Calcium–calmodulin gating of a pH-insensitive isoform of connexin43 gap junctions

2019 ◽  
Vol 476 (7) ◽  
pp. 1137-1148 ◽  
Author(s):  
Siyu Wei ◽  
Christian Cassara ◽  
Xianming Lin ◽  
Richard D. Veenstra
Keyword(s):  
Gap Junctions ◽  
Ventricular Myocytes ◽  
Protein A ◽  
Ph Sensitivity ◽  
Fast Inactivation ◽  
Inhibitory Peptide ◽  
Pipette Solution ◽  
Calmodulin Binding ◽  
Gap Junction Communication ◽  
Gating Mechanism

Abstract Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction communication and the synergism or antagonism between pH and Ca2+ has been questioned for decades. To assess the ability of Ca2+ ions to modulate Cx43 junctional conductance (gj) in the absence of pH-sensitivity, patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full-length Cx43 or the Cx43-M257 (Cx43K258stop) mutant protein, a carboxyl-terminus (CT) truncated version of Cx43 lacking pH-sensitivity. The addition of 1 μM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 gj to zero within 15 min of perfusion. This response was prevented by Ca2+-free saline or addition of 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. Internal addition of a connexin50 cytoplasmic loop calmodulin-binding domain (CaMBD) mimetic peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 μM Gap19 peptide had minimal effect. The investigation of the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 gj, but also noted the abolishment of the previously reported facilitated recovery of gj from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact in its absence. Sequence-specific connexin CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has negligible effect on this chemical gating mechanism.

scholarly journals Calcium-Calmodulin Gating of Connexin43 Gap Junctions in the Absence of the pH Gating Domain

2018 ◽  
Author(s):  
Siyu Wei ◽  
Christian Cassara ◽  
Xianming Lin ◽  
Richard D Veenstra
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ventricular Myocytes ◽  
External Solution ◽  
Inhibitory Peptide ◽  
Pipette Solution ◽  
Calmodulin Binding ◽  
Whole Cell Patch Clamp ◽  
Gating Mechanism ◽  
Mimetic Peptides

AbstractIntracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction channels and the synergism or antagonism between pH and Ca2+ has been questioned for decades. In this study, we assessed whether the calcium gating mechanism occurs independently of the pH gating mechanism by utilizing the Cx43-M257 (Cx43K258stop) mutant, a carboxyl-terminal (CT) truncated version of Cx43 lacking the pH gating domain. Dual whole cell patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full length Cx43 or Cx43-M257 proteins. Addition of 1 μM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 macroscopic gap junction conductance (gj) to zero within 15 min of perfusion, while this response was prevented by omitting 1.8 mM CaCl2 from the external solution or adding 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. The ability of connexin calmodulin binding domain (Cx CaMBD) mimetic peptides and the Gap19 peptide to inhibit the Ca2+/CaM gating response of Cx43 gap junctions was also examined. Internal addition of a Cx50 cytoplasmic loop CaMBD peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 μM Gap19 peptide had no effect. Lastly, the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions were investigated. We confirmed that the fast kinetic inactivation component was absent in Cx43-M257 gap junctions, but also observed that the previously reported facilitated recovery of gj from inactivating potentials was abolished by CT truncation of Cx43. We conclude that CT pH gating domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact. Sequence-specific Cx CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has no effect on this chemical gating mechanism.

scholarly journals Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain

2012 ◽  
Vol 302 (10) ◽  
pp. C1548-C1556 ◽  
Author(s):  
Qin Xu ◽  
Richard F. Kopp ◽  
Yanyi Chen ◽  
Jenny J. Yang ◽  
Michael W. Roe ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Binding Domain ◽  
Cytoplasmic Loop ◽  
Inhibitory Peptide ◽  
Open Probability ◽  
Calmodulin Binding ◽  
Gap Junction Channel ◽  
Whole Cell Patch Clamp

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca2+-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca2+ influx reduced Cx43 gap junction conductance (Gj) by 95%, while increasing cytosolic Ca2+ concentration threefold. By contrast, Cx40 Gj declined by <20%. The Ca2+-induced decline in Cx43 Gj was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca2+-free extracellular solution, if Ca2+ chelation was commenced before complete uncoupling, after which gj was only 60% recoverable. The Cx43 CL136–158 mimetic peptide, but not the scrambled control peptide, or Ca2+/CaM-dependent kinase II 290–309 inhibitory peptide also prevented the Ca2+/CaM-dependent decline of Cx43 Gj. Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca2+/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca2+/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca2+ regulatory properties of Cx43 and Cx40.

