scholarly journals Variants of the KCNMB3 regulatory subunit of maxi BK channels affect channel inactivation

2003 ◽  
Vol 15 (3) ◽  
pp. 191-198 ◽  
Author(s):  
Song Hu ◽  
Malgorzata Z. Labuda ◽  
Massimo Pandolfo ◽  
Greg G. Goss ◽  
Heather E. McDermid ◽  
...  
Keyword(s):  
Time Course ◽  
Bk Channels ◽  
Bk Channel ◽  
Idiopathic Epilepsy ◽  
Regulatory Subunit ◽  
Kinetic Properties ◽  
Regulatory Subunits ◽  
Channel Inactivation ◽  
Current Voltage ◽  
Calcium Activated Potassium Channels

The steady-state and kinetic properties of the KCNMB3 regulatory subunits associated with calcium-activated potassium channels (BK channels) are presented. BK channels containing four sequence variants (V1–V4) in the four different isoforms of the β-subunit (β3a, β3b, β3c, and β3d) were expressed in Xenopus oocytes. Reconstituted BK channel inactivation ranged from none to around 90% inactivation. In particular, channels expressing the β3b-V4 variant displayed a right shift in the potassium current voltage-dependence of activation and inactivated to about 30% of the maximum conductance, compared with wild-type β3b channels that showed no inactivation. When the membrane potential was depolarized, BK channels inactivated with a very rapid time course (∼2–6 ms). This same variant was previously demonstrated to show subtly higher incidence in patients with idiopathic epilepsy (IE) compared with controls, especially when combined with variant V2 (combined heterozygotes). Furthermore, the gene maps to a region containing a susceptibility factor for this disorder. Taken together, these data suggest that neurons expressing BK channels composed of the β3b-V4 variant subunit may experience reduced levels of inhibition and may therefore play permissive roles in high levels of neuronal activity that is characteristic of epilepsy.

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.

scholarly journals Endothelin-1 Stimulates Vasoconstriction Through Rab11A Serine 177 Phosphorylation

2017 ◽  
Vol 121 (6) ◽  
pp. 650-661 ◽  
Author(s):  
Xue Zhai ◽  
M. Dennis Leo ◽  
Jonathan H. Jaggar
Keyword(s):  
Cerebral Arteries ◽  
Phosphorylation Site ◽  
Calcium Sensitivity ◽  
Bk Channels ◽  
Bk Channel ◽  
Recycling Endosomes ◽  
Endothelin 1 ◽  
Current Activation ◽  
Calcium Activated Potassium Channels ◽  
Pkc Protein Kinase C

Rationale: Large-conductance calcium-activated potassium channels (BK) are composed of pore-forming BKα and auxiliary β1 subunits in arterial smooth muscle cells (myocytes). Vasoconstrictors, including endothelin-1 (ET-1), inhibit myocyte BK channels, leading to contraction, but mechanisms involved are unclear. Recent evidence indicates that BKα is primarily plasma membrane localized, whereas the cellular location of β1 can be rapidly altered by Rab11A-positive recycling endosomes. Whether vasoconstrictors regulate the multisubunit composition of surface BK channels to stimulate contraction is unclear. Objective: Test the hypothesis that ET-1 inhibits BK channels by altering BKα and β1 surface trafficking in myocytes, identify mechanisms involved, and determine functional significance in myocytes of small cerebral arteries. Methods and Results: ET-1, through activation of PKC (protein kinase C), reduced surface β1 abundance and the proximity of β1 to surface BKα in myocytes. In contrast, ET-1 did not alter surface BKα, total β1, or total BKα proteins. ET-1 stimulated Rab11A phosphorylation, which reduced Rab11A activity. Rab11A serine 177 was identified as a high-probability PKC phosphorylation site. Expression of a phosphorylation-incapable Rab11A construct (Rab11A S177A) blocked the ET-1–induced Rab11A phosphorylation, reduction in Rab11A activity, and decrease in surface β1 protein. ET-1 inhibited single BK channels and transient BK currents in myocytes and stimulated vasoconstriction via a PKC-dependent mechanism that required Rab11A S177. In contrast, NO-induced Rab11A activation, surface trafficking of β1 subunits, BK channel and transient BK current activation, and vasodilation did not involve Rab11A S177. Conclusions: ET-1 stimulates PKC-mediated phosphorylation of Rab11A at serine 177, which inhibits Rab11A and Rab11A-dependent surface trafficking of β1 subunits. The decrease in surface β1 subunits leads to a reduction in BK channel calcium-sensitivity, inhibition of transient BK currents, and vasoconstriction. We describe a unique mechanism by which a vasoconstrictor inhibits BK channels and identify Rab11A serine 177 as a modulator of arterial contractility.

scholarly journals Molecular mechanism of BK channel activation by the smooth muscle relaxant NS11021

