Abstract 376: Nedd4 Regulated Bk Channels in Diabetes Mellitus

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)

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Impact of Physical Activity on the Incidence of Vascular Diseases in Adults with Type 2 Diabetes Mellitus

10.31661/gmj.v0i0.1549 ◽

2019 ◽

Vol 8 ◽

pp. 1549

Author(s):

Babak Pezeshki ◽

Ehsan Bahramali ◽

Amir Ansari ◽

Aliasghar Karimi ◽

Mojtaba Frajam ◽

...

Keyword(s):

Physical Activity ◽

Diabetes Mellitus ◽

Vascular Function ◽

Vascular Diseases ◽

Major Complication ◽

Control Group ◽

Case Group ◽

Vascular Events ◽

The Relationship

Background: Diabetes mellitus (DM) is a common metabolic disease worldwide and has many complications. The vascular events are the major complication of DM that have an important effect on mortality and disability. The physical activity (PA) enhances the vascular function by several pathways. The aim of this study was to evaluation of the relationship between PA and vascular diseases in patients with DM.Materials and Methods: This research was performed as the case-control study that was extracted from a prospective epidemiological research study in Iran (PERSIAN). The patients with type 2 DM more than six months defined as case group and the non-DM subjects in control group with ratio 1:2, and both groups were matched in the term of age and sex. The MET score was used to evaluate the level of PA and blood glucose, lipid profile, body mass index, overweight, dyslipidemia, glomerular filtration rate, myocardial infarction (MI), unstable angina, and stroke.Results: Overall, 1242 patients with DM were extracted, and 2484 non-diabetic subjects were investigated. In the case group, 355(28.6 %) and 887(71.4%) were men and women, respectively, the and 710 (28.6%) men and 1774(71.4%) women in control group. The mean MET score was 30 and 40.97 in the DM and non-DM groups, respectively (P˂0.001). The frequency of MI, stroke, and cardiac ischemia were 44 (3.5%), 37 (3%), and 267 (21.5%), respectively in DM group, and 54 (2.2%), 43 (1.7%), and 389 (15.7%), respectively in non-DM group.Conclusion: The incidence of vascular events associated with PA level in patients with DM and adherence to regular PA reduce the vascular events and DM complications. [GMJ.2019;inpress:e1549]

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Abstract 971: Inhibition Of Vascular Bk Channel Activity By Caveolae-mediated Angiotensin II Type I Receptor Signaling In Diabetic Vessels

Circulation ◽

10.1161/circ.116.suppl_16.ii_192-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Tong Lu ◽

Xiaoli Wang ◽

Hon-Chi Lee

Keyword(s):

Angiotensin Ii ◽

Tyrosine Kinases ◽

Buoyant Density ◽

Bk Channels ◽

Bk Channel ◽

Hek293 Cells ◽

Type I ◽

Ang Ii ◽

Channel Activity ◽

Channel Inhibition

Angiotensin II (Ang II) type I receptor (ATR 1 ) trafficking into caveolae is essential for Ang II signaling, which is known to be abnormal in diabetic vessels. We have shown that the large conductance Ca 2+ actviated K + (BK) channels are also targeted to caveolae in vascular cells. The potential interaction between Ang II signaling and BK channel function in normal and diabetic vessels is unknown. Using whole-cell patch clamp recordings and molecular biology techniques, we examined the mechanisms through which caveolae targeting facilitates the regulation of BK channels by Ang II signaling. We found that in cultured human coronary arterial smooth muscle cells (CASMC) and in freshly isolated rat CASMC, BK channels, ATR 1 , and Src-family protein tyrosine kinases (Src-PTK) were colocalized and enriched in the low buoyant density, caveolae-rich fractions. 2 μM Ang II inhibited BK channel activity by ∼50% in rat and human CASMC and these effects were completely abolished by 2 μM Losartan (a selective ATR 1 inhibitor), 10 μM PP2 (a selective Src-PTK inhibitor), and by caveolin-1 (cav-1) knockdown using 60 nM siRNA. Similar results were obtained in HEK293 cells coexpressing hSlo, BK-β 1 subunit, ATR 1 , cav-1, and Src-PTK, indicating that inhibition of BK channels by Ang II was mediated through ATR 1 activation of Src-PTK and the integrity of caveolae is critical for Ang II signaling. Culturing human CASMC with high glucose (HG, 22 mM) enhanced Ang II-mediated BK channel inhibition (78.8±16.8% vs. 54.5±15.7% in 5 mM glucose, n=3, p<0.05). Analysis of ATR 1 , Src-PTK, and BK channel distribution by sucrose gradient fractionation and by co-immunoprecipitation with anti-cav-1 antibodies showed that expression of ATR 1 and Src-PTK were up-regulated in human CASMC cultured in HG and in CASMC from streptozotocin-induced diabetic rats. Total BK channel protein in these cells was diminished, but the amount of BK channels co-immunoprecipitated with anti-cav-1 antibody was increased, suggesting increased caveolae targeting of BK channels in diabetes, which leads to enhanced Ang II-mediated BK channel inhibition. These results indicate that Ang II-BK channel interaction is critically dependent upon caveolae targeting under normal conditions and in disease states such as diabetes.