Abstract 1379: Angiotensin II Inhibits the Na+-K+ pump via PKC-dependent Activation of NAD(P)H Oxidase

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Gemma A Figtree ◽  
Caroline N White ◽  
Chia Chi Liu ◽  
Alvaro Garcia ◽  
Elisha J Hamilton ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Myocyte ◽  
Ventricular Myocytes ◽  
Pump Current ◽  
Oxidase Inhibitor ◽  
Ang Ii ◽  
Inhibitory Peptide ◽  
Pipette Solution ◽  
Myocardial Oxidative Stress ◽  
Myocyte Function

Background: The sarcolemmal Na + -K + pump, essentially the only export route for Na + and pivotal for cardiac myocyte function, is inhibited by angiotensin II (Ang II). Since Ang II activates NAD(P)H oxidase we tested the hypothesis that NAD(P)H oxidase mediates Ang II-induced pump inhibition. This is important for our understanding of pathophysiology and treatment of heart failure, a condition with increases in circulating Ang II levels; intracellular myocyte Na + ; NAD(PH oxidase activity; and myocardial oxidative stress. The raised myocyte Na + is believed to contribute to the electro-mechanical phenotype of contractile abnormalities and cardiac arrhythmias. Methods and results: Exposure to 100 nmol/L Ang II increased fluorescence of isolated rabbit ventricular myocytes loaded with a superoxide-sensitive dye. The increase was abolished by pegylated superoxide dismutase (SOD), by the NAD(P)H oxidase inhibitor apocynin and by myristolated inhibitory peptide to ϵ -protein kinase C ( ϵ PKC), previously implicated in Ang II-induced Na + -K + pump inhibition. Exposure of voltage-clamped myocytes to Ang II decreased electrogenic Na + -K + pump current, I p , from 0.48 ± 0.02 pA/pF, N = 11 to 0.36 ± 0.03 pA/pF, N = 11 (P < 0.05). The decrease was abolished (P < 0.05) by inclusion in pipette solution of: SOD, the gp91ds inhibitory peptide that blocks assembly and activation of NAD(P)H oxidase and by ϵ PKC inhibitory peptide. Since co-localization should facilitate NAD(P)H-oxidase dependent regulation of the Na + -K + pump we examined if there is physical association between the pump and NAD(P)H oxidase. Cell lysate was immunoprecipitated with antibody to the α 1 pump subunit. Immunoblotting demonstrated co-immunoprecipitation of the α 1 subunit with caveolin 3 and with membrane-associated p22 phox and cytosolic p47 phox NAD(P)H oxidase subunits at baseline. Ang II had no effect on α 1 /caveolin3 or α 1 /p22 phox interaction, but increased α 1 /p47 phox co-immunoprecipitation. Conclusion: Ang II is known to induce PKC-dependent phosphorylation and translocation of p47 phox resulting in activation of NAD(P)H oxidase, and we propose this activation occurs in a caveolar microdomain and that it mediates the inhibition of the Na + -K + pump by Ang II.

scholarly journals C Terminus L‐type Ca 2+ Channel Calmodulin‐Binding Domains are ‘Auto‐Agonist’ Ligands in Rabbit Ventricular Myocytes

2003 ◽  
Vol 550 (3) ◽  
pp. 731-738 ◽  
Author(s):  
Igor Dzhura ◽  
Yuejin Wu ◽  
Rong Zhang ◽  
Roger J. Colbran ◽  
Susan L. Hamilton ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Calmodulin Binding ◽  
Binding Domains ◽  
C Terminus ◽  
Rabbit Ventricular Myocytes ◽  
Agonist Ligands

Protein kinase Cε and the antiadrenergic action of adenosine in rat ventricular myocytes

2004 ◽  
Vol 287 (4) ◽  
pp. H1721-H1729 ◽  
Author(s):  
Koji Miyazaki ◽  
Satoshi Komatsu ◽  
Mitsuo Ikebe ◽  
Richard A. Fenton ◽  
James G. Dobson
Keyword(s):  
Electrical Stimulation ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Adrenergic Agonist ◽  
Inhibitory Peptide ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Tubular System ◽  
Stimulation Of

