Cellular Biology
Novel Pharmacophores of Connexin43 Based on the “RXP”
Series of Cx43-Binding Peptides
Vandana Verma, Bjarne Due Larsen, Wanda Coombs, Xianming Lin, Gaelle Spagnol, Paul L. Sorgen,
Steven M. Taffet, Mario Delmar
Downloaded from http://circres.ahajournals.org/ by guest on October 2, 2016
Abstract—Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance
intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific
molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-aa peptide
(RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and
action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to
Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development,
remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear
magnetic resonance and patch-clamp strategies to define, for the first time, a unique ensemble of pharmacophores
that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this
family: a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY. These 2
small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers.
Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap
junction channel function. (Circ Res. 2009;105:176-184.)
Key Words: gap junctions 䡲 arrhythmias 䡲 connexin 43
G
and modify its function. Gating of Cx43 relies on an intramolecular particle–receptor interaction between the C-terminal
domain and the cytoplasmic loop.12–15 Using phage display,
we identified a series of peptides containing the sequence
“RXP” (arginine, any amino acid, proline) as a consensus
Cx43CT binding motif and reported that a particular 34-aa
peptide within this RXP series (dubbed RXP-E) binds to
Cx43, prevents heptanol- and low pH–induced gap junction
closure, and prevents action potential propagation block.16,17
Although these studies have shown significant and promising results, further applications of RXP-E are hampered
because of the molecular size and low membrane permeability of this peptide, as well as the metabolic instability and
poor oral bioavailability of peptides in general. Peptide
mimetics, on the other hand, can be developed to retain the
desired biological properties of a peptide. Steps in the design
of mimetic molecules include identification of the essential
active components (or amino acids) of the peptide sequence
(the pharmacophores), determination of their structure/conformation in aqueous solution and finally, development of a
corresponding pharmacophore model.18 Here, we have combined molecular modeling (based on structural analysis of the
RXP series16) and experimental methods to identify the first
ap junctions are intercellular channels formed by oligomerization of connexin proteins. In the heart, the most
abundant connexin is the 43-kDa isotype connexin (Cx)43.
Cardiac gap junctions conduct electric impulses between cells
to maintain normal rhythm, and their closure can be a
substrate for cardiac arrhythmias.1 As such, drugs that selectively open gap junctions may offer a novel strategy for
antiarrhythmic therapy and/or treatment of cardiovascular
disorders.2–5
Gap junction pharmacology is a nascent field (see elsewhere for review6). Recently, hexapeptides such as AAP10
and its stable analog ZP123 (rotigaptide), together with a
novel peptide, GAP134, have been found to modify gap
junctional communication, and to show potential as antiarrhythmic agents.7–10 This accumulated evidence supports the
notion of gap junction modification as a suitable pharmacological target.10 Yet, further development of these molecules
is limited by the fact that their precise molecular target
remains undefined, thus reducing their potential as platforms
for target-specific drug design.11
As an alternative strategy, we have applied knowledge on
the mechanisms of Cx43 chemical gating to design molecules
that bind the carboxyl-terminal domain of Cx43 (Cx43CT)
Original received December 17, 2008; resubmission received May 8, 2009; revised resubmission received June 9, 2009; accepted June 12, 2009.
From the Center for Arrhythmia Research (V.V., X.L., M.D.), Department of Internal Medicine, University of Michigan, Ann Arbor; Zealand Pharma
(B.D.L.), Glostrup, Denmark; Department of Microbiology and Immunology (W.C., S.M.T.), Upstate Medical University, State University of New York,
Syracuse; and Department of Biochemistry and Molecular Biology (G.S., P.L.S.), University of Nebraska Medical Center, Omaha.
Correspondence to Address correspondence to: Mario Delmar, MD, PhD, Center for Arrhythmia Research, University of Michigan Medical School,
5025 Venture Dr, Ann Arbor MI 48104. E-mail mdelmar@umich.edu
© 2009 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.109.200576
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Verma et al
group of pharmacophores (cyclized and linear peptides 6 to 8
aa long), that bind Cx43CT and prevent closure of Cx43
channels. This ensemble of pharmacophores represents a new
platform for future development of small molecules with high
efficacy and affinity that can prevent closure of gap junctions.
Furthermore, we provide the first 3D imprint of a potential
site in Cx43CT that can be used for binding of exogenous
molecules.
Novel Pharmacophores With Cx43 Action
A
V
K
R
H2N-F P-P
R
Y
D
R
6 Asn
5 Tyr
1 Arg
Y
P-P
K
L
F-NH2
Y
Linker
amino acid
H-R
R
A
RB
P-P
C
Materials and Methods
P-P
V
H-R
R
L
H-R
Experimental methods for molecular modeling, electrophysiological
experiments, surface plasmon resonance, and GST-binding assays,
as well as nuclear magnetic resonance experiments followed standard, previously published procedures.16,19,20 Details (including statistical analysis) are provided in the Online Data Supplement at
http://circres.ahajournals.org.