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An unexpected journey: conceptual evolution of mechanoregulated potassium transport in the distal nephron

AJP Cell Physiology ◽

10.1152/ajpcell.00328.2015 ◽

2016 ◽

Vol 310 (4) ◽

pp. C243-C259 ◽

Cited By ~ 23

Author(s):

Rolando Carrisoza-Gaytan ◽

Marcelo D. Carattino ◽

Thomas R. Kleyman ◽

Lisa M. Satlin

Keyword(s):

Flow Rate ◽

Fluid Shear Stress ◽

Collecting Duct ◽

Critical Role ◽

Bk Channels ◽

Bk Channel ◽

Macromolecular Complex ◽

Urinary Flow ◽

Cortical Collecting Duct ◽

Distal Nephron

Flow-induced K secretion (FIKS) in the aldosterone-sensitive distal nephron (ASDN) is mediated by large-conductance, Ca2+/stretch-activated BK channels composed of pore-forming α-subunits (BKα) and accessory β-subunits. This channel also plays a critical role in the renal adaptation to dietary K loading. Within the ASDN, the cortical collecting duct (CCD) is a major site for the final renal regulation of K homeostasis. Principal cells in the ASDN possess a single apical cilium whereas the surfaces of adjacent intercalated cells, devoid of cilia, are decorated with abundant microvilli and microplicae. Increases in tubular (urinary) flow rate, induced by volume expansion, diuretics, or a high K diet, subject CCD cells to hydrodynamic forces (fluid shear stress, circumferential stretch, and drag/torque on apical cilia and presumably microvilli/microplicae) that are transduced into increases in principal (PC) and intercalated (IC) cell cytoplasmic Ca2+ concentration that activate apical voltage-, stretch- and Ca2+-activated BK channels, which mediate FIKS. This review summarizes studies by ourselves and others that have led to the evolving picture that the BK channel is localized in a macromolecular complex at the apical membrane, composed of mechanosensitive apical Ca2+ channels and a variety of kinases/phosphatases as well as other signaling molecules anchored to the cytoskeleton, and that an increase in tubular fluid flow rate leads to IC- and PC-specific responses determined, in large part, by the cell-specific composition of the BK channels.

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β4-Subunit increases Slo responsiveness to physiological Ca2+ concentrations and together with β1 reduces surface expression of Slo in hair cells

AJP Cell Physiology ◽

10.1152/ajpcell.00449.2010 ◽

2011 ◽

Vol 300 (3) ◽

pp. C435-C446 ◽

Cited By ~ 19

Author(s):

Jun-Ping Bai ◽

Alexei Surguchev ◽

Dhasakumar Navaratnam

Keyword(s):

Hair Cells ◽

Critical Role ◽

Relaxation Times ◽

Low Frequency ◽

Surface Expression ◽

Bk Channels ◽

Bk Channel ◽

Operating Voltage ◽

Α Subunit ◽

Voltage Range

Changing kinetics of large-conductance potassium (BK) channels in hair cells of nonmammalian vertebrates, including the chick, plays a critical role in electrical tuning, a mechanism used by these cells to discriminate between different frequencies of sound. BK currents are less abundant in low-frequency hair cells and show large openings in response to a rise in intracellular Ca2+ at a hair cell's operating voltage range (spanning −40 to −60 mV). Although the molecular underpinnings of its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Currents from the α (Slo)-subunit alone do not show dramatic increases in response to changes in Ca2+ concentrations at −50 mV. We have cloned the chick β4- and β1-subunits and show that these subunits are preferentially expressed in low-frequency hair cells, where they decrease Slo surface expression. The β4-subunit in particular is responsible for the BK channel's increased responsiveness to Ca2+ at a hair cell's operating voltage. In contrast, however, the increases in relaxation times induced by both β-subunits suggest additional mechanisms responsible for BK channel function in hair cells.

