Our hypothesis proposes that that the loose, less ordered particle array of coupled junctions is caused by the presence of CaM molecules linked to each of the six connexin of the hemichannel (Figure 13A). Researchers think that in coupled junctions a CaM molecule is bound to each connexin by the C- lobe, while the N-lobe is free, such that there would be six unbound CaM N-lobes per hemichannel (Figure 13A). Due to the negative charge of the CaM lobes, the N-lobes would be repelling each other, such that the channels would be separated (Figure 9A and Figure 10A). In coupled junctions, the channels would be spaced at a ~100 Å center-to-center distance in vertebrates and a ~200 Å in crayfish axons, and the junctions would display loose and irregular channel arrays. Based on the Ca-CaM-cork model, moderate [Ca2+]i rise would cause a CaM’s N-lobe to reversibly close the channel (Figure 13B). This type of “reversible” gating state (Ca-CaM-cork) would not cause gap junction crystallization, as the electrostatic repulsion among the other five N-lobes would prevent it. So, what is causing gap junction crystallization?
We are aware that the mechanism for gap junction crystallization we are proposing is highly speculative and needs to be experimentally tested. Perhaps, future work could test this idea by attempting to detect biochemically the presence of CaM molecule in isolated gap junctions. However, one should realize that if only two CaM molecules are bound to each channel of crystalline gap junction fragments, the CaM will only represent 14.3% of the proteins, as each channel is made of twelve connexin monomers.
- The Calmodulin-Cork Model Is Supported by X-ray Diffraction Images of Isolated Gap Junction Channels in Closed State
In the early 1980s, Makowski and coworkers described the structure of crystalline (hexagonal) gap junctions isolated from mouse liver in a high-resistance configuration (closed channels) by analyzing X-ray diffraction data at 18 Å resolution (Figures 18 and 19B) [120,121]. The channels’ gated condition of these isolated gap junctions was proven by the evidence that the channels were impermeable to sucrose [120]; in their words: “Analysis of diffraction patterns from isolated gap junctions in 50% sucrose shows that the sucrose fills the extracellular gap but fails to enter the channel. It is possible that the channel is closed at both cytoplasmic surfaces, excluding sucrose. This suggests that the isolated junctions are in a high resistance state” [120]. Indeed, the three-dimensional map of the electron density demonstrated that the channels were blocked at both cytoplasmic ends by a small particle; in their words: “Its position blocking the channel suggests that it may comprise a gating structure responsible for the control of channel permeability, X-ray diffraction studies of junctions in varying concentrations of sucrose (Makowski et al. 1984a) [122] indicated that in these preparations the channel was closed to the penetration of sucrose and that a solvent region approximately 100 Å long and centered on the six fold axis remained free of sucrose” [121].
![](/media/common/202112/mceclip0-61b8a25544167.png)
Figure 18. Diagram of the three-dimensional structure of a frozen-hydrated gap junction isolated from mouse liver and solved to 18 Å resolution by X-ray diffraction. The channel “6” in (a,b) is impermeable to 50% sucrose, proving that it is closed at each cytoplasmic end by a particle that prevents sucrose entry. This proves that the channels of isolated (crystalline) junctions are in closed state (Ca-CaM locked state). Reproduced from Ref. [121] with permission from the Journal of Molecular Biology and the Cold Spring Harbor Laboratories.
![](/media/common/202112/mceclip1-61b8a2771be2a.png)
Figure 19. Both the positively charged channel’s mouth and the negatively charged CaM lobes are ~35 Å in size (A). Thus, a CaM lobe could fit well within the positively charged connexon’s mouth (A). Significantly, the three-dimensional structure of gap junctions isolated from mouse liver (B) demonstrates that the channel of isolated (crystalline) junctions is impermeable to 50% sucrose, proving that it is closed at each cytoplasmic end by a particle that prevents sucrose entry (B). Our hypothesis is that the blocking particle is the CaM’s N-lobe (B). Both the CaM and connexon images (A) were provided by Dr. Francesco Zonta (VIMM, University of Padua, Italy. (B) was reproduced from Ref. [121] with permission from the Journal of Molecular Biology and the Cold Spring Harbor Laboratories.
Figure 18. Diagram of the three-dimensional structure of a frozen-hydrated gap junction isolated from mouse liver and solved to 18 Å resolution by X-ray diffraction. The channel “6” in (a,b) is impermeable to 50% sucrose, proving that it is closed at each cytoplasmic end by a particle that prevents sucrose entry. This proves that the channels of isolated (crystalline) junctions are in closed state (Ca-CaM locked state). Reproduced from Ref. [121] with permission from the Journal of Molecular Biology and the Cold Spring Harbor Laboratories.
