion ◽  
Large Body ◽  
Bk Channels ◽  
Bk Channel ◽  
Ang Ii ◽  
Diabetic Mice ◽  
Genetic Ablation ◽  
Channel Function

The large conductance Ca2+-activated K+ (BK) channels, abundantly expressed in coronary artery smooth muscle cells (SMCs), play a pivotal role in regulating coronary circulation. A large body of evidence indicates that coronary arterial BK channel function is diminished in both type 1 and type 2 diabetes. However, the consequence of coronary BK channel dysfunction in diabetes is not clear. We hypothesized that impaired coronary BK channel function exacerbates myocardial ischemia/reperfusion (I/R) injury in streptozotocin-induced diabetic mice. Combining patch-clamp techniques and cellular biological approaches, we found that diabetes facilitated the colocalization of angiotensin II (Ang II) type 1 receptors and BK channel α-subunits (BK-α), but not BK channel β1-subunits (BK-β1), in the caveolae of coronary SMCs. This caveolar compartmentation in vascular SMCs not only enhanced Ang II-mediated inhibition of BK-α but also produced a physical disassociation between BK-α and BK-β1, leading to increased infarct size in diabetic hearts. Most importantly, genetic ablation of caveolae integrity or pharmacological activation of coronary BK channels protected the cardiac function of diabetic mice from experimental I/R injury in both in vivo and ex vivo preparations. Our results demonstrate a vascular ionic mechanism underlying the poor outcome of myocardial injury in diabetes. Hence, activation of coronary BK channels may serve as a therapeutic target for cardiovascular complications of diabetes.

Abstract 1368: BK Channel β1 Subunits Are Specific Effectors Of Cholane-induced Channel Activation

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Anna Bukiya ◽  
Ligia Toro ◽  
Alejandro M Dopico
Keyword(s):  
Smooth Muscle ◽  
Cerebral Artery ◽  
Vascular Tone ◽  
Lithocholic Acid ◽  
Cerebral Arteries ◽  
Bk Channels ◽  
Bk Channel ◽  
Α Subunit ◽  
Steady State Activity ◽  
Inside Out

The activity of large conductance, Ca 2+ - and voltage-gated potassium (BK) channels in smooth muscle critically controls vascular tone. Depolarization-induced Ca 2+ -entry in the myocyte activates BK channels, which generate outward positive current that tends to repolarize the membrane, limit Ca 2+ entry and, thus, oppose contraction. Cholane-derived steroids (e.g., lithocholic acid, LC) reduce vascular tone in isolated, resistance-size rat cerebral arteries by selective activation of myocyte BK channels. In most tissues, native BK channels consist of pore-forming α (encoded by KCNMA1 or Slo1 ) and accessory β1–4 (encoded by KCNMB1–4 ) subunits. Remarkably, KCNMB expression is tissue-specific: while KCNMB1 is highly predominant in smooth muscle, KCNMB2–4 are not. Thus, agents that target BK β1 subunits may be used to selectively modulate myocyte BK channel function. After cloning the BK α subunit from rat cerebral artery myocytes (termed “cbv1”, AY330293 ), we demonstrated that homomeric cbv1 channel steady-state activity (NPo) was not affected by acute LC application. In contrast, heteromeric cbv1+β1 channel NPo was reversibly increased by LC (+290% of control at EC max ~150 μM; EC 50 =46 μM). Whether the other BK β subunits (2–4) can substitute for β1 to evoke LC-sensitivity in the BK channel remains unknown. To test this, we applied 150 μM LC to the intracellular side of inside-out patches excised from Xenopus laevis oocytes expressing cbv1 alone or cbv1 with a given BK β subunit subtype (1–4). Currents were evoked with the membrane clamped at ±20mV and free Ca 2+ i set to 10 μM, a concentration found in the cerebral artery myocyte during contraction. As previously found, LC consistently failed to increase homomeric cbv1 NPo, while drastically enhancing heteromeric cbv1+β1 channel NPo. Remarkably, LC failed to activate cbv1+β2, cbv1+β3 and cbv1+β4 heteromeric channels. In conclusion, the BK β1 (smooth muscle-type) subunit serves as a unique sensor for cholane-derived steroids. Thus, these compounds provide a platform for designing therapeutic agents to treat cardiovascular disease where reduction of vascular tone is required.

