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Special Issue "Chemical Regulation of Gap Junction Channels and Hemichannels"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (15 January 2021) | Viewed by 11038

Special Issue Editor

Prof. Dr. Camillo Peracchia
E-Mail Website
Guest Editor
School of Medicine and Dentistry 601 Elmwood Ave, University of Rochester Medical Center, Rochester, NY 14642, USA
Interests: gap junctions; connexins; cell communication; calmodulin; calcium; channel gating
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Neighboring cells directly exchange small cytosolic molecules via cell–cell channels clustered at gap junctions. Direct cell communication provides a fundamental mechanism for coordinating a host of cellular activities. Conversely, impaired communication causes many diseases. Each channel is formed by the interaction of two hemichannels that create a hydrophilic pathway spanning the two plasma membranes and a narrow extracellular space (gap). In turn, each hemichannel is an oligomer of six proteins (connexins/innexins). Gap junction channels are regulated by a gating mechanism sensitive to changes in cytosolic calcium (Ca2+i) and pHi. Early studies reported that only [Ca2+]i in the micromolar range affects gating concentrations. This would discard the role of Ca2+i as fine modulator of cell coupling. More recently, however, the effectiveness of calmodulin-mediated nanomolar [Ca2+]i in cell–cell channel and hemichannel gating has been widely reported, suggesting that the permeability of connexin/innexin channels and hemichannels is finely modulated. Indeed, in some cells, such as those expressing connexin45, some channels are closed even at resting [Ca2+]i. The relevance of the fine modulation of connexin/innexin channels and hemichannels to cell function in health and disease indicates that detailed knowledge of channel gating mechanisms is extremely important. This is the primary goal of this Special Issue.

Prof. emer Camillo Peracchia
Guest Editor

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Keywords

  • gap junctions
  • connexins
  • channel gating
  • calcium
  • pH
  • calmodulin
  • cell communication
  • cell–cell coupling

Published Papers (8 papers)

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Research

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Article
Urothelium-Specific Deletion of Connexin43 in the Mouse Urinary Bladder Alters Distension-Induced ATP Release and Voiding Behavior
Int. J. Mol. Sci. 2021, 22(4), 1594; https://doi.org/10.3390/ijms22041594 - 05 Feb 2021
Cited by 4 | Viewed by 925
Abstract
Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day–night variations in functional bladder capacity. However, because Cx43 is [...] Read more.
Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day–night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day–night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals’ active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day–night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals’ awake, active phase. Full article
(This article belongs to the Special Issue Chemical Regulation of Gap Junction Channels and Hemichannels)
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Article
A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators
Int. J. Mol. Sci. 2021, 22(3), 1417; https://doi.org/10.3390/ijms22031417 - 31 Jan 2021
Cited by 5 | Viewed by 1634
Abstract
Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. [...] Read more.
Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening. Full article
(This article belongs to the Special Issue Chemical Regulation of Gap Junction Channels and Hemichannels)
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Article
Gap19, a Cx43 Hemichannel Inhibitor, Acts as a Gating Modifier That Decreases Main State Opening While Increasing Substate Gating
Int. J. Mol. Sci. 2020, 21(19), 7340; https://doi.org/10.3390/ijms21197340 - 05 Oct 2020
Cited by 2 | Viewed by 1357
Abstract
Cx43 hemichannels (HCs) are electrically and chemically gated transmembrane pores with low open probability and multiple conductance states, which makes kinetic studies of channel gating in large datasets challenging. Here, we developed open access software, named HemiGUI, to analyze HC gating transitions and [...] Read more.
Cx43 hemichannels (HCs) are electrically and chemically gated transmembrane pores with low open probability and multiple conductance states, which makes kinetic studies of channel gating in large datasets challenging. Here, we developed open access software, named HemiGUI, to analyze HC gating transitions and investigated voltage-induced HC opening based on up to ≈4000 events recorded in HeLa-Cx43-overexpressing cells. We performed a detailed characterization of Cx43 HC gating profiles and specifically focused on the role of the C-terminal tail (CT) domain by recording the impact of adding an EGFP tag to the Cx43 CT end (Cx43-EGFP) or by supplying the Cx43 HC-inhibiting peptide Gap19 that interferes with CT interaction with the cytoplasmic loop (CL). We found that Gap19 not only decreased HC opening activity to the open state (≈217 pS) but also increased the propensity of subconductance (≈80 pS) transitions that additionally became slower as compared to the control. The work demonstrates that large sample transition analysis allows detailed investigations on Cx43 HC gating and shows that Gap19 acts as a HC gating modifier by interacting with the CT that forms a crucial gating element. Full article
(This article belongs to the Special Issue Chemical Regulation of Gap Junction Channels and Hemichannels)
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Article
Calmodulin Binding to Connexin 35: Specializations to Function as an Electrical Synapse
Int. J. Mol. Sci. 2020, 21(17), 6346; https://doi.org/10.3390/ijms21176346 - 01 Sep 2020
Cited by 4 | Viewed by 1482
Abstract
Calmodulin binding is a nearly universal property of gap junction proteins, imparting a calcium-dependent uncoupling behavior that can serve in an emergency to decouple a stressed cell from its neighbors. However, gap junctions that function as electrical synapses within networks of neurons routinely [...] Read more.
Calmodulin binding is a nearly universal property of gap junction proteins, imparting a calcium-dependent uncoupling behavior that can serve in an emergency to decouple a stressed cell from its neighbors. However, gap junctions that function as electrical synapses within networks of neurons routinely encounter large fluctuations in local cytoplasmic calcium concentration; frequent uncoupling would be impractical and counterproductive. We have studied the properties and functional consequences of calmodulin binding to the electrical synapse protein Connexin 35 (Cx35 or gjd2b), homologous to mammalian Connexin 36 (Cx36 or gjd2). We find that specializations in Cx35 calmodulin binding sites make it relatively impervious to moderately high levels of cytoplasmic calcium. Calmodulin binding to a site in the C-terminus causes uncoupling when calcium reaches low micromolar concentrations, a behavior prevented by mutations that eliminate calmodulin binding. However, milder stimuli promote calcium/calmodulin-dependent protein kinase II activity that potentiates coupling without interference from calmodulin binding. A second calmodulin binding site in the end of the Cx35 cytoplasmic loop, homologous to a calmodulin binding site present in many connexins, binds calmodulin with very low affinity and stoichiometry. Together, the calmodulin binding sites cause Cx35 to uncouple only at extreme levels of intracellular calcium. Full article
(This article belongs to the Special Issue Chemical Regulation of Gap Junction Channels and Hemichannels)
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Review