Figure 19. Both the positively charged channel’s mouth and the negatively charged CaM lobes are ~35 Å in size (A). Thus, a CaM lobe could fit well within the positively charged connexon’s mouth (A). Significantly, the three-dimensional structure of gap junctions isolated from mouse liver (B) demonstrates that the channel of isolated (crystalline) junctions is impermeable to 50% sucrose, proving that it is closed at each cytoplasmic end by a particle that prevents sucrose entry (B). Our hypothesis is that the blocking particle is the CaM’s N-lobe (B). Both the CaM and connexon images (A) were provided by Dr. Francesco Zonta (VIMM, University of Padua, Italy. (B) was reproduced from Ref. [121] with permission from the Journal of Molecular Biology and the Cold Spring Harbor Laboratories.
Significantly, the blocking particle, spherical in shape, is approximately 30–35 Ǻ in diameter [121] (Figures 18 and 19B), which is remarkably similar in size to a CaM lobe (Figure 19A). Indeed, in their words: “The channel has a diameter of 20–30 Ǻ along most of its length but appears to narrow to a minimum diameter of about 15 Ǻ in the extracellular half of the bilayer … Both the sucrose results and the three-dimensional map are consistent with the idea that a structure located near the cytoplasmic surface of the membraned is blocking the channel in these preparations” [121]. These studies support our evidence that gap junctions with crystalline (hexagonal) channel arrays are in an uncoupled (gated) state (Ca-CaM locked gate, Figures 13-16) [11,17,18,127–130] and suggest that a CaM lobe is gating the channel.
- Conclusions and Future Perspectives
This article has reviewed four decades of data supporting the direct role of CaM in gap junction channel gating. The Ca-CaM-cork gating mechanism [113], proposed over two decades ago [112], is based on evidence from the effect of CaM inhibitors, the inhibition of CaM expression, the expression of a CaM mutant (CaMCC) with higher Ca2+-sensitivity, CaM-connexin co-localization at gap junctions, the presence of high-affinity CaM-binding sites in connexins, the expression of connexin mutants, the gating effect of repeated large Vj pulses, data at the single channel level, the recovery of lost gating competency by addition of Ca-CaM to internally perfused crayfish axons and, finally, X-ray diffraction data on isolated gap junction fragments.
One may ask: why is it important to understand in detail the gating mechanism of gap junction channels? It is important not only because cell–cell uncoupling is more than just a safety mechanism for protecting healthy cells from damaged neighbors (healing over), but because the gating sensitivity to [Ca2+]i in the high nanomolar range indicates that the fine modulation of direct cell–cell communication provides cells with the means for regulating tissue homeostasis. In addition, and more importantly, the field should be encouraged to test the effect of recently discovered disease-causing CaM mutants on gap junction function [135].
Indeed, recent evidence of diseases caused by CaM mutations [136–141] suggests the potential role of CaM mutants in diseases affecting gap junction function. Almost two dozen CaM mutations have been found to cause cardiac malfunctions, most of which occur in the CaM’s C-lobe, one in the N-lobe, and one in the linker between the C- and N-lobes. In most cases the electrocardiogram (ECG) demonstrates the presence of Long QT Syndrome (LQTS), a change that affects the electrical activity of the heart, which is often associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) phenotype and Idiopathic Ventricular Fibrillation (IVF). CPVT patients manifest ventricular tachycardia that can lead to death by ventricular fibrillation. In most cases, cardiac malfunctions have been attributed to the effect of CaM mutations on the ryanodine receptor (RyR2) and the cardiac L-type voltage-gated Ca2+ channel. However, other membrane channels, potential targets of CaM mutants, have also been suggested. Curiously, however, in spite of strong evidence for the direct CaM role in gap junction channel regulation, the potential consequences of these CaM mutants on direct cell–cell communication—a mechanism fundamental for the function of virtually all vertebrate and invertebrate organs—have not yet been addressed. Therefore, it is clear that future efforts should be aimed at testing the effect of these CaM mutants, and the future discovered CaM mutants, on gap junction mediated communication [135].
Author Contributions: C.P. is the principal investigator. L.M.L.P., Research Assistant, has performed the general lab work and the technical work for preparation and injection of oocytes
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflicts of interest.
We are aware that the mechanism for gap junction crystallization we are proposing is highly speculative and needs to be experimentally tested. Perhaps, future work could test this idea by attempting to detect biochemically the presence of CaM molecule in isolated gap junctions. However, one should realize that if only two CaM molecules are bound to each channel of crystalline gap junction fragments, the CaM will only represent 14.3% of the proteins, as each channel is made of twelve connexin monomers.