scholarly journals Functional and Molecular Evidence for Impairment of Calcium-Activated Potassium Channels in Type-1 Diabetic Cerebral Artery Smooth Muscle Cells

2007 ◽  
Vol 28 (2) ◽  
pp. 377-386 ◽  
Author(s):  
Ling Dong ◽  
Yun-Min Zheng ◽  
Dee Van Riper ◽  
Rakesh Rathore ◽  
Qing-Hua Liu ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Type 1 Diabetes ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cerebral Arteries ◽  
Bk Channels ◽  
Bk Channel ◽  
Elevated Blood ◽  
Diabetic Mice

Cerebral vascular dysfunction and associated diseases often occur in type-1 diabetes, but the underlying mechanisms are largely unknown. In this study, we sought to determine whether big-conductance, Ca2+-activated K+ (BK) channels were impaired in vascular (cerebral artery) smooth muscle cells (CASMCs) from streptozotocin-induced type-1 diabetic mice using patch clamp, molecular biologic, and genetic approaches. Our data indicate that the frequency and amplitude of spontaneous transient outward currents (STOCs) are significantly decreased, whereas the activity of spontaneous Ca2+ sparks is increased, in diabetic CASMCs. The sensitivity of BK channels to voltage, Ca2+, and the specific inhibitor iberiotoxin are all reduced in diabetic myocytes. Diabetic mice show increased myogenic tone and decreased contraction in response to iberiotoxin in cerebral arteries and elevated blood pressure. The expression of the BK channel β1, but not α-subunit protein, is markedly decreased in diabetic cerebral arteries. Diabetic impairment of BK channel activity is lost in CASMCs from BK channel β1-subunit gene deletion mice. In conclusion, the BK channel β1-subunit is impaired in type-1 diabetic vascular SMCs, resulting in increased vasoconstriction and elevated blood pressure, thereby contributing to vascular diseases in type-1 diabetes.

scholarly journals Testosterone decreases urinary bladder smooth muscle excitability via novel signaling mechanism involving direct activation of the BK channels

2016 ◽  
Vol 311 (6) ◽  
pp. F1253-F1259 ◽  
Author(s):  
Kiril L. Hristov ◽  
Shankar P. Parajuli ◽  
Aaron Provence ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Bladder Pressure ◽  
Resting Membrane Potential ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Inside Out

In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large-conductance voltage- and Ca2+-activated K+ (BK) channels with paxilline (1 μM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of −20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a nongenomic mechanism. Live-cell Ca2+ imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca2+ concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent from genomic testosterone receptors, and thus regulates DSM excitability.

Deglycosylation of the β1-subunit of the BK channel changes its biophysical properties

2006 ◽  
Vol 291 (4) ◽  
pp. C750-C756 ◽  
Author(s):  
Brian M. Hagen ◽  
Kenton M. Sanders
Keyword(s):  
Smooth Muscle ◽  
Molecular Mass ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Dependent Manner ◽  
Biophysical Properties ◽  
Single Channel Recordings ◽  
Inside Out ◽  
Α Subunits

Large-conductance Ca2+-activated potassium (BK) channels are composed of pore-forming α-subunits and auxiliary β-subunits. The α-subunits are widely expressed in many cell types, whereas the β-subunits are more tissue specific and influence diverse aspects of channel function. In the current study, we identified the presence of the smooth muscle-specific β1-subunit in murine colonic tissue using Western blotting. The native β1-subunits migrated in SDS-PAGE as two molecular mass bands. Enzymatic removal of N-linked glycosylations from the β1-subunit resulted in a single band that migrated at a lower molecular mass than the native β1-subunit bands, suggesting that the native β1-subunit exists in either a core glycosylated or highly glycosylated form. We investigated the functional consequence of deglycosylating the β1-subunit during inside-out single-channel recordings. During inside-out single-channel recordings, with N-glycosidase F in the pipette solution, the open probability ( Po) and mean open time of BK channels increased in a time-dependent manner. Deglycosylation of BK channels did not affect the conductance but shifted the steady-state voltage of activation toward more positive potentials without affecting slope when Ca2+ concentration was <1 μM. Treatment of myocytes lacking the β1-subunits of the BK channel with N-glycosidase F had no effect. These data suggest that glycosylations on the β1-subunit in smooth muscle cells can modify the biophysical properties of BK channels.