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Review
Regulation of Connexin Gap Junctions and Hemichannels by Calcium and Calcium Binding Protein Calmodulin
Int. J. Mol. Sci. 2020, 21(21), 8194; https://doi.org/10.3390/ijms21218194 - 02 Nov 2020
Cited by 9 | Viewed by 1500
Abstract
Connexins are the structural components of gap junctions and hemichannels that mediate the communication and exchange of small molecules between cells, and between the intracellular and extracellular environment, respectively. Connexin (Cx) 46 is predominately expressed in lens fiber cells, where they function in [...] Read more.
Connexins are the structural components of gap junctions and hemichannels that mediate the communication and exchange of small molecules between cells, and between the intracellular and extracellular environment, respectively. Connexin (Cx) 46 is predominately expressed in lens fiber cells, where they function in maintaining the homeostasis and transparency of the lens. Cx46 mutations are associated with impairment of channel function, which results in the development of congenital cataracts. Cx46 gap junctions and hemichannels are closely regulated by multiple mechanisms. Key regulators of Cx46 channel function include Ca2+ and calmodulin (CaM). Ca2+ plays an essential role in lens homeostasis, and its dysregulation causes cataracts. Ca2+ associated CaM is a well-established inhibitor of gap junction coupling. Recent studies suggest that elevated intracellular Ca2+ activates Cx hemichannels in lens fiber cells and Cx46 directly interacts with CaM. A Cx46 site mutation (Cx46-G143R), which is associated with congenital Coppock cataracts, shows an increased Cx46-CaM interaction and this interaction is insensitive to Ca2+, given that depletion of Ca2+ reduces the interaction between CaM and wild-type Cx46. Moreover, inhibition of CaM function greatly reduces the hemichannel activity in the Cx46 G143R mutant. These research findings suggest a new regulatory mechanism by which enhanced association of Cx46 with CaM leads to the increase in hemichannel activity and dysregulation may lead to cataract development. In this review, we will first discuss the involvement of Ca2+/CaM in lens homeostasis and pathology, and follow by providing a general overview of Ca2+/CaM in the regulation of Cx46 gap junctions. We discuss the most recent studies concerning the molecular mechanism of Ca2+/CaM in regulating Cx46 hemichannels. Finally, we will offer perspectives of the impacts of Ca2+/CaM and dysregulation on Cx46 channels and vice versa. Full article
(This article belongs to the Special Issue Chemical Regulation of Gap Junction Channels and Hemichannels)
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Review
Lens Connexin Channels Show Differential Permeability to Signaling Molecules
Int. J. Mol. Sci. 2020, 21(18), 6943; https://doi.org/10.3390/ijms21186943 - 22 Sep 2020
Cited by 7 | Viewed by 779
Abstract
Gap junction channels mediate the direct intercellular passage of small ions as well as larger solutes such as second messengers. A family of proteins called connexins make up the subunits of gap junction channels in chordate animals. Each individual connexin forms channels that [...] Read more.
Gap junction channels mediate the direct intercellular passage of small ions as well as larger solutes such as second messengers. A family of proteins called connexins make up the subunits of gap junction channels in chordate animals. Each individual connexin forms channels that exhibit distinct permeability to molecules that influence cellular signaling, such as calcium ions, cyclic nucleotides, or inositol phosphates. In this review, we examine the permeability of connexin channels containing Cx43, Cx46, and Cx50 to signaling molecules and attempt to relate the observed differences in permeability to possible in vivo consequences that were revealed by studies of transgenic animals where these connexin genes have been manipulated. Taken together, these data suggest that differences in the permeability of individual connexin channels to larger solutes like 3′,5′-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) could play a role in regulating epithelial cell division, differentiation, and homeostasis in organs like the ocular lens. Full article
(This article belongs to the Special Issue Chemical Regulation of Gap Junction Channels and Hemichannels)
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Review
Calmodulin-Cork Model of Gap Junction Channel Gating—One Molecule, Two Mechanisms
Int. J. Mol. Sci. 2020, 21(14), 4938; https://doi.org/10.3390/ijms21144938 - 13 Jul 2020
Cited by 8 | Viewed by 1324
Abstract
The Calmodulin-Cork gating model is based on evidence for the direct role of calmodulin (CaM) in channel gating. Indeed, chemical gating of cell-to-cell channels is sensitive to nanomolar cytosolic calcium concentrations [Ca2+]i. Calmodulin inhibitors and inhibition of CaM expression [...] Read more.
The Calmodulin-Cork gating model is based on evidence for the direct role of calmodulin (CaM) in channel gating. Indeed, chemical gating of cell-to-cell channels is sensitive to nanomolar cytosolic calcium concentrations [Ca2+]i. Calmodulin inhibitors and inhibition of CaM expression prevent chemical gating. CaMCC, a CaM mutant with higher Ca2+-sensitivity greatly increases chemical gating sensitivity (in CaMCC the NH2-terminal EF-hand pair (res. 9–76) is replaced by the COOH-terminal pair (res. 82–148). Calmodulin colocalizes with connexins. Connexins have high-affinity CaM binding sites. Several connexin mutants paired to wild-type connexins have a high gating sensitivity that is eliminated by inhibition of CaM expression. Repeated transjunctional voltage (Vj) pulses slowly and progressively close a large number of channels by the chemical/slow gate (CaM lobe). At the single-channel level, the chemical/slow gate closes and opens slowly with on-off fluctuations. The model proposes two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, gating involves Ca2+-induced CaM-activation. In the second, gating takes place without [Ca2+]i rise. The Ca-CaM-Cork gating is only reversed by a return of [Ca2+]i to resting values, while the CaM-Cork gating is reversed by Vj positive at the gated side. Full article
(This article belongs to the Special Issue Chemical Regulation of Gap Junction Channels and Hemichannels)
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Other

