Identification of a Novel Peptide That Interferes With the Chemical Regulation of Connexin43
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Junko Shibayama, Rebecca Lewandowski, Fa
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the Departments of Pharmacology (J.S., R.L., W.C., S.S., M.D.) and Microbiology/Immunology (S.M.T.), State University of New York, Upstate Medical University, Syracuse
Department of Biochemistry and Molecular Biology (F.K., P.L.S.), University of Nebraska Medical Center, Omaha.
Abstract
The carboxyl-terminal domain of connexin43 (Cx43CT) is involved in various intra- and intermolecular interactions that regulate gap junctions. Here, we used phage display to identify novel peptidic sequences that bind Cx43CT and modify Cx43 regulation. We found that Cx43CT binds preferentially to peptides containing a sequence RXP, where X represents any amino acid and R and P correspond to the amino acids arginine and proline, respectively. A biased "RXP library" led to the identification of a peptide (dubbed "RXP-E") that bound Cx43CT with high affinity. Nuclear magnetic resonance data showed RXP-E–induced shifts in the resonance peaks of residues 343 to 346 and 376 to 379 of Cx43CT. Patch-clamp studies revealed that RXP-E partially prevented octanol-induced and acidification-induced uncoupling in Cx43-expressing cells. Moreover, RXP-E increased mean open time of Cx43 channels. The full effect of RXP-E was dependent on the integrity of the CT domain. These data suggest that RXP-based peptides could serve as tools to help determine the role of Cx43 as a regulator of function in conditions such as ischemia-induced arrhythmias.
Key Words: Cx43CT connexin particle-receptor interaction gap junctions
Introduction
Connexins are integral membrane proteins that oligomerize to form intercellular channels called gap junctions. The most abundant gap junction protein in a number of mammalian systems is connexin43 (Cx43). Our previous work has suggested that regulation of Cx43 channels results from the association of the carboxyl-terminal (CT) domain, acting as a gating particle, and a separate region of the connexin molecule, acting as a receptor for the gating particle.1,2 Additional studies have shown that this intramolecular interaction can be modulated by other intermolecular interactions in the microenvironment of the gap junction plaque.3 Thus, the emerging picture of a gap junction plaque is that of a macromolecular complex in which proteins act in concert to modulate intercellular communication. At the center of these interactions is the CT domain, which acts as a substrate for a number of kinases,4 a ligand for noncatalytic proteins, and a gating particle to modify coupling between cells.5
As a key player in the regulation of gap junctions, CT presents itself as a target of chemical6,7 or genetic manipulation intended to modify function.8 Here, we sought to disrupt the regulation of Cx43 by chemical means. Our rationale was based on the knowledge that Cx43CT is capable of interacting with other proteins. We reasoned that this "stickiness" of Cx43CT can be used to "adhere" peptidic sequences to it. We further speculated that the interaction of Cx43CT with small peptides can modify the behavior of the gap junction channel. This rationale was supported by previous work showing that peptides can modify both the chemical and voltage-gating behavior of Cx43.6,7 In the present study, we used a high-throughput phage display screening to find peptidic sequences that bind Cx43CT. Further analysis using a combination of surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and dual patch clamp led to the identification of a specific peptide that binds to Cx43CT with high affinity, affects residues 343 to 346 and 376 to 379 of Cx43, and partially prevents octanol-induced and acidification-induced uncoupling. These results support the feasibility of a peptide-based strategy to manipulate Cx43 regulation in native tissues. These peptides can be used as tools to characterize the specific role of gap junction regulation in health and disease.
Materials and Methods
Phage Display
Recombinant Cx43CT (amino acids 255 to 382 of rCx43) was produced as previously described1 and used as "bait" for peptide screening. For peptide presentation, we used a "library" of bacteriophage, displaying &55 copies of 2.7x109 random 12-mer peptides (Ph.D.-12 Phage display peptide library kit; New England BioLabs Inc). Details of the protein production and screening procedures are outlined in the online data supplement available at http://circres.ahajournals.org.
Biased Phage Display
We used the Ph.D. Peptide Display Cloning System (New England BioLabs Inc) to create a biased phage display library of sequence XXXXRXPXXXX, where X is any amino acid flanking an arginine and a proline with an additional random residue at the center. The randomized peptides were followed by a GGGS spacer. These phages were analyzed for titer and sequence before screening for Cx43CT.
Surface Plasmon Resonance
SPR is a spectroscopic method to determine binding amplitude and kinetics in real time.9,10 Recombinant Cx43CT was covalently bound to a carboxylmethyl dextran matrix.10 Dissociation constants (KD) were calculated from the time course of binding and unbinding of the ligand, using a 1:1 (Langmuir) association and dissociation kinetic model (Biacore software package). In both phases (association and dissociation), the first 5 to 8 seconds of recording were not included in the fit, as to avoid artifacts resulting from peptide distribution within the flow cells.10
Nuclear Magnetic Resonance
All NMR data were acquired on a Varian INOVA 600-MHz NMR spectrometer using a cryoprobe11; the sample temperature was maintained at 7°C. Gradient-enhanced 2D 1H-15N HSQC experiments12 were used to observe all backbone amide resonances in 15N-labeled Cx43CT. Methodological details are presented in the online data supplement.
Electrophysiological Analysis
Experiments were conducted on N2a (neuroblastoma) cells. Cx43 was expressed in a lac-switch stable system (induced by 0.1 to 1.0 mmol/L IPTG; see Zhong et al13) or transiently using an IRES plasmid coding for eGFP.7 M257 (a mutant of Cx43 truncated at amino acid 257; see Morley et al2) was transiently expressed in N2a cells also using an eGFP-containing IRES plasmid. Synthetic peptides were diluted in the pipette solution to a final concentration of 0.1 mmol/L as in previous work.7 For some experiments, octanol (2.0 mmol/L) was superfused during recording. Methods for single-channel recordings and criteria for inclusion and analysis were as reported before,7,14 and as outlined in online data supplement.
Results
Nonbiased Phage Display
Initial control experiments were conducted to standardize the phage display assay. The library was presented to purified streptavidin, a protein known to bind preferentially to peptides containing an HPQ consensus motif.15,16 After 3 rounds of enrichment, 14 phage plaques were selected for sequencing. All 14 showed preservation of the HPQ motif, indicating that the experimental conditions were adequate for appropriate peptide recognition by the target.
