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.

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.

scholarly journals Chronic Prenatal Hypoxia Down-Regulated BK Channel Β1 Subunits in Mesenteric Artery Smooth Muscle Cells of the Offspring

2018 ◽  
Vol 45 (4) ◽  
pp. 1603-1616 ◽  
Author(s):  
Bailin Liu ◽  
Yanping Liu ◽  
Ruixiu Shi ◽  
Xueqin Feng ◽  
Xiang Li ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Bk Channels ◽  
Bk Channel ◽  
Mesenteric Arteries ◽  
Prenatal Hypoxia ◽  
Pregnant Rats ◽  
Α Subunit

Background/Aims: Chronic hypoxia in utero could impair vascular functions in the offspring, underlying mechanisms are unclear. This study investigated functional alteration in large-conductance Ca2+-activated K+ (BK) channels in offspring mesenteric arteries following prenatal hypoxia. Methods: Pregnant rats were exposed to normoxic control (21% O2, Con) or hypoxic (10.5% O2, Hy) conditions from gestational day 5 to 21, their 7-month-old adult male offspring were tested for blood pressure, vascular BK channel functions and expression using patch clamp and wire myograh technique, western blotting, and qRT-PCR. Results: Prenatal hypoxia increased pressor responses and vasoconstrictions to phenylephrine in the offspring. Whole-cell currents density of BK channels and amplitude of spontaneous transient outward currents (STOCs), not the frequency, were significantly reduced in Hy vascular myocytes. The sensitivity of BK channels to voltage, Ca2+, and tamoxifen were reduced in Hy myocytes, whereas the number of channels per patch and the single-channel conductance were unchanged. Prenatal hypoxia impaired NS1102- and tamoxifen-mediated relaxation in mesenteric arteries precontracted with phenylephrine in the presence of Nω-nitro-L-arginine methyl ester. The mRNA and protein expression of BK channel β1, not the α-subunit, was decreased in Hy mesenteric arteries. Conclusions: Impaired BK channel β1-subunits in vascular smooth muscle cells contributed to vascular dysfunction in the offspring exposed to prenatal hypoxia.

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 Chloroform Extract ofArtemisia annuaL. Relaxes Mouse Airway Smooth Muscle

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Jun Huang ◽  
Li-Qun Ma ◽  
Yongle Yang ◽  
Nana Wen ◽  
Wan Zhou ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Single Channel ◽  
Artemisia Annua ◽  
Chloroform Extract ◽  
Bk Channels ◽  
Bk Channel ◽  
Airway Smooth Muscle Cells ◽  
Patch Clamp Technique ◽  
Channel Currents

Artemisia annuaL. belongs to the Asteraceae family, which is indigenous to China. It has valuable pharmacological properties, such as antimalarial, anti-inflammatory, and anticancer properties. However, whether it possesses antiasthma properties is unknown. In the current study, chloroform extract ofArtemisia annuaL. (CEAA) was prepared, and we found that CEAA completely eliminated acetylcholine (ACh) or high K+-elicited (80 mM) contractions of mouse tracheal rings (TRs). Patch-clamp technique and ion channel blockers were employed to explore the underlying mechanisms of the relaxant effect of CEAA. In whole-cell current recording, CEAA almost fully abolished voltage-dependent Ca2+channel (VDCC) currents and markedly enhanced large conductance Ca2+-activated K+(BK) channel currents on airway smooth muscle cells (ASMCs). In single channel current recording, CEAA increased the opening probability but had no effect on the single channel conductance of BK channels. However, under paxilline-preincubated (a selective BK channel blocker) conditions, CEAA only slightly increased BK channel currents. These results indicate that CEAA may contain active components with potent antiasthma activity. The abolished VDCCs by CEAA may mainly contribute to the underlying mechanism through which it acts as an effective antiasthmatic compound, but the enhanced BK currents might play a less important role in the antiasthmatic effects.

scholarly journals Intracellular Mg2+ Enhances the Function of Bk-Type Ca2+-Activated K+ Channels

2001 ◽  
Vol 118 (5) ◽  
pp. 589-606 ◽  
Author(s):  
Jingyi Shi ◽  
Jianmin Cui
Keyword(s):  
Binding Sites ◽  
Neurotransmitter Release ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Dependent Manner ◽  
Physiological Conditions ◽  
Channel Function ◽  
Open Conformation ◽  
Voltage Dependent

