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GAP JUNCTIONS CONTAIN CHANNELS that allow the passage of ions and small molecules between adjacent cells. This intercellular communication has been implicated in the coordination of cellular responses to intracellular signaling molecules. Calcium and inositol phosphates are among the second messengers that can pass through gap junction channels (15). Stimulation of a cell can elicit a rise in calcium that is then propagated as a "wave" to many surrounding cells. Studies of tracheal epithelial cells have suggested that these calcium waves are mediated by gap junction-mediated intercellular passage of inositol trisphosphate (3, 16, 17). Interventions such as the delivery of antibodies directed against gap junction proteins (connexins) have strongly supported the role of gap junctions in this process (2). The current paper by Boitano and Evans (4) provides further evidence toward this conclusion. Propagated calcium waves have been extensively investigated in many different cell types (5, 7; reviewed in Ref. 18). Although in some cases intercellular calcium waves are mediated by gap junctions, in others they appear to be mediated by passage of extracellular messengers (e.g., interaction of ATP released from a stimulated cell with P2 purinergic receptors on adjacent cells leads to increased intracellular calcium and further ATP secretion by the adjacent cells, producing a propagated ATP-induced calcium wave) (13).
The most interesting aspect of the Boitano and Evans (4)
paper is their use of short peptides corresponding to sequences within
the extracellular loops of connexins as inhibitors of intercellular communication. This approach is based on our current understanding of
connexin structure and topology. Hydropathy plots originally suggested
that a connexin contained four-transmembrane-spanning regions and two
short extracellular loops (14). The disposition of the
first and second extracellular regions was confirmed by the production
of anti-peptide antibodies that were used for immunolocalization of the
corresponding epitopes on split gap junctions (10, 21). To
form complete gap junction channels, the extracellular domains of
hemichannels contributed by two adjacent cells must "dock" with
each other. Therefore, it was hypothesized that reagents that
interfered with the interactions of these extracellular domains might
impair cell-to-cell communication. Unfortunately, although the
antibodies against the extracellular loop amino acid sequences were
useful for immunocytochemical studies, they were of limited utility as
reagents for blocking cell-to-cell communication (12). An
alternative approach was developed with the hypothesis that small
peptides corresponding to these extracellular connexin domains might
impair the interactions of the extracellular loops by binding to their
recognition sites in a hemichannel (Fig.
1). Dahl et al. (8) examined
the effectiveness of peptides as inhibitors of cell-cell channel
formation produced by connexin (Cx) 32 expressed in paired
Xenopus oocytes. They found that peptides representing the
two extracellular loops of Cx32 as well as five smaller peptides corresponding to subsets of these peptides (Table
1) were quite effective.
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A number of subsequent papers have used a similar strategy, with the intent of blocking the function of endogenous connexins in various systems. These studies have identified peptides that are effective inhibitors of intercellular communication or of various physiological processes likely mediated by gap junction channels. (The peptides used by the different authors and the connexins of origin are listed in Table 1.) Because of differences in the experimental systems studied, several questions arise. These issues can be considered in terms of the suggested mechanisms of action of the inhibitory peptides as discussed by Chaytor et al. (6).
A major difference between these studies is the status of cellular
interactions when inhibitory peptides are applied. In some cases
(8, 9, 11, 20), inhibitory peptides are applied to cells
before the cells have time to interact with each other. These cases are
consistent with a mechanism by which the inhibitory peptides might
block docking of two preformed hemichannels that had not yet formed a
complete channel (Fig. 2A). In
other studies, inhibitory peptides were applied to tissues
(6) or cultured cells (4) with established
interactions. This leads to a second, alternative possible mechanism.
The peptides might facilitate the breaking apart of previously formed
gap junction channels. Indeed, if the docking of channels were
disrupted progressively beginning at the periphery of a gap junction,
this might lead to an "unzipping" of gap junction plaques (Fig.