The library was presented to Cx43CT. After 3 rounds of selection and amplification, DNA of 156 phage plaques was purified and sequenced. Of the estimated 2.7x109 different sequences in the library, 48 unique sequences were recovered. One particular sequence (PRPTMGNLPDVL), recovered from 45 different plaques, showed homology with a 10 amino acid segment (RATLLNVPDL) of the second PDZ domain of zonula ocludens-1 (ZO-1). When aligned, 5 amino acids were identical and 4 were conserved; binding between the second PDZ domain of ZO-1 and Cx43CT in vivo and in vitro has been well documented.17,18 The results supported the notion that the phage display method can be helpful at recognizing Cx43CT binding sequences.
The "RXP" Motif and Biased Phage Display
Further analysis of the 12-mer peptides revealed that 16 of 48 unique sequences shared a motif RXP (where X represents any amino acid). The RXP motif was not detected in phage that bound to streptavidin. The motif was shown in 11 peptides in the N- to C-terminal orientation and 5 of them in the reverse direction. Specific sequences are presented in Table 1. The RXP sequence occurred in different positions within the 12-mer peptide, thus preventing proper alignments to determine the frequency of other amino acids at specific positions relative to RXP. To overcome this limitation and search for peptides with higher binding affinity (see section on SPR, below), we generated a biased phage display library where the RXP motif was forced to the center of the sequence, flanked at each side by 4 randomized amino acids. Sixty clones containing an insert (of an estimated 3 to 4x104 total clones in the library) were chosen at random for sequencing before exposure of the library to Cx43CT. All inserts coded for an 11-mer peptide with the RXP motif in the appropriate location. In 3 independent runs, the same library was presented to Cx43CT. The binding step was performed at pH 6.5 in 2 of the runs (runs 1 and 2) and at pH 7.4 in 1 additional run (run 3). The total number of phage plaques containing a full-coding insert that were recovered after the binding step were 120, 119, and 163 for runs 1, 2, and 3, respectively. Surprisingly, 89% of all sequences corresponded to "doublets," that is, peptides where 2 RXP 11-mers had been inserted in tandem (see supplemental Table I and Discussion). Although we did not detect a clear preference for specific amino acids in a given position relative to the RXP core, we did observe a preponderance of basic residues in sequences recovered from all screenings (see supplemental Figure I and related text). Furthermore, 5 specific peptides were recovered from all 3 runs and represented a large fraction of the repeats (Table 2). The absence of doublets in the initial library before screening and their high frequency after the binding step strongly suggest that these peptides, although rare, were highly selected for Cx43CT binding.
In Vitro Binding Detected by SPR
Phage display allows for the rapid screening of thousands of peptides, but the characteristics of binding cannot be properly defined. Furthermore, the peptides are part of a capsid protein; the latter may affect the ability of the peptide to properly interact with the target protein. We, therefore, selected some of the peptides identified by phage screening to characterize their ability to bind Cx43CT using SPR. Recombinant Cx43CT was covalently bound to a carboxymethyl dextran matrix and synthetic peptides presented for binding. A total of six 12-mer peptides from the nonbiased screening (peptides labeled RXP-1 to RXP-6; Table 1) and 5 peptides from the biased screening (labeled RXP-A to RXP-E; Table 2) were tested. RXP-2, RXP-3, RXP-5, and RXP-6 showed no significant binding (<100 resonance units of maximal response at peptide concentration of 1 mmol/L). Among the peptides obtained from the nonbiased search, RXP-4 caused the largest deflection in SPR signal, and a small, near-negligible signal was obtained from exposure of Cx43CT to RXP-1. Examples are shown in Figure 1A. RXP-4 (blue line) or RXP-1 (red line) was added at time 0 (peptide concentration was 250 μmol/L). Washout was initiated after 2 minutes of exposure. The peptide dissociated rapidly and completely, suggesting poor binding affinity to Cx43CT. We tested higher concentrations of the peptide but could not detect an asymptotic maximum response. In addition, dissociation rates were too fast to reliably use them to calculate the kinetics of binding. As such, these results do show Cx43CT–RXP-4 binding, but the interaction between the 2 molecules was too weak to allow for proper calculation of kinetic values. A different result emerged from the testing of the doublets. RXP-A and RXP-E showed significant binding to Cx43CT. In particular, peptide RXP-E generated a large, concentration-dependent deflection followed by a slow dissociation on washout (Figure 1B). The transitions were well fit by exponential functions and the rates of association and dissociation were used to estimate kinetic parameters (see Materials and Methods). A full range of concentrations was tested in 3 separate occasions and at 2 different pH values of the solvent (6.5 and 7.4). No differences were observed as a function of pH. From these studies, we calculated a dissociation constant (KD) of 3.9 μmol/L for the Cx43CT-RXP-E interaction. This value is similar to that measured for the association of Cx43CT to binding partners such as the SH3 domain of c-src,3,17 the second PDZ domain of ZO-1,3,17 or the binding of Aquaporin-0 to Cx45.6.19 Overall, these results show that different RXP peptides are able to interact with Cx43CT in vitro with various degrees of affinity. The possibility of structural modifications caused on Cx43CT as a result of RXP interactions (and hence an initial approach to the possible location of the binding site) was assessed next.
Nuclear Magnetic Resonance
Peptides RXP-1, RXP-4, and RXP-E were tested for their ability to modify the structure of Cx43CT. The peptides were diluted in PBS (pH 5.8) containing 15N-Cx43CT, and 15N-HSQC spectra were acquired. An example of the resonance shifts caused by the presence of RXP-E is shown in Figure 2. Resonance contours for Cx43CT in control conditions (Cx43CT alone; no peptides added) are depicted in black. Contours obtained in the presence of RXP-E are labeled red. In previous studies, we have assigned the specific resonance peaks that correspond to each amino acid in the Cx43CT sequence.20 Accordingly, shifts in the resonance assignments directly reveal the identity of the amino acids whose position in space is modified by the presence of the peptide. Addition of RXP-E peptide strongly affected the resonance peaks of residues R376, D378, and D379 of Cx43CT (bottom panels). In addition, there was a minor shift in amino acids 343 to 346 (middle). These residues are part of the -helical domains of Cx43CT and may be involved in intramolecular interactions relevant for Cx43 regulation17 (see also Discussion). Similar results were obtained from the exposure of Cx43CT to RXP-4 (supplemental Figure II). Resonance peaks for G291, A323, and I358 are presented as examples of residues whose position was unaffected by the peptides. It is important to note that residues P375 and P377 do not provide an identifiable resonance peak because they do not contain an amide bond; yet these residues may also be a part of the structural modification caused by the presence of the peptide. In contrast, no resonance shifts were observed when Cx43CT was exposed to RXP-1 (data not shown). This is consistent with SPR experiments showing very weak (almost undetectable) interaction between RXP-1 and Cx43CT (Figure 1A). Overall, the data show that RXP-4 and RXP-E alter the conformation of Cx43 residues 343 to 346 and 376 to 379.