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

Physiological Genomics ◽

10.1152/physiolgenomics.00110.2003 ◽

2003 ◽

Vol 15 (3) ◽

pp. 191-198 ◽

Cited By ~ 25

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.

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Coronary Large Conductance Ca2+-Activated K+ Channel Dysfunction in Diabetes Mellitus

Frontiers in Physiology ◽

10.3389/fphys.2021.750618 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tong Lu ◽

Hon-Chi Lee

Keyword(s):

Diabetes Mellitus ◽

Vascular Tone ◽

Microvascular Complications ◽

Bk Channels ◽

Bk Channel ◽

K Channel ◽

Organ Perfusion ◽

Biophysical Properties ◽

Diabetic Vasculopathy ◽

Channel Expression

Diabetes mellitus (DM) is an independent risk of macrovascular and microvascular complications, while cardiovascular diseases remain a leading cause of death in both men and women with diabetes. Large conductance Ca2+-activated K+ (BK) channels are abundantly expressed in arteries and are the key ionic determinant of vascular tone and organ perfusion. It is well established that the downregulation of vascular BK channel function with reduced BK channel protein expression and altered intrinsic BK channel biophysical properties is associated with diabetic vasculopathy. Recent efforts also showed that diabetes-associated changes in signaling pathways and transcriptional factors contribute to the downregulation of BK channel expression. This manuscript will review our current understandings on the molecular, physiological, and biophysical mechanisms that underlie coronary BK channelopathy in diabetes mellitus.

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Bidirectional control of BK channel open probability by CAMKII and PKC in medial vestibular nucleus neurons

Journal of Neurophysiology ◽

10.1152/jn.00058.2011 ◽

2011 ◽

Vol 105 (4) ◽

pp. 1651-1659 ◽

Cited By ~ 35

Author(s):

Ingrid van Welie ◽

Sascha du Lac

Keyword(s):

Single Channel ◽

Vestibular Nucleus ◽

Critical Role ◽

Bk Channels ◽

Bk Channel ◽

Medial Vestibular Nucleus ◽

Intrinsic Excitability ◽

Action Potential Firing ◽

Open Probability ◽

Single Channel Currents

Large conductance K+ (BK) channels are a key determinant of neuronal excitability. Medial vestibular nucleus (MVN) neurons regulate eye movements to ensure image stabilization during head movement, and changes in their intrinsic excitability may play a critical role in plasticity of the vestibulo-ocular reflex. Plasticity of intrinsic excitability in MVN neurons is mediated by kinases, and BK channels influence excitability, but whether endogenous BK channels are directly modulated by kinases is unknown. Double somatic patch-clamp recordings from MVN neurons revealed large conductance potassium channel openings during spontaneous action potential firing. These channels displayed Ca2+ and voltage dependence in excised patches, identifying them as BK channels. Recording isolated single channel currents at physiological temperature revealed a novel kinase-mediated bidirectional control in the range of voltages over which BK channels are activated. Application of activated Ca2+/calmodulin-dependent kinase II (CAMKII) increased BK channel open probability by shifting the voltage activation range towards more hyperpolarized potentials. An opposite shift in BK channel open probability was revealed by inhibition of phosphatases and was occluded by blockade of protein kinase C (PKC), suggesting that active PKC associated with BK channel complexes in patches was responsible for this effect. Accordingly, direct activation of endogenous PKC by PMA induced a decrease in BK open probability. BK channel activity affects excitability in MVN neurons and bidirectional control of BK channels by CAMKII, and PKC suggests that cellular signaling cascades engaged during plasticity may dynamically control excitability by regulating BK channel open probability.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00073-10 ◽

2010 ◽

Vol 30 (14) ◽

pp. 3646-3660 ◽

Cited By ~ 29

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.

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Glu496Ala polymorphism of human P2X7receptor does not affect its electrophysiological phenotype

AJP Cell Physiology ◽

10.1152/ajpcell.00042.2002 ◽

2003 ◽

Vol 284 (3) ◽

pp. C749-C756 ◽

Cited By ~ 53

Author(s):

Wolfgang Boldt ◽

Manuela Klapperstück ◽

Cora Büttner ◽

Sven Sadtler ◽

Günther Schmalzing ◽

...