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Hypothesis
Connexin/Innexin Channels in Cytoplasmic Organelles. Are There Intracellular Gap Junctions? A Hypothesis!
Int. J. Mol. Sci. 2020, 21(6), 2163; https://doi.org/10.3390/ijms21062163 - 21 Mar 2020
Cited by 3 | Viewed by 1452
Abstract
This paper proposes the hypothesis that cytoplasmic organelles directly interact with each other and with gap junctions forming intracellular junctions. This hypothesis originated over four decades ago based on the observation that vesicles lining gap junctions of crayfish giant axons contain electron-opaque particles, [...] Read more.
This paper proposes the hypothesis that cytoplasmic organelles directly interact with each other and with gap junctions forming intracellular junctions. This hypothesis originated over four decades ago based on the observation that vesicles lining gap junctions of crayfish giant axons contain electron-opaque particles, similar in size to junctional innexons that often appear to directly interact with junctional innexons; similar particles were seen also in the outer membrane of crayfish mitochondria. Indeed, vertebrate connexins assembled into hexameric connexons are present not only in the membranes of the Golgi apparatus but also in those of the mitochondria and endoplasmic reticulum. It seems possible, therefore, that cytoplasmic organelles may be able to exchange small molecules with each other as well as with organelles of coupled cells via gap junctions. Full article
(This article belongs to the Special Issue Chemical Regulation of Gap Junction Channels and Hemichannels)
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