Effect of RXP-E on Cx43 Channels
The ability of RXP-E to bind Cx43CT led us to propose that this peptide may also alter the behavior of Cx43 channels. Gap junction currents were recorded from N2a cells transfected with Cx43. To reduce macroscopic currents, cell pairs were superfused with 2 mmol/L octanol.7 To our surprise, the presence of RXP-E in the internal pipette solution prevented octanol-induced uncoupling (ie, reduction of Gj to 0) in all 8 pairs tested. Results are presented in Figure 3A. Data were obtained from cell pairs recorded in control conditions (closed circles; N=7) or when the internal pipette solution contained either RXP-E (solid triangles; N=8), RXP-1 (open squares; N=6) or a peptide containing the same amino acids as RXP-E but in a randomized sequence (open triangles; N=6). Peptide concentration in all cases was 0.1 mmol/L. The plot correlates percentage of junctional conductance (relative to control) as a function of time after onset of octanol superfusion. Clearly, octanol exposure led to a rapid drop in electrical coupling either in control or in the presence of RXP-1 or scrambled RXP-E peptides. However, all cell pairs recorded in the presence of RXP-E remained electrically coupled (ie, Gj did not reach 0) throughout continuous octanol superfusion (supplemental Figure III). The average Gj decreased only to 58.32±6.33% of control, and there was a statistically significant difference between the average Gj measured after 10 minutes of recording in control or in the presence of RXP-E (P<0.001).
Effect of RXP-E on Truncated Cx43 Channels
The results presented in Figures 1 and 2 show that RXP-E binds to Cx43CT. We therefore assessed whether the effect of RXP-E on octanol-induced uncoupling requires the integrity of the CT domain. To assess this hypothesis, we tested the effect of octanol on junctional communication between cells expressing a truncated form of Cx43 lacking most of CT domain (mutant M257; see Morley et al2). Results are shown in Figure 3B. Data obtained from cell pairs exposed to RXP-E (triangles; N=8) and compared with control (circles; N=13). In this case, octanol led to complete uncoupling regardless of the presence of the peptide (see supplemental Figure IIIB). Though there was a delay in the time course (Figure 3B, triangles; 50% decrease in 3.2 minutes versus 1.2 minutes in control), the average reduction in Gj recorded after 10 minutes of octanol superfusion was not statistically different from control (P>0.05). The results suggest that an effect of RXP-E may be preserved after truncation of the CT domain, although the full effect is largely dependent on the integrity of the CT domain.
RXP-E Partially Prevents Acidification-Induced Uncoupling
Next, we assessed whether RXP-E can interfere with the extent and time course of uncoupling induced by reduced intracellular pH (pHi). Patch pipettes were filled with a 2(N-morpholino)ethanesulfonic acid (MES)-containing solution, buffered to a pH of 6.2. Junctional current (Ij) was measured immediately after patch break and every 20 seconds thereafter. Figure 3C shows these results. In the absence of RXP-E, Gj decreased progressively, reaching 12.08±3.48% of control within 15 minutes after patch break (solid circles; N=5). In the presence of RXP-E (solid triangles; N=5), a decrease in Gj was also observed, but it was significantly dampened; after 15 minutes, average Gj decreased only to 45.28±10.89% of the initial value. This value was significantly different from that recorded in control (P<0.05). Interestingly, the presence of the scrambled RXP-E did not disrupt acidification-induced uncoupling (open triangles; N=5; P>0.05 when compared with control). Overall, the data show that RXP-E partially prevented the closure of Cx43 channels consequent to a reduction in pHi.
Effect of RXP-E on Single-Channel Activity
We were able to record spontaneous single-channel activity from 4 Cx43-expressing cell pairs exposed to RXP-E. Results were compared with those obtained in control. Examples of single-channel traces are shown in Figure 4A (Vj=60 mV). Top trace corresponds to control (pipettes filled with normal internal pipette solution); bottom trace was recorded from a different pair, using patch pipettes containing RXP-E (0.1 mmol/L). The control recording shows the characteristic properties of Cx43 channels: 3 states (open, closed, and residual), fast transitions between open and residual, and a unitary conductance of &100 pS in the open state. In contrast, in the presence of RXP-E, the events recorded showed a unitary conductance similar to that of the wild-type channel, but open times were greatly prolonged and the residual state was absent. All-events histograms of unitary conductance are shown in Figure 4B. The data collected from cell pairs in the absence of RXP peptides was best described by 2 Gaussian functions, centered at 80.7 pS and 104.5 pS (left panel labeled "control"; N=5; n=383; where N is number of cell pairs and n represents number of events). These peaks correspond to conductances calculated from transitions between residual and open and closed and open states, respectively. The histogram on the right was obtained from cell pairs recorded with patch pipettes containing RXP-E. Consistent with the loss of the residual state, data were best described by a single Gaussian, centered at 100.5 pS (N=4; n=110). In addition, a prolongation of open time was observed. As shown in Figure 4C, data describing the frequency distribution of dwell open times in control conditions were best fit (least squares minimum) by a monoexponential function with a time constant (t1) of 0.12±0.05 seconds (N=3; n=475; Vj=60 mV). These values were consistent with those previously reported.14 Data collected in the presence of RXP-E were also best fit by a single exponential, but the value of t1 was prolonged (1.27±0.17 seconds; N=3; n=368; Vj=60mV). Overall, the data suggest that RXP-E increases the stability of the open state of the channel, without modifying its unitary conductance.
Discussion
We have conducted a high-throughput assay to identify peptidic sequences that bind Cx43CT. We identified a group of peptides sharing an RXP motif and an excess of basic residues within the sequence. The binding kinetics and structural modification of CT were studied in 3 of these peptides (RXP-1, RXP-4, and RXP-E); 1 peptide (RXP-E) showed a clear effect on Cx43 channel function. Before discussing the implications of our study, some technical issues need to be addressed.
Technical Considerations
Phage display is a powerful screening technique; yet, like any high-throughput method, it is prone to both false-positive and false-negative results. It is possible that, because of experimental conditions, we failed to identify sequences that would be of relevance. Similarly, it is possible that some of the sequences identified do not represent good Cx43CT binders when isolated from the phage. Despite this limitation, the system did provide us with the ability to recognize a group of molecules with the potential to significantly modify channel behavior (Figures 3 and 4).
