EDITORIAL
Peptide inhibitors of intercellular communication


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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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Fig. 1.   Hypothetical basis for the inhibitory peptide strategy. Connexin extracellular loops from 2 apposed hemichannels interact within the extracellular space to form a complete channel (arrow 1). Peptides corresponding to portions of these loops (~) might bind to the same sites and prevent docking between hemichannels (arrow 2).


                              
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Table 1.   Peptide inhibitors of intercellular communication

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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Fig. 2.   Proposed mechanisms of action of inhibitory peptides. A: inhibition of docking. Gap junction channels are formed by docking of hemichannels from 2 adjacent cells (arrow 1). In the presence of extracellular loop peptides (~), docking is prevented (arrow 2). B: undocking of gap junction channels. Addition of inhibitory peptides (~) induces destabilization of established gap junction channels, leading to the undocking of hemichannels. This mechanism presumes a steady-state balance between hemichannel docking and undocking. To successfully compete with the extracellular loops of the apposed hemichannel, either the affinity of the inhibitory peptide for its recognition sequence must be greater than that between the extracellular loops or the concentration of the peptide within the junctional extracellular space must be high enough to displace the bound extracellular loop from the apposed hemichannel.

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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REFERENCES

1.   Beyer, EC, Paul DL, and Goodenough DA. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105: 2621-2629, 1987[Abstract].

2.   Boitano, S, Dirksen ER, and Evans WH. Sequence-specific antibodies to connexins block intercellular calcium signaling through gap junctions. Cell Calcium 23: 1-9, 1998[ISI][Medline].

3.   Boitano, S, Dirksen ER, and Sanderson MJ. Intercellular propagation of calcium waves mediated by inositol trisphosphate. Science 258: 292-295, 1992[ISI][Medline].

4.   Boitano, S, and Evans WH. Connexin mimetic peptides reversibly inhibit Ca2+ signaling through gap junctions in airway cells. Am J Physiol Lung Cell Mol Physiol 279: L623-L630, 2000[Abstract/Free Full Text].

5.   Cao, D, Lin G, Westphale EM, Beyer EC, and Steinberg TH. Mechanisms for the coordination of intercellular calcium signalling in insulin-secreting cells. J Cell Sci 110: 497-504, 1997[Abstract/Free Full Text].

6.   Chaytor, AT, Evans WH, and Griffith TM. Peptides homologous to extracellular loop motifs of connexin 43 reversibly abolish rhythmic contractile activity in rabbit arteries. J Physiol (Lond) 503: 99-110, 1997[Abstract].

7.   Cornell-Bell, AH, Finkbeiner SM, Cooper MS, and Smith SJ. Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science 247: 470-473, 1990[ISI][Medline].

8.   Dahl, G, Nonner W, and Werner R. Attempts to define functional domains of gap junction proteins with synthetic peptides. Biophys J 67: 1816-1822, 1994[Abstract].

9.   Eugenín, EA, González H, Sáez CG, and Sáez JC. Gap junctional communication coordinates vasopressin-induced glycogenolysis in rat hepatocytes. Am J Physiol Gastrointest Liver Physiol 274: G1109-G1116, 1998[Abstract/Free Full Text].

10.   Goodenough, DA, Paul DL, and Jesaitis L. Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions. J Cell Biol 107: 1817-1824, 1988[Abstract].

11.   Kwak, BR, and Jongsma HJ. Selective inhibition of gap junction channel activity by synthetic peptides. J Physiol (Lond) 516: 679-685, 1999[Abstract/Free Full Text].

12.   Meyer, RA, Laird DW, Revel JP, and Johnson RG. Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies. J Cell Biol 119: 179-189, 1992[Abstract].

13.   Osipchuk, Y, and Calahan M. Cell-to-cell spread of calcium signals mediated by ATP receptors in mast cells. Nature 359: 241-244, 1992[ISI][Medline].

14.   Paul, DL. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol 103: 123-134, 1986[Abstract].

15.   Saez, JC, Connor JA, Spray DC, and Bennett MV. Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-trisphosphate, and to calcium ions. Proc Natl Acad Sci USA 86: 2708-2712, 1989[Abstract].

16.   Sanderson, MJ, Charles AC, and Dirksen ER. Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells. Cell Regul 1: 585-596, 1990[ISI][Medline].

17.   Sanderson, MJ, Chow I, and Dirksen ER. Intercellular communication between ciliated cells in culture. Am J Physiol Cell Physiol 254: C63-C74, 1988[Abstract/Free Full Text].

18.   Scemes, E, Suadicani SO, and Spray DC. Intercellular calcium wave communication via gap junction dependent and independent mechanisms. In: Gap Junctions: Molecular Basis of Cell Communication in Health and Disease, edited by Peracchia C.. San Diego, CA: Academic, 2000, p. 145-173.

19.   Unger, VM, Kumar NM, Gilula NB, and Yeager M. Three-dimensional structure of a recombinant gap junction membrane channel. Science 283: 1176-1180, 1999[Abstract/Free Full Text].

20.   Warner, A, Clements DK, Parikh S, Evans WH, and DeHaan RL. Specific motifs in the external loops of connexin proteins can determine gap junction formation between chick heart myocytes. J Physiol (Lond) 488: 721-728, 1995[Abstract].

21.   Zimmer, DB, Green CR, Evans WH, and Gilula NB. Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures. J Biol Chem 262: 7751-7763, 1987[Abstract/Free Full Text].

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)


Am J Physiol Lung Cell Mol Physiol 279(4):L619-L622
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society