2020 ◽  
Vol 152 (6) ◽  
Author(s):  
Michael E. Rockman ◽  
Alexandre G. Vouga ◽  
Brad S. Rothberg
Keyword(s):  
Smooth Muscle ◽  
Muscle Relaxant ◽  
Time Course ◽  
Bk Channels ◽  
Bk Channel ◽  
Neurological Deficits ◽  
Open Probability ◽  
Channel Activation ◽  
Voltage Sensors ◽  
Smooth Muscle Relaxant

Large-conductance Ca2+-activated K+ channels (BK channels) are activated by cytosolic calcium and depolarized membrane potential under physiological conditions. Thus, these channels control electrical excitability in neurons and smooth muscle by gating K+ efflux and hyperpolarizing the membrane in response to Ca2+ signaling. Altered BK channel function has been linked to epilepsy, dyskinesia, and other neurological deficits in humans, making these channels a key target for drug therapies. To gain insight into mechanisms underlying pharmacological modulation of BK channel gating, here we studied mechanisms underlying activation of BK channels by the biarylthiourea derivative, NS11021, which acts as a smooth muscle relaxant. We observe that increasing NS11021 shifts the half-maximal activation voltage for BK channels toward more hyperpolarized voltages, in both the presence and nominal absence of Ca2+, suggesting that NS11021 facilitates BK channel activation primarily by a mechanism that is distinct from Ca2+ activation. 30 µM NS11021 slows the time course of BK channel deactivation at −200 mV by ∼10-fold compared with 0 µM NS11021, while having little effect on the time course of activation. This action is most pronounced at negative voltages, at which the BK channel voltage sensors are at rest. Single-channel kinetic analysis further shows that 30 µM NS11021 increases open probability by 62-fold and increases mean open time from 0.15 to 0.52 ms in the nominal absence of Ca2+ at voltages less than −60 mV, conditions in which BK voltage sensors are largely in the resting state. We could therefore account for the major activating effects of NS11021 by a scheme in which the drug primarily shifts the pore-gate equilibrium toward the open state.

scholarly journals Regulation of BK Channels by Beta and Gamma Subunits

2019 ◽  
Vol 81 (1) ◽  
pp. 113-137 ◽  
Author(s):  
Vivian Gonzalez-Perez ◽  
Christopher J. Lingle
Keyword(s):  
Functional Properties ◽  
Splice Variants ◽  
Bk Channels ◽  
Bk Channel ◽  
Diverse Range ◽  
Α Subunit ◽  
Regulatory Subunits ◽  
Gamma Subunits ◽  
Specific Alternative ◽  
Alternative Splice Variants

Ca2+- and voltage-gated K+ channels of large conductance (BK channels) are expressed in a diverse variety of both excitable and inexcitable cells, with functional properties presumably uniquely calibrated for the cells in which they are found. Although some diversity in BK channel function, localization, and regulation apparently arises from cell-specific alternative splice variants of the single pore–forming α subunit ( KCa1.1, Kcnma1, Slo1) gene, two families of regulatory subunits, β and γ, define BK channels that span a diverse range of functional properties. We are just beginning to unravel the cell-specific, physiological roles served by BK channels of different subunit composition.

Abstract 376: Nedd4 Regulated Bk Channels in Diabetes Mellitus

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
DAI-MIN ZHANG ◽  
Shao-liang Chen ◽  
Yanrong Zhu ◽  
Peng Ye
Keyword(s):  
Diabetes Mellitus ◽  
Current Density ◽  
Knockout Mice ◽  
Vascular Function ◽  
Critical Role ◽  
Vascular Diseases ◽  
Bk Channels ◽  
Bk Channel ◽  
Hek293 Cells ◽  
Kinetic Properties

Big conductance calcium activated potassium(BK) channel plays a critical role in pathophysiological regulation of vascular function. Recent studies indicated that the expression reduction of BK channels in high glucose condition exacerbated vessel dilation, and led to coronary artery diseases, while BK channel expression was reserved in A-kinase anchoring protein(AKAP) knockout mice at same condition. Here, We are to investigate heterologous co-expression of Nedd4 ligase, ubiquitin protein ligase, and KCa1.1 in HEK293 cells. The result shown that co-expression reduced BK current density without modulation of kinetic properties as measured by path clamp techniques. Modulation of current density was dependent on ligase activity and was lost in AKAP knockout mice with diabetes mellitus. Taken together, our data disclose a novel mechanism of KCa1.1 channel regulation that NEDD4 decreased BK channels expression in diabetes mellitus depending on AKAP signal complexity. These findings provide a new insight into potential therapeutic target in vascular diseases, especially in diabetes mellitus.This work was supported by the National Natural Science Foundation of China(Grant No. 8137034)

scholarly journals Goblet cell LRRC26 regulates BK channel activation and protects against colitis in mice