Our screening of the biased prebound library failed to identify any double sequences. Yet, the majority of bound RXP peptides corresponded to doublets, which suggests that Cx43CT had a strong preference for longer sequences. Whether this is consequent to increased number of RXP motifs, increased number of basic residues (note that at least some of the linkers were also rich on basic amino acids), increased size of the peptide, or any combination of the above remains to be determined. What was clear is that among the "double RXP" series, we found 1 peptide with a high-binding affinity and significant effect on channel function.
The formation of doublets can be explained based on the analysis of the sequences of the linkers. To produce phage with random inserts, a single-stranded oligonucleotide containing a randomized sequence must be replicated using the Klenow fragment of DNA polymerase. This forms a double-stranded DNA fragment with blunt ends. Restriction digestion puts "sticky ends" on this fragment for insertion into phage DNA. In rare cases, 1 of these sticky ends was either not produced because of incomplete digestion or filled after digestion because of continued activity of the DNA polymerase. This led to blunt ends capable of ligation, rather than mismatched sticky ends. A more detailed explanation of this mechanism can be found in supplemental Figure IV. These events were rare enough as to not be detected in the sampling of the library sequences performed before screening.
It is worth noting that the peptides most frequently repeated in the phage display screening were not the ones with the highest binding affinity when tested by SPR. This apparent discrepancy may result from the fact that codon distribution (and consequently, transfer RNA [tRNA] availability) is not equal in the bacteria used to amplify the phage. Accordingly, even if a peptide binds with high affinity, its amplification may be limited by the presence of rare codons in the phage. Consistent with this hypothesis, RXP-E contained the least represented codon in E. coli, likely acting as a limiting factor in its production. The latter emphasizes not only this specific limitation of the phage display method but also the importance of using alternative in vitro techniques to assess binding by the peptides identified through the screening process.
Structural Modifications in Cx43CT
Structural analysis of Cx43CT revealed that both RXP-4 and RXP-E caused a shift in the resonance peaks of amino acids within the 375 to 379 region. Interestingly, this region is within the PDZ binding domain17,21 and near areas relevant for Cx43 phosphorylation.4 Moreover, both peptides also modified the position of residues within the second -helical domain of Cx43CT,22 which is involved in pH-dependent dimerization of the protein.22 By modifying the position of relevant amino acids in space, RXP peptides may interfere with both intra- and intermolecular interactions that could regulate the function of gap junctions. Yet, we are still short of demonstrating a causal link between the specific resonance shifts and the functional effects. Moreover, which residues were modified as a result of a direct physical interaction with the peptide (ie, a binding site) and which ones shifted as a result of a distant secondary effect remain to be determined.
Octanol-Induced Uncoupling and the Effect of RXP-E
RXP-E prevented octanol-induced uncoupling, and the full effect required the integrity of the CT domain. The molecular mechanism by which octanol causes gap junction closure is not completely understood. It has been proposed that volatile anesthetics and long-chain alcohols exert their effects on membrane channels (including gap junctions) via nonspecific actions on the lipid bilayers.23–25 However, recent studies on ligand-gated channels show that n-alkanols exert their functional effect by interacting directly with specific binding pockets in the channel proteins.26 Our results suggest a direct interaction between octanol and Cx43. However, the structural involvement of the CT in this process is unclear, because this domain is not part of the pore-forming region of the channel. Yet, it is important to note that Cx43CT interacts with regions of the channel affiliated with the pore.1,7 It is therefore possible that RXP-E may use the CT as a "scaffolding," from which it interacts with pore-forming or pore-vestibular regions (including the CL domain),1,7 thereby holding the channel in its open state. An RXP-E-dependent delay in octanol-induced uncoupling observed in M257 channels (Figure 3B) suggests a possible interaction of RXP-E with a domain of Cx43 different from the CT.
Previous studies have shown that unsaturated free fatty acids (FFAs) are generated during ischemia and can have an arrhythmogenic effect.27 Other authors have shown that FFAs can cause gap junction channel closure,28 perhaps by a mechanism similar to that of octanol-induced uncoupling.28 Whether RXP peptides can interfere with the arrhythmogenic effects of FFAs remains to be determined.
Effect of RXP-E on pH Gating of Cx43
Also relevant to pathophysiology is the observation that RXP-E partially prevented acidification-induced uncoupling; the latter is considered 1 of the possible substrates for ventricular arrhythmias following myocardial ischemia or infarction.29 Yet, the extent to which pH gating of Cx43 is beneficial or deleterious to function in the ischemic heart remains undetermined. By interfering with gap junction regulation, RXP-E (or future derivatives of it) may serve as a tool to dissect the specific role that gap junction regulation plays in the electrophysiology of the ischemic heart.
Future Implications
Peptides or peptide-derived molecules have been used in the past in an attempt to regulate Cx43. Sequence analysis shows that none of the Cx-modifying peptides previously described carries an RXP motif. A number of those sequences have been derived from Cx43 itself.6,7 Others, although apparently capable of preserving intercellular coupling under certain conditions,30,31 seem to act indirectly via modulation of molecules (such as kinases) that in turn regulate Cx43.32,33 The latter carries a high risk of connexin-unrelated effects on cell function, as kinases are likely to interact with a variety of molecules, not only Cx43. Here we present the first demonstration of a peptidic molecule, exogenous to the cell, that can prevent a form of chemically induced uncoupling likely by direct interaction with Cx43CT. Insofar, RXP-E delivery has required the use of patch pipettes. Yet, our preliminary studies show that RXP-E can be introduced into intact cells by use of peptide transfer domains.34 These sequences are now used widely in the development of peptide-based pharmacophores targeted to intracellular proteins. Much remains to be learned about the effects of RXP peptides on Cx43, other connexins or other channels. The present description opens the door for future development of chemical tools to regulate the function of Cx43-containing gap junctions both in health and in disease.
Acknowledgments
A patent application for the use of RXP peptides on gap junction regulation was filed as US Serial no. 60/758,886 on January 13, 2006.
This work was supported by NIH grants GM57691, HL080602, GM072631, and HL39707 and American Heart Association Grants 0560050Z and 0515623T. The technical assistance of Li Gao, Chris Burrer, and Fang Lu is very much appreciated.
Footnotes
Both authors contributed equally to this study.
