Gap junction regulation by calmodulin

Zou, Juan; Salarian, Mani; Chen, Yanyi; Veenstra, Richard D.; Louis, Charles F.; Yang, Jenny J.

doi:10.1016/j.febslet.2014.01.003

Cited by 53 publications

(66 citation statements)

References 92 publications

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“…Indeed, our ITC data also shows that this interaction is primarily driven by an enthalpic change, meaning an electrostatic change. Previous papers suggest the electrostatic interactions between Cx44 (ovine ortholog of Cx46) and CaM (16,27). The ITC experimental data demonstrated that the stoichiometry between Cx46MT and CaM is one to one, which is consistent with predicated interaction between Cx44 and CaM.…”

Section: Discussionsupporting

confidence: 68%

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Cataract-associated connexin 46 mutation alters its interaction with calmodulin and function of hemichannels

Riquelme²,

Wang

et al. 2018

Journal of Biological Chemistry

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Connexin channels help maintain eye lens homeostasis and transparency. The G143R missense substitution in connexin (Cx) 46 is associated with congenital Coppock cataracts; however, the underlying molecular mechanism is largely unknown. Here, we report that compared with wild type (WT) Cx46, the G143R substitution abolishes hemichannel conductance in Xenopus oocytes and in HeLa cells. Moreover, this substitution is dominant-negative and inhibits conductance of WT Cx46. CD analysis indicated that the substitution greatly reduces the α-helical structure of the intracellular Cx46 loop domain. Protein pulldown assays and isothermal titration calorimetry (ITC) revealed that this Cx46 domain directly interacts with calmodulin (CaM) in a Ca 2+ -dependent fashion, an observation confirmed by immunofluorescent co-localization of Cx46 with CaM. Interestingly, the G143R substitution enhanced the Cx46-CaM interaction and attenuated its abolishment by Ca 2+ depletion. Moreover, Cx46 increased dye influx, and the G143R substitution augmented this effect. Inhibition of Ca 2+ -mediated CaM activation blocked hemichannel permeability. The membrane potential plays a crucial role in Cx46 membrane permeability. We found that the activity of hemichannels is detectable under rest and hyperpolarization conditions, but is eliminated with depolarization. These results suggested that the G143R substitution impairs voltage-dependent electrical conductance and alters membrane permeability mediated by Cx46 hemichannels. The latter likely is caused by the substitution-induced structural changes of the intracellular loop domain associated with the increased interaction with CaM and reduced Ca 2+ sensitivity. The data suggest that the G143R-induced enhancement of the CaMCx46 interaction results in altered hemichannel activities and might be related to cataract formation.The lens is a clear part of the anterior eye that focuses light and images onto the retina. Cataracts, referring to the clouding of the lens, are responsible for 51% of world blindness according to WHO data in 2010 (1). The lens has three main parts; the capsule, the epithelial layer in the anterior portion, and fiber cells which comprise the bulk of lens (2). Lens fiber cells communicate to each other and to the surface of the lens by gap junction channels and hemichannels. These allow small molecules (< 1.2 kDa) like ions, second messenger and metabolites to pass through adjacent cells (3-5). Lens is an avascular organ and thus relies in part upon gap junction channels and hemichannels to maintain its metabolic homeostasis and transparency. Hemichannels, also called connexins, are undocked http://www.jbc.org/cgi

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Section: Discussionsupporting

confidence: 68%

“…It has been shown that α-helicity of the IL domain of connexins contributes to the binding of CaM and helps stabilize protein conformation (27). Surprisingly, α-helical content was greatly reduced in the IL domain containing G143R mutation despite tighter binding to CaM.…”

Section: Discussionmentioning

confidence: 99%

Cataract-associated connexin 46 mutation alters its interaction with calmodulin and function of hemichannels