2020 ◽  
Author(s):  
Vivian Gonzalez-Perez ◽  
Pedro L. Martinez-Espinosa ◽  
Monica Sala-Rabanal ◽  
Nikhil Bharadwaj ◽  
Xiao-Ming Xia ◽  
...  
Keyword(s):  
Intestinal Epithelium ◽  
Goblet Cells ◽  
Bk Channels ◽  
Bk Channel ◽  
Intestinal Lumen ◽  
Regulatory Subunit ◽  
Secretory Cells ◽  
Mucus Layer ◽  
Α Subunit ◽  
Genetic Ablation

AbstractGoblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich-repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca2+-and voltage-activated K+ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2-negative cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK-pore forming α-subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate (DSS). These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice.SignificanceA primary function of goblet cells (GCs) of the intestinal epithelium is to generate a protective mucus layer lining the intestinal lumen. GC dysfunction is linked to Inflammatory Bowel Disease (IBD). GC mucus secretion is thought to be dependent on contributions of an ensemble of anion and cation fluxes, although understanding remains limited. Here, it is shown in mouse colon that the Ca2+- and voltage-dependent BK-type K+ channel, specifically in association with the LRRC26 regulatory subunit, plays a critical role in normal GC function, protecting mice against chemically-induced colitis. The results demonstrate that normal K+ fluxes mediated by LRRC26-containing BK channels are required for normal GC function, potentially providing insights into the potential role of BK channels in IBD.

scholarly journals MiR-339-3p aggravates rat vascular inflammation induced by AT1R autoantibodies by down-regulating BKα protein expression

2021 ◽  
Author(s):  
Huirong Liu ◽  
Yang Li ◽  
Yan Sun ◽  
Mingming Yue ◽  
Ming Gao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Protein Level ◽  
Vascular Inflammation ◽  
Bk Channels ◽  
Bk Channel ◽  
The Body ◽  
Neural Precursor Cells ◽  
Α Subunit ◽  
Channel Protein ◽  
Calcium Activated Potassium Channels

The abnormality of large-conductance calcium-activated potassium channels (BK channels) is an important factor in inducing vascular inflammation. BK channel agonists can readily recover BK channel function and improve vascular inflammation. However, it is not clear how to improve BK dysfunction caused by downregulation of BK channel protein expression. This study found that angiotensin II-1 receptor autoantibodies (AT1-AA), which are widely present in the body of various types of cardiovascular diseases, can down-regulate the expression of BK channel protein and induce vascular inflammation. Further research found that the elevated neural precursor cells expressed developmentally downregulated 4-like (NEDD4L) protein level is involved in the down-regulation of BK channel α subunit (BKα) protein level by AT1-AA. Bioinformatics analysis and experiments have confirmed that miR-339-3p plays an irreplaceable role in the high expression of NEDD4L and the low expression of BKα, and aggravates the vascular inflammation induced by AT1-AA. Overall, AT1-AA increased miR-339-3p expression (targeting BKα via the miR-339-3p/NEDD4L axis or miR-339-3p alone), reduced BKα protein expression in VSMCs, and induced vascular inflammation. The results of the study indicate that miR-339-3p may become a new target for reversing vascular inflammation in AT1-AA-positive patients.

scholarly journals Dynamics of Signaling between Ca2+ Sparks and Ca2+- Activated K+ Channels Studied with a Novel Image-Based Method for Direct Intracellular Measurement of Ryanodine Receptor Ca2+ Current

2000 ◽  
Vol 116 (6) ◽  
pp. 845-864 ◽  
Author(s):  
Ronghua ZhuGe ◽  
Kevin E. Fogarty ◽  
Richard A. Tuft ◽  
Lawrence M. Lifshitz ◽  
Kemal Sayar ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
High Speed ◽  
Time Course ◽  
Imaging System ◽  
Release Site ◽  
Bk Channels ◽  
Bk Channel ◽  
Global Changes ◽  
Channel Kinetics ◽  
K Channels