Original received December 9, 2005; revision received April 21, 2006; accepted May 1, 2006.
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the Departments of Pharmacology (J.S., R.L., W.C., S.S., M.D.) and Microbiology/Immunology (S.M.T.), State University of New York, Upstate Medical University, Syracuse
Department of Biochemistry and Molecular Biology (F.K., P.L.S.), University of Nebraska Medical Center, Omaha.
Abstract
The carboxyl-terminal domain of connexin43 (Cx43CT) is involved in various intra- and intermolecular interactions that regulate gap junctions. Here, we used phage display to identify novel peptidic sequences that bind Cx43CT and modify Cx43 regulation. We found that Cx43CT binds preferentially to peptides containing a sequence RXP, where X represents any amino acid and R and P correspond to the amino acids arginine and proline, respectively. A biased "RXP library" led to the identification of a peptide (dubbed "RXP-E") that bound Cx43CT with high affinity. Nuclear magnetic resonance data showed RXP-E–induced shifts in the resonance peaks of residues 343 to 346 and 376 to 379 of Cx43CT. Patch-clamp studies revealed that RXP-E partially prevented octanol-induced and acidification-induced uncoupling in Cx43-expressing cells. Moreover, RXP-E increased mean open time of Cx43 channels. The full effect of RXP-E was dependent on the integrity of the CT domain. These data suggest that RXP-based peptides could serve as tools to help determine the role of Cx43 as a regulator of function in conditions such as ischemia-induced arrhythmias.
Key Words: Cx43CT connexin particle-receptor interaction gap junctions
Introduction
Connexins are integral membrane proteins that oligomerize to form intercellular channels called gap junctions. The most abundant gap junction protein in a number of mammalian systems is connexin43 (Cx43). Our previous work has suggested that regulation of Cx43 channels results from the association of the carboxyl-terminal (CT) domain, acting as a gating particle, and a separate region of the connexin molecule, acting as a receptor for the gating particle.1,2 Additional studies have shown that this intramolecular interaction can be modulated by other intermolecular interactions in the microenvironment of the gap junction plaque.3 Thus, the emerging picture of a gap junction plaque is that of a macromolecular complex in which proteins act in concert to modulate intercellular communication. At the center of these interactions is the CT domain, which acts as a substrate for a number of kinases,4 a ligand for noncatalytic proteins, and a gating particle to modify coupling between cells.5
As a key player in the regulation of gap junctions, CT presents itself as a target of chemical6,7 or genetic manipulation intended to modify function.8 Here, we sought to disrupt the regulation of Cx43 by chemical means. Our rationale was based on the knowledge that Cx43CT is capable of interacting with other proteins. We reasoned that this "stickiness" of Cx43CT can be used to "adhere" peptidic sequences to it. We further speculated that the interaction of Cx43CT with small peptides can modify the behavior of the gap junction channel. This rationale was supported by previous work showing that peptides can modify both the chemical and voltage-gating behavior of Cx43.6,7 In the present study, we used a high-throughput phage display screening to find peptidic sequences that bind Cx43CT. Further analysis using a combination of surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and dual patch clamp led to the identification of a specific peptide that binds to Cx43CT with high affinity, affects residues 343 to 346 and 376 to 379 of Cx43, and partially prevents octanol-induced and acidification-induced uncoupling. These results support the feasibility of a peptide-based strategy to manipulate Cx43 regulation in native tissues. These peptides can be used as tools to characterize the specific role of gap junction regulation in health and disease.
Materials and Methods
Phage Display
Recombinant Cx43CT (amino acids 255 to 382 of rCx43) was produced as previously described1 and used as "bait" for peptide screening. For peptide presentation, we used a "library" of bacteriophage, displaying &55 copies of 2.7x109 random 12-mer peptides (Ph.D.-12 Phage display peptide library kit; New England BioLabs Inc). Details of the protein production and screening procedures are outlined in the online data supplement available at http://circres.ahajournals.org.
Biased Phage Display
We used the Ph.D. Peptide Display Cloning System (New England BioLabs Inc) to create a biased phage display library of sequence XXXXRXPXXXX, where X is any amino acid flanking an arginine and a proline with an additional random residue at the center. The randomized peptides were followed by a GGGS spacer. These phages were analyzed for titer and sequence before screening for Cx43CT.
Surface Plasmon Resonance
SPR is a spectroscopic method to determine binding amplitude and kinetics in real time.9,10 Recombinant Cx43CT was covalently bound to a carboxylmethyl dextran matrix.10 Dissociation constants (KD) were calculated from the time course of binding and unbinding of the ligand, using a 1:1 (Langmuir) association and dissociation kinetic model (Biacore software package). In both phases (association and dissociation), the first 5 to 8 seconds of recording were not included in the fit, as to avoid artifacts resulting from peptide distribution within the flow cells.10
Nuclear Magnetic Resonance
All NMR data were acquired on a Varian INOVA 600-MHz NMR spectrometer using a cryoprobe11; the sample temperature was maintained at 7°C. Gradient-enhanced 2D 1H-15N HSQC experiments12 were used to observe all backbone amide resonances in 15N-labeled Cx43CT. Methodological details are presented in the online data supplement.
Electrophysiological Analysis
Experiments were conducted on N2a (neuroblastoma) cells. Cx43 was expressed in a lac-switch stable system (induced by 0.1 to 1.0 mmol/L IPTG; see Zhong et al13) or transiently using an IRES plasmid coding for eGFP.7 M257 (a mutant of Cx43 truncated at amino acid 257; see Morley et al2) was transiently expressed in N2a cells also using an eGFP-containing IRES plasmid. Synthetic peptides were diluted in the pipette solution to a final concentration of 0.1 mmol/L as in previous work.7 For some experiments, octanol (2.0 mmol/L) was superfused during recording. Methods for single-channel recordings and criteria for inclusion and analysis were as reported before,7,14 and as outlined in online data supplement.
Results
Nonbiased Phage Display
Initial control experiments were conducted to standardize the phage display assay. The library was presented to purified streptavidin, a protein known to bind preferentially to peptides containing an HPQ consensus motif.15,16 After 3 rounds of enrichment, 14 phage plaques were selected for sequencing. All 14 showed preservation of the HPQ motif, indicating that the experimental conditions were adequate for appropriate peptide recognition by the target.