Riquelme²,

Wang

et al. 2018

Journal of Biological Chemistry

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“…Conversely, Cx50 exhibited a significant decrease in hydrodynamic radius of 5.3 Å that is consistent with a collapsed complex formation [49]. This result further suggested that binding of Cx45p 164–186 to CaM involves an extended conformation that is different from α-connexin Cx50 with an embraced compact form [34,49]. …”

Section: Resultsmentioning

confidence: 89%

“…Various past studies have proposed that CaM may play an active role in the regulation of gap junctions comprised of all three connexin subfamilies [34–37]. Evidence of CaM’s role in regulation of Cx45 was first reported by Peracchia et al by monitoring the sensitivity of Cx45 channels to CO 2 and inhibiting CaM expression in oocytes [38].…”

Section: Introductionmentioning

confidence: 99%

Direct visualization of interaction between calmodulin and connexin45

Zou

Salarian

Chen

et al. 2017

Biochemical Journal

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Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of γ-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer. The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM-binding site at the cytosolic loop (residues 164–186) of Cx45 using a peptide model. The strong binding (Kd ~ 5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+-dependent. Furthermore, high-resolution nuclear magnetic resonance spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N-labeled CaM, but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differ from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+ sensitivity of CaM, especially at the N-terminal domain. Results of the present study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM’s modulation of connexins.

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“…2A and B). In addition, the CaM binding sites of several intracellular domains of connexins blockade gapjunction channels (Zou et al, 2014b). Overall, the interaction between GJIC and calcium pathways has an important role in Cd-induced cytotoxicity.…”

Section: Discussionmentioning

confidence: 99%

Alpha-lipoic acid protects against cadmium-induced hepatotoxicity via calcium signalling and gap junctional intercellular communication in rat hepatocytes

Zou

Han

et al. 2015

J. Toxicol. Sci.

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-This study investigated the protective effect of alpha-lipoic acid (LA) on cadmium (Cd)-induced hepatotoxicity in BRL 3A rat liver cells. We demonstrated that LA ameliorated Cd-induced cellular injury in cell viability and nuclear fragmentation in BRL 3A cells. Furthermore, LA markedly ameliorated Cd-induced gap junctional intercellular communication (GJIC) inhibition and Cx43 mRNA expression decrease, as well as disassembly of gap junctions. The gap junction blocker carbenoxolone disodium (CBX) as well as LA protected healthy cells from Cd-exposed cells in Transwell co-culture system. LA also protected BRL 3A cells against Cd-induced elevation of the intracellular concentration of free calcium ([Ca 2+ ] i ). Pretreatment with a chelater of intracellular Ca 2+ BAPTA-AM or chelater of extracellular Ca 2+ EGTA attenuated Cd-induced cytotoxicity and GJIC inhibition. CBX exacerbated the decrease in cell viability and further elevated the increase in [Ca 2+ ] i induced by Cd, whereas BAPTA-AM partly attenuated these phenomena, while EGTA had little effects. These results suggested that Cd-induced hepatotoxicity via GJIC inhibition and [Ca 2+ ] i elevation, which originates mainly from intracellular stores. GJIC inhibition has dual effects: (i) it restricts release of Ca 2+ from the cell, which exacerbates the [Ca 2+ ] i elevation and cytotoxicity induced by Cd; and (ii) it protects healthy cells from their dangerous neighbors by blocking intercellular communication. Above all, our results indicated that LA partly prevented Cd-induced cytotoxicity via GJIC and calcium signaling in BRL 3A rat liver cells.

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Gap junction regulation by calmodulin

Cited by 53 publications

References 92 publications

Cataract-associated connexin 46 mutation alters its interaction with calmodulin and function of hemichannels

Cataract-associated connexin 46 mutation alters its interaction with calmodulin and function of hemichannels

Direct visualization of interaction between calmodulin and connexin45

Alpha-lipoic acid protects against cadmium-induced hepatotoxicity via calcium signalling and gap junctional intercellular communication in rat hepatocytes

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