Ca2+ sparks are highly localized cytosolic Ca2+ transients caused by a release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RyRs); they are the elementary events underlying global changes in Ca2+ in skeletal and cardiac muscle. In smooth muscle and some neurons, Ca2+ sparks activate large conductance Ca2+-activated K+ channels (BK channels) in the spark microdomain, causing spontaneous transient outward currents (STOCs) that regulate membrane potential and, hence, voltage-gated channels. Using the fluorescent Ca2+ indicator fluo-3 and a high speed widefield digital imaging system, it was possible to capture the total increase in fluorescence (i.e., the signal mass) during a spark in smooth muscle cells, which is the first time such a direct approach has been used in any system. The signal mass is proportional to the total quantity of Ca2+ released into the cytosol, and its rate of rise is proportional to the Ca2+ current flowing through the RyRs during a spark (ICa(spark)). Thus, Ca2+ currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of ICa(spark) in different sparks varies more than fivefold, Ca2+ sparks appear to be caused by the concerted opening of a number of RyRs. Sparks with the same underlying Ca2+ current cause STOCs, whose amplitudes vary more than threefold, a finding that is best explained by variability in coupling ratio (i.e., the ratio of RyRs to BK channels in the spark microdomain). The time course of STOC decay is approximated by a single exponential that is independent of the magnitude of signal mass and has a time constant close to the value of the mean open time of the BK channels, suggesting that STOC decay reflects BK channel kinetics, rather than the time course of [Ca2+] decline at the membrane. Computer simulations were carried out to determine the spatiotemporal distribution of the Ca2+ concentration resulting from the measured range of ICa(spark). At the onset of a spark, the Ca2+ concentration within 200 nm of the release site reaches a plateau or exceeds the [Ca2+]EC50 for the BK channels rapidly in comparison to the rate of rise of STOCs. These findings suggest a model in which the BK channels lie close to the release site and are exposed to a saturating [Ca2+] with the rise and fall of the STOCs determined by BK channel kinetics. The mechanism of signaling between RyRs and BK channels may provide a model for Ca2+ action on a variety of molecular targets within cellular microdomains.

scholarly journals Highly Specific Alternative Splicing of Transcripts Encoding BK Channels in the Chicken's Cochlea Is a Minor Determinant of the Tonotopic Gradient

2010 ◽  
Vol 30 (14) ◽  
pp. 3646-3660 ◽  
Author(s):  
Soledad Miranda-Rottmann ◽  
Andrei S. Kozlov ◽  
A. J. Hudspeth
Keyword(s):  
Cellular Response ◽  
Single Channel ◽  
Electrical Potential ◽  
Sensory Epithelium ◽  
Bk Channels ◽  
Bk Channel ◽  
Kinetic Properties ◽  
Auditory Hair Cells ◽  
Specific Alternative ◽  
Single Channel Currents

ABSTRACT The frequency sensitivity of auditory hair cells in the inner ear varies with their longitudinal position in the sensory epithelium. Among the factors that determine the differential cellular response to sound is the resonance of a hair cell's transmembrane electrical potential, whose frequency correlates with the kinetic properties of the high-conductance Ca2+-activated K+ (BK) channels encoded by a Slo (kcnma1) gene. It has been proposed that the inclusion of specific alternative axons in the Slo transcripts along the cochlea underlies the gradient of BK-channel kinetics. By analyzing the complete sequences of chicken Slo gene (cSlo) cDNAs from the chicken's cochlea, we show that most transcripts lack alternative exons. Transcripts with more than one alternative exon constitute only 10% of the total. Although the fraction of transcripts containing alternative exons increases from the cochlear base to the apex, the combination of alternative exons is not regulated. There is also a clear increase in the expression of BK transcripts with long carboxyl termini toward the apex. When long and short BK transcripts are expressed in HEK-293 cells, the kinetics of single-channel currents differ only slightly, but they are substantially slowed when the channels are coexpressed with the auxiliary β subunit that occurs more widely at the apex. These results argue that the tonotopic gradient is not established by the selective inclusion of highly specific cSlo exons. Instead, a gradient in the expression of β subunits slows BK channels toward the low-frequency apex of the cochlea.

scholarly journals LINGO1 is a regulatory subunit of large conductance, Ca2+-activated potassium channels

2020 ◽  
Vol 117 (4) ◽  
pp. 2194-2200 ◽  
Author(s):  
Srikanth Dudem ◽  
Roddy J. Large ◽  
Shruti Kulkarni ◽  
Heather McClafferty ◽  
Irina G. Tikhonova ◽  
...  
Keyword(s):  
Human Brain ◽  
Transmembrane Protein ◽  
Membrane Surface ◽  
Surface Expression ◽  
Bk Channels ◽  
Bk Channel ◽  
Regulatory Subunit ◽  
Motor Disorders ◽  
Genetic Ablation ◽  
Functional Knockdown

LINGO1 is a transmembrane protein that is up-regulated in the cerebellum of patients with Parkinson’s disease (PD) and Essential Tremor (ET). Patients with additional copies of the LINGO1 gene also present with tremor. Pharmacological or genetic ablation of large conductance Ca2+-activated K+ (BK) channels also result in tremor and motor disorders. We hypothesized that LINGO1 is a regulatory BK channel subunit. We show that 1) LINGO1 coimmunoprecipitated with BK channels in human brain, 2) coexpression of LINGO1 and BK channels resulted in rapidly inactivating BK currents, and 3) LINGO1 reduced the membrane surface expression of BK channels. These results suggest that LINGO1 is a regulator of BK channels, which causes a “functional knockdown” of these currents and may contribute to the tremor associated with increased LINGO1 levels.

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