The library was presented to Cx43CT. After 3 rounds of selection and amplification, DNA of 156 phage plaques was purified and sequenced. Of the estimated 2.7x109 different sequences in the library, 48 unique sequences were recovered. One particular sequence (PRPTMGNLPDVL), recovered from 45 different plaques, showed homology with a 10 amino acid segment (RATLLNVPDL) of the second PDZ domain of zonula ocludens-1 (ZO-1). When aligned, 5 amino acids were identical and 4 were conserved; binding between the second PDZ domain of ZO-1 and Cx43CT in vivo and in vitro has been well documented.17,18 The results supported the notion that the phage display method can be helpful at recognizing Cx43CT binding sequences.
The "RXP" Motif and Biased Phage Display
Further analysis of the 12-mer peptides revealed that 16 of 48 unique sequences shared a motif RXP (where X represents any amino acid). The RXP motif was not detected in phage that bound to streptavidin. The motif was shown in 11 peptides in the N- to C-terminal orientation and 5 of them in the reverse direction. Specific sequences are presented in Table 1. The RXP sequence occurred in different positions within the 12-mer peptide, thus preventing proper alignments to determine the frequency of other amino acids at specific positions relative to RXP. To overcome this limitation and search for peptides with higher binding affinity (see section on SPR, below), we generated a biased phage display library where the RXP motif was forced to the center of the sequence, flanked at each side by 4 randomized amino acids. Sixty clones containing an insert (of an estimated 3 to 4x104 total clones in the library) were chosen at random for sequencing before exposure of the library to Cx43CT. All inserts coded for an 11-mer peptide with the RXP motif in the appropriate location. In 3 independent runs, the same library was presented to Cx43CT. The binding step was performed at pH 6.5 in 2 of the runs (runs 1 and 2) and at pH 7.4 in 1 additional run (run 3). The total number of phage plaques containing a full-coding insert that were recovered after the binding step were 120, 119, and 163 for runs 1, 2, and 3, respectively. Surprisingly, 89% of all sequences corresponded to "doublets," that is, peptides where 2 RXP 11-mers had been inserted in tandem (see supplemental Table I and Discussion). Although we did not detect a clear preference for specific amino acids in a given position relative to the RXP core, we did observe a preponderance of basic residues in sequences recovered from all screenings (see supplemental Figure I and related text). Furthermore, 5 specific peptides were recovered from all 3 runs and represented a large fraction of the repeats (Table 2). The absence of doublets in the initial library before screening and their high frequency after the binding step strongly suggest that these peptides, although rare, were highly selected for Cx43CT binding.
In Vitro Binding Detected by SPR
Phage display allows for the rapid screening of thousands of peptides, but the characteristics of binding cannot be properly defined. Furthermore, the peptides are part of a capsid protein; the latter may affect the ability of the peptide to properly interact with the target protein. We, therefore, selected some of the peptides identified by phage screening to characterize their ability to bind Cx43CT using SPR. Recombinant Cx43CT was covalently bound to a carboxymethyl dextran matrix and synthetic peptides presented for binding. A total of six 12-mer peptides from the nonbiased screening (peptides labeled RXP-1 to RXP-6; Table 1) and 5 peptides from the biased screening (labeled RXP-A to RXP-E; Table 2) were tested. RXP-2, RXP-3, RXP-5, and RXP-6 showed no significant binding (<100 resonance units of maximal response at peptide concentration of 1 mmol/L). Among the peptides obtained from the nonbiased search, RXP-4 caused the largest deflection in SPR signal, and a small, near-negligible signal was obtained from exposure of Cx43CT to RXP-1. Examples are shown in Figure 1A. RXP-4 (blue line) or RXP-1 (red line) was added at time 0 (peptide concentration was 250 μmol/L). Washout was initiated after 2 minutes of exposure. The peptide dissociated rapidly and completely, suggesting poor binding affinity to Cx43CT. We tested higher concentrations of the peptide but could not detect an asymptotic maximum response. In addition, dissociation rates were too fast to reliably use them to calculate the kinetics of binding. As such, these results do show Cx43CT–RXP-4 binding, but the interaction between the 2 molecules was too weak to allow for proper calculation of kinetic values. A different result emerged from the testing of the doublets. RXP-A and RXP-E showed significant binding to Cx43CT. In particular, peptide RXP-E generated a large, concentration-dependent deflection followed by a slow dissociation on washout (Figure 1B). The transitions were well fit by exponential functions and the rates of association and dissociation were used to estimate kinetic parameters (see Materials and Methods). A full range of concentrations was tested in 3 separate occasions and at 2 different pH values of the solvent (6.5 and 7.4). No differences were observed as a function of pH. From these studies, we calculated a dissociation constant (KD) of 3.9 μmol/L for the Cx43CT-RXP-E interaction. This value is similar to that measured for the association of Cx43CT to binding partners such as the SH3 domain of c-src,3,17 the second PDZ domain of ZO-1,3,17 or the binding of Aquaporin-0 to Cx45.6.19 Overall, these results show that different RXP peptides are able to interact with Cx43CT in vitro with various degrees of affinity. The possibility of structural modifications caused on Cx43CT as a result of RXP interactions (and hence an initial approach to the possible location of the binding site) was assessed next.
Nuclear Magnetic Resonance
Peptides RXP-1, RXP-4, and RXP-E were tested for their ability to modify the structure of Cx43CT. The peptides were diluted in PBS (pH 5.8) containing 15N-Cx43CT, and 15N-HSQC spectra were acquired. An example of the resonance shifts caused by the presence of RXP-E is shown in Figure 2. Resonance contours for Cx43CT in control conditions (Cx43CT alone; no peptides added) are depicted in black. Contours obtained in the presence of RXP-E are labeled red. In previous studies, we have assigned the specific resonance peaks that correspond to each amino acid in the Cx43CT sequence.20 Accordingly, shifts in the resonance assignments directly reveal the identity of the amino acids whose position in space is modified by the presence of the peptide. Addition of RXP-E peptide strongly affected the resonance peaks of residues R376, D378, and D379 of Cx43CT (bottom panels). In addition, there was a minor shift in amino acids 343 to 346 (middle). These residues are part of the -helical domains of Cx43CT and may be involved in intramolecular interactions relevant for Cx43 regulation17 (see also Discussion). Similar results were obtained from the exposure of Cx43CT to RXP-4 (supplemental Figure II). Resonance peaks for G291, A323, and I358 are presented as examples of residues whose position was unaffected by the peptides. It is important to note that residues P375 and P377 do not provide an identifiable resonance peak because they do not contain an amide bond; yet these residues may also be a part of the structural modification caused by the presence of the peptide. In contrast, no resonance shifts were observed when Cx43CT was exposed to RXP-1 (data not shown). This is consistent with SPR experiments showing very weak (almost undetectable) interaction between RXP-1 and Cx43CT (Figure 1A). Overall, the data show that RXP-4 and RXP-E alter the conformation of Cx43 residues 343 to 346 and 376 to 379.