Adenosine-induced antiadrenergic effects in the heart are mediated by adenosine A1 receptors (A1R). The role of PKCε in the antiadrenergic action of adenosine was explored with adult rat ventricular myocytes in which PKCε was overexpressed. Myocytes were transfected with a pEGFP-N1 vector in the presence or absence of a PKCε construct and compared with normal myocytes. The extent of myocyte shortening elicited by electrical stimulation of quiescent normal and transfected myocytes was recorded with video imaging. PKCε was found localized primarily in transverse tubules. The A1R agonist chlorocyclopentyladenosine (CCPA) at 1 μM rendered an enhanced localization of PKCε in the t-tubular system. The β-adrenergic agonist isoproterenol (Iso; 0.4 μM) elicited a 29–36% increase in myocyte shortening in all three groups. Although CCPA significantly reduced the Iso-produced increase in shortening in all three groups, the reduction caused by CCPA was greatest with PKCε overexpression. The CCPA reduction of the Iso-elicited shortening was eliminated in the presence of a PKCε inhibitory peptide. These results suggest that the translocation of PKCε to the t-tubular system plays an important role in A1R-mediated antiadrenergic actions in the heart.

Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium

2000 ◽  
Vol 278 (5) ◽  
pp. H1662-H1670 ◽  
Author(s):  
Jeffrey E. Saffitz ◽  
Karen G. Green ◽  
William J. Kraft ◽  
Kenneth B. Schechtman ◽  
Kathryn A. Yamada
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ventricular Myocytes ◽  
Intercellular Junctions ◽  
Ventricular Myocardium ◽  
Electron Microscopic ◽  
Confocal Immunofluorescence Microscopy ◽  
Normal Number ◽  
Gap Junction Channel ◽  
Junction Protein

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/− mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/− mice was due almost entirely to a reduction in the number of individual gap junctions (226 ± 52 vs. 150 ± 32 individual gap junctions/field in Cx43 +/+ and +/− ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.

pH Sensitivity of Non-Synaptic Field Bursts in the Dentate Gyrus

2000 ◽  
Vol 84 (2) ◽  
pp. 927-933 ◽  
Author(s):  
Jeffrey S. Schweitzer ◽  
Haiwei Wang ◽  
Zhi-Qi Xiong ◽  
Janet L. Stringer
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Granule Cell ◽  
Low Ph ◽  
Ph Sensitivity ◽  
Extracellular Ph ◽  
Granule Cell Layer ◽  
Ph Changes

Under conditions of low [Ca2+]o and high [K+]o, the rat dentate granule cell layer in vitro develops recurrent spontaneous prolonged field bursts that resemble an in vivo phenomenon called maximal dentate activation. To understand how pH changes in vivo might affect this phenomenon, the slices were exposed to different extracellular pH environments in vitro. The field bursts were highly sensitive to extracellular pH over the range 7.0–7.6 and were suppressed at low pH and enhanced at high pH. Granule cell resting membrane potential, action potentials, and postsynaptic potentials were not significantly altered by pH changes within the range that suppressed the bursts. The pH sensitivity of the bursts was not altered by pharmacologic blockade of N-methyl-d-aspartate (NMDA), non-NMDA, and GABAA receptors at concentrations of these agents sufficient to eliminate both spontaneous and evoked synaptic potentials. Gap junction patency is known to be sensitive to pH, and agents that block gap junctions, including octanol, oleamide, and carbenoxolone, blocked the prolonged field bursts in a manner similar to low pH. Perfusion with gap junction blockers or acidic pH suppressed field bursts but did not block spontaneous firing of single and multiple units, including burst firing. These data suggest that the pH sensitivity of seizures and epileptiform phenomena in vivo may be mediated in large part through mechanisms other than suppression of NMDA-mediated or other excitatory synaptic transmission. Alterations in electrotonic coupling via gap junctions, affecting field synchronization, may be one such process.

scholarly journals Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells.