- The Calmodulin-Cork Model Is Supported by X-ray Diffraction Images of Isolated Gap Junction Channels in Closed State
In the early 1980s, Makowski and coworkers described the structure of crystalline (hexagonal) gap junctions isolated from mouse liver in a high-resistance configuration (closed channels) by analyzing X-ray diffraction data at 18 Å resolution (Figures 18 and 19B) [120,121]. The channels’ gated condition of these isolated gap junctions was proven by the evidence that the channels were impermeable to sucrose [120]; in their words: “Analysis of diffraction patterns from isolated gap junctions in 50% sucrose shows that the sucrose fills the extracellular gap but fails to enter the channel. It is possible that the channel is closed at both cytoplasmic surfaces, excluding sucrose. This suggests that the isolated junctions are in a high resistance state” [120]. Indeed, the three-dimensional map of the electron density demonstrated that the channels were blocked at both cytoplasmic ends by a small particle; in their words: “Its position blocking the channel suggests that it may comprise a gating structure responsible for the control of channel permeability, X-ray diffraction studies of junctions in varying concentrations of sucrose (Makowski et al. 1984a) [122] indicated that in these preparations the channel was closed to the penetration of sucrose and that a solvent region approximately 100 Å long and centered on the six fold axis remained free of sucrose” [121].
Significantly, the blocking particle, spherical in shape, is approximately 30–35 Ǻ in diameter [121] (Figures 18 and 19B), which is remarkably similar in size to a CaM lobe (Figure 19A). Indeed, in their words: “The channel has a diameter of 20–30 Ǻ along most of its length but appears to narrow to a minimum diameter of about 15 Ǻ in the extracellular half of the bilayer … Both the sucrose results and the three-dimensional map are consistent with the idea that a structure located near the cytoplasmic surface of the membraned is blocking the channel in these preparations” [121]. These studies support our evidence that gap junctions with crystalline (hexagonal) channel arrays are in an uncoupled (gated) state (Ca-CaM locked gate, Figures 13-16) [11,17,18,127–130] and suggest that a CaM lobe is gating the channel.
- Conclusions and Future Perspectives
This article has reviewed four decades of data supporting the direct role of CaM in gap junction channel gating. The Ca-CaM-cork gating mechanism [113], proposed over two decades ago [112], is based on evidence from the effect of CaM inhibitors, the inhibition of CaM expression, the expression of a CaM mutant (CaMCC) with higher Ca2+-sensitivity, CaM-connexin co-localization at gap junctions, the presence of high-affinity CaM-binding sites in connexins, the expression of connexin mutants, the gating effect of repeated large Vj pulses, data at the single channel level, the recovery of lost gating competency by addition of Ca-CaM to internally perfused crayfish axons and, finally, X-ray diffraction data on isolated gap junction fragments.
One may ask: why is it important to understand in detail the gating mechanism of gap junction channels? It is important not only because cell–cell uncoupling is more than just a safety mechanism for protecting healthy cells from damaged neighbors (healing over), but because the gating sensitivity to [Ca2+]i in the high nanomolar range indicates that the fine modulation of direct cell–cell communication provides cells with the means for regulating tissue homeostasis. In addition, and more importantly, the field should be encouraged to test the effect of recently discovered disease-causing CaM mutants on gap junction function [135].
Indeed, recent evidence of diseases caused by CaM mutations [136–141] suggests the potential role of CaM mutants in diseases affecting gap junction function. Almost two dozen CaM mutations have been found to cause cardiac malfunctions, most of which occur in the CaM’s C-lobe, one in the N-lobe, and one in the linker between the C- and N-lobes. In most cases the electrocardiogram (ECG) demonstrates the presence of Long QT Syndrome (LQTS), a change that affects the electrical activity of the heart, which is often associated with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) phenotype and Idiopathic Ventricular Fibrillation (IVF). CPVT patients manifest ventricular tachycardia that can lead to death by ventricular fibrillation. In most cases, cardiac malfunctions have been attributed to the effect of CaM mutations on the ryanodine receptor (RyR2) and the cardiac L-type voltage-gated Ca2+ channel. However, other membrane channels, potential targets of CaM mutants, have also been suggested. Curiously, however, in spite of strong evidence for the direct CaM role in gap junction channel regulation, the potential consequences of these CaM mutants on direct cell–cell communication—a mechanism fundamental for the function of virtually all vertebrate and invertebrate organs—have not yet been addressed. Therefore, it is clear that future efforts should be aimed at testing the effect of these CaM mutants, and the future discovered CaM mutants, on gap junction mediated communication [135].
Author Contributions: C.P. is the principal investigator. L.M.L.P., Research Assistant, has performed the general lab work and the technical work for preparation and injection of oocytes
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflicts of interest.
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