Effect of RXP-E on Cx43 Channels
The ability of RXP-E to bind Cx43CT led us to propose that this peptide may also alter the behavior of Cx43 channels. Gap junction currents were recorded from N2a cells transfected with Cx43. To reduce macroscopic currents, cell pairs were superfused with 2 mmol/L octanol.7 To our surprise, the presence of RXP-E in the internal pipette solution prevented octanol-induced uncoupling (ie, reduction of Gj to 0) in all 8 pairs tested. Results are presented in Figure 3A. Data were obtained from cell pairs recorded in control conditions (closed circles; N=7) or when the internal pipette solution contained either RXP-E (solid triangles; N=8), RXP-1 (open squares; N=6) or a peptide containing the same amino acids as RXP-E but in a randomized sequence (open triangles; N=6). Peptide concentration in all cases was 0.1 mmol/L. The plot correlates percentage of junctional conductance (relative to control) as a function of time after onset of octanol superfusion. Clearly, octanol exposure led to a rapid drop in electrical coupling either in control or in the presence of RXP-1 or scrambled RXP-E peptides. However, all cell pairs recorded in the presence of RXP-E remained electrically coupled (ie, Gj did not reach 0) throughout continuous octanol superfusion (supplemental Figure III). The average Gj decreased only to 58.32±6.33% of control, and there was a statistically significant difference between the average Gj measured after 10 minutes of recording in control or in the presence of RXP-E (P<0.001).
Effect of RXP-E on Truncated Cx43 Channels
The results presented in Figures 1 and 2 show that RXP-E binds to Cx43CT. We therefore assessed whether the effect of RXP-E on octanol-induced uncoupling requires the integrity of the CT domain. To assess this hypothesis, we tested the effect of octanol on junctional communication between cells expressing a truncated form of Cx43 lacking most of CT domain (mutant M257; see Morley et al2). Results are shown in Figure 3B. Data obtained from cell pairs exposed to RXP-E (triangles; N=8) and compared with control (circles; N=13). In this case, octanol led to complete uncoupling regardless of the presence of the peptide (see supplemental Figure IIIB). Though there was a delay in the time course (Figure 3B, triangles; 50% decrease in 3.2 minutes versus 1.2 minutes in control), the average reduction in Gj recorded after 10 minutes of octanol superfusion was not statistically different from control (P>0.05). The results suggest that an effect of RXP-E may be preserved after truncation of the CT domain, although the full effect is largely dependent on the integrity of the CT domain.
RXP-E Partially Prevents Acidification-Induced Uncoupling
Next, we assessed whether RXP-E can interfere with the extent and time course of uncoupling induced by reduced intracellular pH (pHi). Patch pipettes were filled with a 2(N-morpholino)ethanesulfonic acid (MES)-containing solution, buffered to a pH of 6.2. Junctional current (Ij) was measured immediately after patch break and every 20 seconds thereafter. Figure 3C shows these results. In the absence of RXP-E, Gj decreased progressively, reaching 12.08±3.48% of control within 15 minutes after patch break (solid circles; N=5). In the presence of RXP-E (solid triangles; N=5), a decrease in Gj was also observed, but it was significantly dampened; after 15 minutes, average Gj decreased only to 45.28±10.89% of the initial value. This value was significantly different from that recorded in control (P<0.05). Interestingly, the presence of the scrambled RXP-E did not disrupt acidification-induced uncoupling (open triangles; N=5; P>0.05 when compared with control). Overall, the data show that RXP-E partially prevented the closure of Cx43 channels consequent to a reduction in pHi.
Effect of RXP-E on Single-Channel Activity
We were able to record spontaneous single-channel activity from 4 Cx43-expressing cell pairs exposed to RXP-E. Results were compared with those obtained in control. Examples of single-channel traces are shown in Figure 4A (Vj=60 mV). Top trace corresponds to control (pipettes filled with normal internal pipette solution); bottom trace was recorded from a different pair, using patch pipettes containing RXP-E (0.1 mmol/L). The control recording shows the characteristic properties of Cx43 channels: 3 states (open, closed, and residual), fast transitions between open and residual, and a unitary conductance of &100 pS in the open state. In contrast, in the presence of RXP-E, the events recorded showed a unitary conductance similar to that of the wild-type channel, but open times were greatly prolonged and the residual state was absent. All-events histograms of unitary conductance are shown in Figure 4B. The data collected from cell pairs in the absence of RXP peptides was best described by 2 Gaussian functions, centered at 80.7 pS and 104.5 pS (left panel labeled "control"; N=5; n=383; where N is number of cell pairs and n represents number of events). These peaks correspond to conductances calculated from transitions between residual and open and closed and open states, respectively. The histogram on the right was obtained from cell pairs recorded with patch pipettes containing RXP-E. Consistent with the loss of the residual state, data were best described by a single Gaussian, centered at 100.5 pS (N=4; n=110). In addition, a prolongation of open time was observed. As shown in Figure 4C, data describing the frequency distribution of dwell open times in control conditions were best fit (least squares minimum) by a monoexponential function with a time constant (t1) of 0.12±0.05 seconds (N=3; n=475; Vj=60 mV). These values were consistent with those previously reported.14 Data collected in the presence of RXP-E were also best fit by a single exponential, but the value of t1 was prolonged (1.27±0.17 seconds; N=3; n=368; Vj=60mV). Overall, the data suggest that RXP-E increases the stability of the open state of the channel, without modifying its unitary conductance.
Discussion
We have conducted a high-throughput assay to identify peptidic sequences that bind Cx43CT. We identified a group of peptides sharing an RXP motif and an excess of basic residues within the sequence. The binding kinetics and structural modification of CT were studied in 3 of these peptides (RXP-1, RXP-4, and RXP-E); 1 peptide (RXP-E) showed a clear effect on Cx43 channel function. Before discussing the implications of our study, some technical issues need to be addressed.
Technical Considerations
Phage display is a powerful screening technique; yet, like any high-throughput method, it is prone to both false-positive and false-negative results. It is possible that, because of experimental conditions, we failed to identify sequences that would be of relevance. Similarly, it is possible that some of the sequences identified do not represent good Cx43CT binders when isolated from the phage. Despite this limitation, the system did provide us with the ability to recognize a group of molecules with the potential to significantly modify channel behavior (Figures 3 and 4).
