Calmodulin Binds to Chick Lens Gap Junction Protein in a Calcium-Independent Manner

Welsh, Michael J.; Aster, Jon C.; Ireland, Mark E.; Alcala, Jose; Maisel, H.

doi:10.1126/science.6280283

Cited by 92 publications

(39 citation statements)

References 25 publications

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“…Since calmodulin (CaM) binding to gap junctions was discovered [6][7][8][9], a vast amount of studies have indicated that CaM is, indeed, involved in the calciumdependent regulation of gap junction function. CaM has even been shown to be able to pass through a gap junction, possibly due to its elongated shape [10,11].…”

Section: Introductionmentioning

confidence: 99%

Complex formation between calmodulin and a peptide from the intracellular loop of the gap junction protein connexin43: Molecular conformation and energetics of binding

Myllykoski

Kuczera

Kursula

2009

Biophysical Chemistry

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. Complex formation between calmodulin and a peptide from the intracellular loop of the gap junction protein connexin43: Molecular conformation and energetics of binding. Biophysical Chemistry, Elsevier, 2009, 144 (3) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT AbstractGap junctions are formed by a family of transmembrane proteins, connexins.Connexin43 is a widely studied member of the family, being ubiquitously expressed in a variety of tissues and a target of a large number of disease mutations. The intracellular loop of connexin43 has been shown to include a calmodulin binding domain, but detailed 3-dimensional data on the structure of the complex are not available. In this study, we used a synthetic peptide from this domain to reveal the conformation of the calmodulin-peptide complex by small angle X-ray scattering.Upon peptide binding, calmodulin lost its dumbbell shape, adopting a more globular conformation. We also studied the energetics of the interaction using calorimetry and computational methods. All our data indicate that calmodulin binds to the peptide from cx43 in the classical 'collapsed' conformation.

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Section: Introductionmentioning

confidence: 99%

Complex formation between calmodulin and a peptide from the intracellular loop of the gap junction protein connexin43: Molecular conformation and energetics of binding

Myllykoski

Kuczera

Kursula

2009

Biophysical Chemistry

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“…Increased cytosolic calcium concentration (Ca i 2ϩ ) in lens cells has been correlated with lens cataract development (9,20,28,45) and changes in gap junction permeability (8,10,14). Furthermore, the ubiquitous calcium-binding protein calmodulin (CaM) has been proposed to physically interact with gap junctions (18,40,41) and has been proposed to modulate fiber cell coupling in intact rat lenses (14).…”

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confidence: 99%

Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells

Lurtz

Louis

2003

American Journal of Physiology-Cell Physiology

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The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca2+ (Cai2+) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Cai2+ (to micromolar concentrations) observed after the addition of ionophore plus Ca2+ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Cai2+, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Cai2+ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Cai2+ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Cai2+.

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“…Calmodulin inhibitors such as calmidazolium or phenothiazines, e.g. chlorpromazine, inhibit electrical uncoupling in Xenopus embryos [14,15] and in epidermal cells [16]. Calmodulin antagonists and antibodies inhibit cell proliferation [17], an effect which may be related to efficacy of gap-junction-mediated communication, since there is increasing evidence that there is a direct correlation between the functional expression of connexins and growth control in cells transfected with connexin genes [18].…”

Section: Introductionmentioning

confidence: 99%

Connexin 32 of gap junctions contains two cytoplasmic calmodulin-binding domains

Török

Stauffer

Evans

1997

Biochemical Journal

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A fluorescent calmodulin derivative, 2-chloro-[4-(ϵ-amino-Lys75)]-[6-(4-diethylaminophenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-calmodulin) [Török and Trentham (1994) Biochemistry 33, 12807–12820], and equilibrium fluorescence methods were used to identify calmodulin-binding domains of connexin subunits of gap junctions. Synthetic peptides corresponding to six extramembrane regions of connexin 32, a major component of rat liver gap junctions, and peptides derived from connexin 43 and 26, were tested. Two cytoplasmically oriented peptides that correspond to an N-terminal 21-amino-acid sequence and a 15-amino-acid sequence at the C-terminal tail of connexin 32 bound TA-calmodulin in a Ca2+-dependent manner. The dissociation constants (Kd) of TA-calmodulin binding to GAP 10 (MNWTGLYTLLSGVNRHSTAIG, residues 1–21) and GAP 8M (ACARRAQRRSNPPSR, residues 216–230) were 27 nM and 1.2 μM respectively at 150 mM ionic strength, 2 mM MgCl2, 100 μM CaCl2, pH 7.0 and 21 °C. Both halves of each peptide were required for calmodulin binding. Substitution of Trp3 present in all connexins by Tyr increasedKd for TA-calmodulin by 40-fold. Liver gap junctions (whose connexons contain mainly connexin 32) and recombinant connexons constructed of connexin 26 expressed by baculovirus-infected insect cells exhibited weaker binding of TA-calmodulin with variable Ca2+-dependence. These studies identify two calmodulin-binding amino-acid sequences in connexin 32, and provide independent evidence that calmodulin may function as an intracellular ligand, regulating Ca2+-dependent intercellular communication across gap junctions.

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Calmodulin Binds to Chick Lens Gap Junction Protein in a Calcium-Independent Manner

Cited by 92 publications

References 25 publications

Complex formation between calmodulin and a peptide from the intracellular loop of the gap junction protein connexin43: Molecular conformation and energetics of binding

Complex formation between calmodulin and a peptide from the intracellular loop of the gap junction protein connexin43: Molecular conformation and energetics of binding

Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells

Connexin 32 of gap junctions contains two cytoplasmic calmodulin-binding domains

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