IK channels are involved in the regulatory volume decrease in human epithelial cells

2003 ◽  
Vol 284 (1) ◽  
pp. C77-C84 ◽  
Author(s):  
Jun Wang ◽  
Shigeru Morishima ◽  
Yasunobu Okada
Keyword(s):  
Epithelial Cells ◽  
Regulatory Volume Decrease ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Blockers ◽  
Hypotonic Stress ◽  
Human Epithelial Cells ◽  
Ik Channel ◽  
Inside Out ◽  
Volume Decrease

Parallel activation of Ca2+-dependent K+ channels and volume-sensitive Cl− channels is known to be responsible for KCl efflux during regulatory volume decrease (RVD) in human epithelial Intestine 407 cells. The present study was performed to identify the K+ channel type. RT-PCR demonstrated mRNA expression of Ca2+-activated, intermediate conductance K+(IK), but not small conductance K+ (SK1) or large conductance K+ (BK) channels in this cell line. Whole cell recordings showed that ionomycin or hypotonic stress activated inwardly rectifying K+ currents that were reversibly blocked by IK channel blockers [clotrimazole (CLT) and charybdotoxin] but not by SK and BK channel blockers (apamin and iberiotoxin). Inside-out recordings revealed the existence of CLT-sensitive single K+-channel activity, which exhibited an intermediate unitary conductance (30 pS at −100 mV). The channel was activated by cytosolic Ca2+ in inside-out patches and by a hypotonic challenge in cell-attached patches. The RVD was suppressed by CLT, but not by apamin or iberiotoxin. Thus we conclude that the IK channel is involved in the RVD process in these human epithelial cells.

Rotundic Acid Regulates the Effects of Let-7f-5p on Caco2 Cell Proliferation

2020 ◽  
Vol 20 ◽  
Author(s):  
Yuan Feng ◽  
Xinran Liu ◽  
Yueqing Han ◽  
Mantian Chen ◽  
Lin Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Colony Formation ◽  
Cancer Cell Line ◽  
Human Colon ◽  
Colon Cancer Cell Line ◽  
Inhibitory Effect ◽  
Human Colon Cancer ◽  
Caco2 Cell ◽  
Study Results ◽  
Rotundic Acid

Background & Objective: Nowadays, the interaction between natural products and microRNAs provides a promising field for exploring the chemo preventive agents for various cancers.As a member of microRNAs, the expression of let-7f-5p is universally down regulated in colorectal cancer (CRC). The present study aimed to uncover the function of let-7f-5p in the proliferation of human colon cancer cell line Caco2 and explored chemo preventive agents from natural resources that can prevent the development of CRC. Methods: Herein, Caco2 cells were transfected with let-7f-5p mimic and inhibitor to manipulate let-7f-5p levels, and the expression of let-7f-5p wasper formed by RT‑qPCR. Next, we determined how let-7f-5p regulates Caco2 cell proliferation by using MTT, wound-healing, cell cycle,and colony formation assays.Besides, to further understand the effect of let-7f-5p, we evaluated the protein level of AMER3 and SLC9A9 by using western blotting assays. Results: The results showed a suppressive function of let-7f-5p on Caco2 cell proliferation and then put forward a triterpenoid (rotundic acid, RA) which significant antagonized the effect of cell proliferation, restitution after wounding,and colony formation caused by let-7f-5p. Moreover, the western blot results further indicated that the inhibitory effect of RA might be due to its suppressive role in let-7f-5p-targeted AMER3 and SLC9A9 regulation. Conclusion: Our validation study results confirmed that let-7f-5p was a potent tumor suppressor gene of Caco2 cell proliferation,and RA showed as a regulator of the effect oflet-7f-5p on cell proliferation and then could be a potential chemo preventive agent for CRC treatment.

Sign in / Sign up

Export Citation Format

Share Document