Our screening of the biased prebound library failed to identify any double sequences. Yet, the majority of bound RXP peptides corresponded to doublets, which suggests that Cx43CT had a strong preference for longer sequences. Whether this is consequent to increased number of RXP motifs, increased number of basic residues (note that at least some of the linkers were also rich on basic amino acids), increased size of the peptide, or any combination of the above remains to be determined. What was clear is that among the "double RXP" series, we found 1 peptide with a high-binding affinity and significant effect on channel function.
The formation of doublets can be explained based on the analysis of the sequences of the linkers. To produce phage with random inserts, a single-stranded oligonucleotide containing a randomized sequence must be replicated using the Klenow fragment of DNA polymerase. This forms a double-stranded DNA fragment with blunt ends. Restriction digestion puts "sticky ends" on this fragment for insertion into phage DNA. In rare cases, 1 of these sticky ends was either not produced because of incomplete digestion or filled after digestion because of continued activity of the DNA polymerase. This led to blunt ends capable of ligation, rather than mismatched sticky ends. A more detailed explanation of this mechanism can be found in supplemental Figure IV. These events were rare enough as to not be detected in the sampling of the library sequences performed before screening.
It is worth noting that the peptides most frequently repeated in the phage display screening were not the ones with the highest binding affinity when tested by SPR. This apparent discrepancy may result from the fact that codon distribution (and consequently, transfer RNA [tRNA] availability) is not equal in the bacteria used to amplify the phage. Accordingly, even if a peptide binds with high affinity, its amplification may be limited by the presence of rare codons in the phage. Consistent with this hypothesis, RXP-E contained the least represented codon in E. coli, likely acting as a limiting factor in its production. The latter emphasizes not only this specific limitation of the phage display method but also the importance of using alternative in vitro techniques to assess binding by the peptides identified through the screening process.
Structural Modifications in Cx43CT
Structural analysis of Cx43CT revealed that both RXP-4 and RXP-E caused a shift in the resonance peaks of amino acids within the 375 to 379 region. Interestingly, this region is within the PDZ binding domain17,21 and near areas relevant for Cx43 phosphorylation.4 Moreover, both peptides also modified the position of residues within the second -helical domain of Cx43CT,22 which is involved in pH-dependent dimerization of the protein.22 By modifying the position of relevant amino acids in space, RXP peptides may interfere with both intra- and intermolecular interactions that could regulate the function of gap junctions. Yet, we are still short of demonstrating a causal link between the specific resonance shifts and the functional effects. Moreover, which residues were modified as a result of a direct physical interaction with the peptide (ie, a binding site) and which ones shifted as a result of a distant secondary effect remain to be determined.
Octanol-Induced Uncoupling and the Effect of RXP-E
RXP-E prevented octanol-induced uncoupling, and the full effect required the integrity of the CT domain. The molecular mechanism by which octanol causes gap junction closure is not completely understood. It has been proposed that volatile anesthetics and long-chain alcohols exert their effects on membrane channels (including gap junctions) via nonspecific actions on the lipid bilayers.23–25 However, recent studies on ligand-gated channels show that n-alkanols exert their functional effect by interacting directly with specific binding pockets in the channel proteins.26 Our results suggest a direct interaction between octanol and Cx43. However, the structural involvement of the CT in this process is unclear, because this domain is not part of the pore-forming region of the channel. Yet, it is important to note that Cx43CT interacts with regions of the channel affiliated with the pore.1,7 It is therefore possible that RXP-E may use the CT as a "scaffolding," from which it interacts with pore-forming or pore-vestibular regions (including the CL domain),1,7 thereby holding the channel in its open state. An RXP-E-dependent delay in octanol-induced uncoupling observed in M257 channels (Figure 3B) suggests a possible interaction of RXP-E with a domain of Cx43 different from the CT.
Previous studies have shown that unsaturated free fatty acids (FFAs) are generated during ischemia and can have an arrhythmogenic effect.27 Other authors have shown that FFAs can cause gap junction channel closure,28 perhaps by a mechanism similar to that of octanol-induced uncoupling.28 Whether RXP peptides can interfere with the arrhythmogenic effects of FFAs remains to be determined.
Effect of RXP-E on pH Gating of Cx43
Also relevant to pathophysiology is the observation that RXP-E partially prevented acidification-induced uncoupling; the latter is considered 1 of the possible substrates for ventricular arrhythmias following myocardial ischemia or infarction.29 Yet, the extent to which pH gating of Cx43 is beneficial or deleterious to function in the ischemic heart remains undetermined. By interfering with gap junction regulation, RXP-E (or future derivatives of it) may serve as a tool to dissect the specific role that gap junction regulation plays in the electrophysiology of the ischemic heart.
Future Implications
Peptides or peptide-derived molecules have been used in the past in an attempt to regulate Cx43. Sequence analysis shows that none of the Cx-modifying peptides previously described carries an RXP motif. A number of those sequences have been derived from Cx43 itself.6,7 Others, although apparently capable of preserving intercellular coupling under certain conditions,30,31 seem to act indirectly via modulation of molecules (such as kinases) that in turn regulate Cx43.32,33 The latter carries a high risk of connexin-unrelated effects on cell function, as kinases are likely to interact with a variety of molecules, not only Cx43. Here we present the first demonstration of a peptidic molecule, exogenous to the cell, that can prevent a form of chemically induced uncoupling likely by direct interaction with Cx43CT. Insofar, RXP-E delivery has required the use of patch pipettes. Yet, our preliminary studies show that RXP-E can be introduced into intact cells by use of peptide transfer domains.34 These sequences are now used widely in the development of peptide-based pharmacophores targeted to intracellular proteins. Much remains to be learned about the effects of RXP peptides on Cx43, other connexins or other channels. The present description opens the door for future development of chemical tools to regulate the function of Cx43-containing gap junctions both in health and in disease.
Acknowledgments
A patent application for the use of RXP peptides on gap junction regulation was filed as US Serial no. 60/758,886 on January 13, 2006.
This work was supported by NIH grants GM57691, HL080602, GM072631, and HL39707 and American Heart Association Grants 0560050Z and 0515623T. The technical assistance of Li Gao, Chris Burrer, and Fang Lu is very much appreciated.
Footnotes
Both authors contributed equally to this study.
Original received December 9, 2005; revision received April 21, 2006; accepted May 1, 2006.
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