BK channels modulate neurotransmitter release due to their activation by voltage and Ca2+. Intracellular Mg2+ also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg2+ blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg2+ activates mslo1 BK channels independently of Ca2+ and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca2+ binding sites in the tail, is not activated by Mg2+. However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg2+ sensitivity similar to mslo1 channels, indicating that Mg2+ sites differ from Ca2+ sites. We discovered that Mg2+ also binds to Ca2+ sites and competitively inhibits Ca2+-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg2+ under physiological conditions is to enhance BK channel function.

scholarly journals State-dependent inhibition of BK channels by the opioid agonist loperamide

2021 ◽  
Vol 153 (9) ◽  
Author(s):  
Alexandre G. Vouga ◽  
Michael E. Rockman ◽  
Jiusheng Yan ◽  
Marlene A. Jacobson ◽  
Brad S. Rothberg
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Open Probability ◽  
Opioid Agonist ◽  
State Dependent ◽  
Dependent Inhibition ◽  
Cytosolic Side ◽  
Relative Stabilization ◽  
Α Subunits

Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.

scholarly journals Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca2+-activated K+ channel

2012 ◽  
Vol 302 (9) ◽  
pp. C1361-C1370 ◽  
Author(s):  
Wenkuan Xin ◽  
Qiuping Cheng ◽  
Rupal P. Soder ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
Dependent Manner ◽  
Detrusor Smooth Muscle ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Pde Inhibition

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca2+-activated K+ (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ∼3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ∼2-fold and hyperpolarized the DSM cell resting membrane potential by ∼6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca2+ sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.

scholarly journals Lack of negative slope in I-V plots for BK channels at positive potentials in the absence of intracellular blockers

2013 ◽  
Vol 141 (4) ◽  
pp. 493-497 ◽  
Author(s):  
Yanyan Geng ◽  
Xiaoyu Wang ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Negative Slope ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Linear Current ◽  
Joint Application ◽  
Current Voltage ◽  
Α Subunits

Large-conductance, voltage- and Ca2+-activated K+ (BK) channels display near linear current–voltage (I-V) plots for voltages between −100 and +100 mV, with an increasing sublinearity for more positive potentials. As is the case for many types of channels, BK channels are blocked at positive potentials by intracellular Ca2+ and Mg2+. This fast block progressively reduces single-channel conductance with increasing voltage, giving rise to a negative slope in the I-V plots beyond about +120 mV, depending on the concentration of the blockers. In contrast to these observations of pronounced differences in the magnitudes and shapes of I-V plots in the absence and presence of intracellular blockers, Schroeder and Hansen (2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) have reported identical I-V plots in the absence and presence of blockers for BK channels, with both plots having reduced conductance and negative slopes, as expected for blockers. Schroeder and Hansen included both Ca2+ and Mg2+ in the intracellular solution rather than a single blocker, and they also studied BK channels expressed from α plus β1 subunits, whereas most previous studies used only α subunits. Although it seems unlikely that these experimental differences would account for the differences in findings between previous studies and those of Schroeder and Hansen, we repeated the experiments using BK channels comprised of α plus β1 subunits with joint application of 2.5 mM Ca2+ plus 2.5 mM Mg2+, as Schroeder and Hansen did. In contrast to the findings of Schroeder and Hansen of identical I-V plots, we found marked differences in the single-channel I-V plots in the absence and presence of blockers. Consistent with previous studies, we found near linear I-V plots in the absence of blockers and greatly reduced currents and negative slopes in the presence of blockers. Hence, studies of conductance mechanisms for BK channels should exclude intracellular Ca2+/Mg2+, as they can reduce conductance and induce negative slopes.

scholarly journals Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

2014 ◽  
Vol 306 (5) ◽  
pp. C460-C470 ◽  
Author(s):  
Kiril L. Hristov ◽  
Amy C. Smith ◽  
Shankar P. Parajuli ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Regulation ◽  
Pkc Activation

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility.

scholarly journals Regulatory γ1 subunits defy symmetry in functional modulation of BK channels

2018 ◽  
Vol 115 (40) ◽  
pp. 9923-9928 ◽  
Author(s):  
Vivian Gonzalez-Perez ◽  
Manu Ben Johny ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Single Channel ◽  
Regulatory Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bk Channels ◽  
Bk Channel ◽  
Single Type ◽  
Regulatory Subunits ◽  
And Function

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, β and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four β subunits per channel, with each β subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+channel.

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