2B). The studies that have used this experimental strategy
also differ in the duration of the application of inhibitory peptides
before their effects are examined. In some instances, the effects of
inhibitory peptides were examined at least 2 h after their
application, and the peptides were shown to have inhibited gap junction
function, although not completely, at the time of examination (8,
9, 11). Because in these studies peptides were applied to
dissociated cells, the observed peptide inhibitory effects are
consistent with the inhibition of formation of new gap junction
channels, probably by impairing hemichannel docking. In other studies
(4, 20), the inhibitory effects on gap junction function
were examined within minutes of peptide application, and the effects
were observed at least 1 h after peptide application. Because most
connexins have half-lives of 1.5-5 h, only partial uncoupling
would be expected after 1 h of peptide application due to
impairment of hemichannel docking. Therefore, this mechanism may not
fully explain the time course of inhibition in these studies. Assuming
that connexin turnover is not altered, extensive uncoupling within
1 h or less must imply disruption or blockade of existing
channels. In yet other cases, the effects of the inhibitory peptides
and their reversibility were studied within minutes after peptide
application. The time course of both processes was remarkably short
with some of the peptides used (15-20 min) (4, 6).
The effectiveness of short-term peptide application suggests that
inhibitory peptides bind to hemichannels participating in formed gap
junction channels, leading to "undocking." The reversibility of the
effects suggests that hemichannels that have bound peptides are not
targeted for degradation. A third, alternative, hypothetical mechanism
of action for these peptides was also discussed by Chaytor et al.
(6); extracellular loop peptides might bind to connexin
molecules in intact channels, inducing a conformational change that
leads to channel closure. Assuming that such conformational changes
would be fast, the time course of the effects in this hypothesis would
depend mainly on access of the peptide to its recognition sequence(s).
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The specificity of these extracellular loop peptides is unclear. As shown in Table 1, several studies have used very similar peptide sequences and have blocked functional gap junction coupling in a wide variety of systems. It is likely that different connexins (or more than one connexin) are expressed in these cells. Nevertheless, these common inhibitors appear effective at decreasing communication in these diverse systems. It is possible that these peptides are promiscuous inhibitors of multiple different connexins or that the formation of heteromeric channels allows a sequence that recognizes only one connexin to be effective at substantially reducing communication. However, in their study, Kwak and Jongsma (11) designed peptides to the most divergent regions of extracellular loop 2 in Cx43 and Cx40, and they found that the Cx40-based peptide reduced the frequency of 150-pS single-channel events, whereas the Cx43-based sequence decreased the frequency of 80-pS single-channel events. Thus it may be possible to achieve selective inhibition of cell-to-cell communication by careful choice of inhibitory peptides based on the knowledge of the connexins expressed in the system under study.
The different studies using extracellular sequence-based peptides do suggest a blockade of gap junction function based on multiple different physiological examinations. However, these studies include few biochemical or immunochemical analyses. Specifically, no evidence has been provided for the direct binding of peptides to connexin proteins. The half-life of peptides in the different systems has not been examined. Neither the relative abundance nor the cellular localization of connexins has been examined in the presence and absence of peptides. It would be interesting to know whether there is a substantial number of hemichannels at the gap junction membrane or whether peptide-bound connexins are targeted for degradation. In this respect, connexin turnover has not been determined in these treated cells. The possible disruption of gap junctional plaques might be detected by biochemical or ultrastructural methods.
Further examination of connexin sequences recognized by inhibitory peptides may give insights into which amino acid sequences from the extracellular loops interact to form gap junction channels. However, these interactions are likely to be complex. The precise secondary structure within the gap has not yet been defined, but all models based on three-dimensional structural studies suggest that hemichannel docking involves multivalent interactions of subunits between hemichannels (19).
Moreover, if this technology is to be widely adapted for use in blocking gap junction function, further hurdles must be overcome. The block produced by these inhibitors has been complete in only a few instances in the systems examined to date. Peptide stability must be ensured and new delivery methods must be developed if these inhibitors are to be effective in systems other than in vitro models.
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Viviana M. Berthoud, Eric C. Beyer, Department of Pediatrics University of Chicago Chicago, Illinois 60637-1470 | |||||
Kyung Hwan Seul, Chonbuk National University Medical School Chonju, Republic of Korea American Journal of Physiology- Lung Cellular and Molecular Physiology October 2000, Volume 279 (23) |