Keyword(s):

Amino Acid ◽

Critical Role ◽

Amino Acid Position ◽

Functional Expression ◽

Hek293 Cells ◽

Kinetic Properties ◽

Loss Of Function ◽

Ion Channel Properties ◽

Gated Channel ◽

Channel Properties

A glutamate to alanine exchange at amino acid position 496 of the human P2X7 receptor was recently shown to be associated with a loss of function in human B lymphocytes in terms of ATP-induced ethidium+ uptake, Ba2+ influx, and induction of apoptosis (Gu BJ, Zhang WY, Worthington RA, Sluyter R, Dao-Ung P, Petrou S, Barden JA, and Wiley JS. J Biol Chem 276: 11135–11142, 2001). Here we analyzed the effect of the Glu496 to Ala exchange on the channel properties of the human P2X7 receptor expressed in Xenopus oocytes with the two-microelectrode voltage-clamp technique. The amplitudes of ATP-induced whole cell currents characteristic of functional expression, kinetic properties including ATP concentration dependence, and permeation behavior were not altered by this amino acid exchange. Also in HEK293 cells, the Ala496 mutant mediated typical P2X7 receptor-dependent currents like the parent Glu496 hP2X7 receptor. Because the function of the P2X7 receptor as an ATP-gated channel for small cations including Ba2+ remained unaffected by this mutation, we conclude that Glu496 plays a critical role in pore formation but does not determine the ion channel properties of the human P2X7 receptor.

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Direct Regulation of BK Channels by Phosphatidylinositol 4,5-Bisphosphate as a Novel Signaling Pathway

The Journal of General Physiology ◽

10.1085/jgp.200709913 ◽

2008 ◽

Vol 132 (1) ◽

pp. 13-28 ◽

Cited By ~ 66

Author(s):

Thirumalini Vaithianathan ◽

Anna Bukiya ◽

Jianxi Liu ◽

Penchong Liu ◽

Maria Asuncion-Chin ◽

...

Keyword(s):

Membrane Surface ◽

Acyl Chain ◽

Bk Channels ◽

Bk Channel ◽

Water Soluble ◽

Hek293 Cells ◽

Channel Activity ◽

Smooth Muscle Contractility ◽

Acyl Chain Length ◽

Vascular Myocytes

Large conductance, calcium- and voltage-gated potassium (BK) channels are ubiquitous and critical for neuronal function, immunity, and smooth muscle contractility. BK channels are thought to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) only through phospholipase C (PLC)–generated PIP2 metabolites that target Ca2+ stores and protein kinase C and, eventually, the BK channel. Here, we report that PIP2 activates BK channels independently of PIP2 metabolites. PIP2 enhances Ca2+-driven gating and alters both open and closed channel distributions without affecting voltage gating and unitary conductance. Recovery from activation was strongly dependent on PIP2 acyl chain length, with channels exposed to water-soluble diC4 and diC8 showing much faster recovery than those exposed to PIP2 (diC16). The PIP2–channel interaction requires negative charge and the inositol moiety in the phospholipid headgroup, and the sequence RKK in the S6–S7 cytosolic linker of the BK channel-forming (cbv1) subunit. PIP2-induced activation is drastically potentiated by accessory β1 (but not β4) channel subunits. Moreover, PIP2 robustly activates BK channels in vascular myocytes, where β1 subunits are abundantly expressed, but not in skeletal myocytes, where these subunits are barely detectable. These data demonstrate that the final PIP2 effect is determined by channel accessory subunits, and such mechanism is subunit specific. In HEK293 cells, cotransfection of cbv1+β1 and PI4-kinaseIIα robustly activates BK channels, suggesting a role for endogenous PIP2 in modulating channel activity. Indeed, in membrane patches excised from vascular myocytes, BK channel activity runs down and Mg-ATP recovers it, this recovery being abolished by PIP2 antibodies applied to the cytosolic membrane surface. Moreover, in intact arterial myocytes under physiological conditions, PLC inhibition on top of blockade of downstream signaling leads to drastic BK channel activation. Finally, pharmacological treatment that raises PIP2 levels and activates BK channels dilates de-endothelized arteries that regulate cerebral blood flow. These data indicate that endogenous PIP2 directly activates vascular myocyte BK channels to control vascular tone.

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