1992 ◽  
Vol 118 (6) ◽  
pp. 1501-1510 ◽  
Author(s):  
K A Birch ◽  
J S Pober ◽  
G B Zavoico ◽  
A R Means ◽  
B M Ewenstein
Keyword(s):  
Signal Transduction ◽  
Endothelial Cells ◽  
Phorbol Esters ◽  
Umbilical Vein ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Inhibitory Peptide ◽  
Permeabilized Cells ◽  
Calmodulin Binding ◽  
Umbilical Vein Endothelial Cells

Thrombin stimulates cultured endothelial cells (EC) to secrete stored von Willebrand factor (vWF), but the signal transduction pathways are poorly defined. Thrombin is known to elevate the concentration of intracellular calcium ([Ca2+]i) and to activate protein kinase C (PKC) in EC. Since both calcium ionophores and phorbol esters release vWF, both second messenger pathways have been postulated to participate in vWF secretion in response to naturally occurring agonists. We find that in intact human EC, vWF secretion stimulated by either thrombin or by a thrombin receptor activating peptide, TR(42-55), can be correlated with agonist-induced elevations of [Ca2+]i. Further evidence implicating calcium in the signal transduction pathway is suggested by the finding that MAPTAM, a cell-permeant calcium chelator, in combination with the extracellular calcium chelator EGTA, can inhibit thrombin-stimulated secretion. In contrast, the observation that staurosporine (a pharmacological inhibitor of PKC) blocks phorbol ester- but not thrombin-stimulated secretion provides evidence against PKC-mediated signal transduction. To examine further the signal transduction pathway initiated by thrombin, we developed novel conditions for minimal permeabilization of EC with saponin (4-8 micrograms/ml for 5-15 min at 37 degrees C) which allow the introduction of small extracellular molecules without the loss of large intracellular proteins and which retain thrombin-stimulated secretion. These minimally permeabilized cells secrete vWF in response to exogenous calcium, and EGTA blocks thrombin-induced secretion. Moreover, in these cells, thrombin-stimulated secretion is blocked by a calmodulin-binding inhibitory peptide but not by a PKC inhibitory peptide. Taken together, these findings demonstrate that thrombin-stimulated vWF secretion is transduced by a rise in [Ca2+]i and provide the first evidence for the role of calmodulin in this process.

scholarly journals Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart.

1993 ◽  
Vol 101 (5) ◽  
pp. 629-650 ◽  
Author(s):  
T C Hwang ◽  
M Horie ◽  
D C Gadsby
Keyword(s):  
Steady State ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Ventricular Myocytes ◽  
Protein Phosphatases ◽  
Pipette Solution ◽  
Whole Cell ◽  
Endogenous Protein ◽  
High Level ◽  
Cl Conductance

The regulation of cardiac Cl- conductance by cAMP-dependent protein kinase (PKA) and cellular phosphatases was studied in isolated guinea pig ventricular myocytes by using wide-tipped, perfused pipettes to record whole-cell currents. Exposure to forskolin (Fsk) or isoproterenol (Iso) elicits a Cl- conductance that results exclusively from PKA-dependent phosphorylation because it can be completely abolished, or its activation fully prevented, by switching to pipette solution containing PKI, a synthetic peptide inhibitor of PKA. The Cl- conductance activated by micromolar concentrations of either agonist reached its steady-state amplitude in 1-2 min and was deactivated promptly and entirely, usually within 2 min, upon washing out the agonist, implying a continuous high level of activity of endogenous protein phosphatases. Accordingly, intracellular application of okadaic acid or microcystin, both potent inhibitors of protein phosphatases 1 and 2A, during exposure to Fsk enhanced the steady-state Cl- conductance and slowed its deactivation after washing out the Fsk. Maximal potentiation of the conductance, by approximately 60%, was obtained with pipette concentrations of approximately 10 microM okadaic acid (or approximately 5 microM microcystin) and did not result from an increase in the apparent affinity for Fsk. In the presence of maximally effective concentrations of okadaic acid and/or microcystin, deactivation of the enhanced Cl- conductance upon washout of agonist was incomplete, with about half of the conductance persisting indefinitely. That residual conductance did not reflect continued action of PKA because it was insensitive to PKI, but was identified as a fraction of the activated Cl- conductance by its biophysical characteristics. The results suggest that complete deactivation of the PKA-regulated cardiac Cl- conductance requires dephosphorylation by a type 1 and/or 2A phosphatase, but that partial deactivation can be accomplished by activity of some other phosphatase(s). These findings are consistent with sequential phosphorylation of a protein, probably the Cl- channel itself, at two different kinds of sites. The resulting phosphoproteins can be distinguished on the basis of their different contributions to whole-cell Cl- conductance.

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