B
P-P
H-R
177
P-P
Y-NH 2
2 Arg
4 Pro
3 Pro
E
Y
Results
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Cyclized Hexapeptides Based on Analysis of the
RXP Series
Molecular Modeling
Our previous studies revealed that 12-mer peptides that bind
Cx43CT share at least 2 common features (see the previous
report,16 including table 1 and online figure I of that article):
the (1) presence of an RXP motif and (2) a predominance of
basic amino acids. Thus, for identification of a core active
element with Cx43CT binding activity, we modeled the
structure of a peptide that fulfilled the following criteria: (1)
was capable of binding Cx43CT, (2) showed an abundance of
RXP sites, (3) was strongly basic, and (4) its primary
sequence suggested a high-ordered secondary structure. A
peptide labeled “RXP-4” by Shibayama et al16 best fulfilled
these criteria. Analysis of its sequence (RRPPYRVPPKLF)
showed 4 different RXP motifs (one of them in C-to-N
direction, another placed terminally), a balance of charge of
⫹4 at physiological pH (7.4), and a combination of 2
proline–proline repeats likely to induce turns in the peptide,
thus facilitating a helix-like conformation. We therefore used
RXP-4 as a starting point to identify a novel Cx43CT-binding
platform. Molecular modeling predicted that, in an ␣-helical
conformation, the proline–proline repeats in RXP-4 would
face opposite directions (Figure 1A). If extended, this ␣-helix
yielded 2 horseshoe conformations (Figure 1B), both containing RXP motifs. Separating the 2 horseshoe-like sequences
between tyrosine in position 5 and arginine in position 6 (red
dotted line in Figure 1B) yielded 2 shorter peptides, both
having terminal RXP motifs and a high content of basic
residues (RRPPY and RVPPKLF; see Figure 1B). Given that
RRP is a terminal RXP motif in the original RXP-4 peptide,
the RRPPY sequence was chosen for further analysis.
In a pentapeptide of sequence RRPPY, amino acids R1 and
Y5 are placed at the opposite terminal ends, and the horseshoe conformation is anticipated to be at equilibrium with a
corresponding “open” linear conformation (see Figure 1C).
Thus, to stabilize the horseshoe conformation and keep the
correct distance between R1 and Y5, the peptide was
backbone-cyclized using an asparagine (N6) as a linker
amino acid between R1 and Y5. The cyclic peptide (cycloRRPPYN) is illustrated in Figure 1D (small numbers mark
R
R
Figure 1. Strategy for identification of Cx43-binding pharmacophores based on RXP series.16 A, Position of amino acids in
peptide RXP-4 when modeled as an ␣-helix. B, Representation
of ␣-helix in A as 2 horseshoe-like structures. Red dotted line
marks site where sequence was separated for further study. C,
Representation of 2 likely configurations of peptide RRPPY, as a
horseshoe, or as linearized molecule. D, Placement of asparagine between first arginine and last tyrosine fixed the R1–Y5
spacing into a cyclic conformation (compound CyRP-61). E,
Molecular modeling results comparing position of arginines and
tyrosine residues in RXP-4 with those in CyRP-61.
the position given to each amino acid). This peptide was
dubbed “CyRP-61” because it was the first cyclized, RXPderived hexapeptide in this new series (see Table). Molecular
modeling predicted an excellent correlation of the position in
space of side chains R1R2Y5 in CyRP-61 (green in Figure
1E) with the equivalent R1R2Y5 side chains of RXP-4 (red in
Figure 1E). Similar results were obtained by the conservative
substitution N-Q at position 6 (CyRP-62; see the Table). This
design strategy, previously used to transform peptide AAP10
to a more potent tripeptide analog, was predicted to generate
more stable, smaller peptides with a core active structure
capable of binding Cx43CT. This prediction was tested by
surface plasmon resonance (SPR) experiments, as described
below.
Table.
Sequence of Cyclic RXP-Derived Peptides
Name
Molecular Mass (Da)
Sequence
CyRP-61
783.89
Cyclo-RRPPYN
CyRP-62
797.92
Cyclo-RRPPYQ
CyRP-63
806.93
Cyclo-RRPPWN
CyRP-71
954.11
Cyclo-RRPPYRQ
CyRP-72
961.10
Cyclo-RRPPYRN
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July 17, 2009
Resonance Units
CyRP-62
C
Normalized Gj
-40 -20 0
1.4
1.2
1.0mM
500µM
250µM
125µM
62.5µM
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Time (sec)
CyRP-63
Control
1.0
0.8
0.6
0.4
0.2
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0.0
CyRP-62
Control
0
20 40 60 80 100 120
D
Normalized Gj
300
250
200
150
100
50
0
-50
-100
Normalized Gj
B
A
100 200 300 400 500 600
Time (sec)
1.2
CyRP-61
Control
1.0
0.8
0.6
0.4
Figure 2. CyRP peptides bind to Cx43CT and interfere with octanol-induced uncoupling of Cx43 channels. A, Surface plasmon resonance results. Change
in angle of incidence of resonance (“resonance
units”) plotted against time after onset of peptide
exposure. Upward and downward arrows indicate
onset and washout of CyRP-62 superfusion. B
through D, Patch-clamp results from N2A cells
expressing Cx43. Peptides were diluted in patch
pipettes (100 ␮mol/L). Time zero: onset of octanol
superfusion. Red and black symbols indicate,
respectively, results from cells exposed to a given
peptide or those kept as control. Significance values
were P⬍0.001, P⫽0.148, and P⫽0.019 for data in
B, C, and D, respectively. (Each data set compared
to results obtained without peptide.)
0.2
0.0
0
100 200 300 400 500 600
Time (sec)
0
100
200
300
400
500
600
Time (sec)
CyRP-62 Binds to Cx43CT
SPR allows for assessment of ligand–analyte binding in real
time and was used for characterization of the RXP series.16
Cx43CT was covalently bound to the matrix of a sensor chip.
Figure 2A shows plots of angle of incidence of resonance (in
“response units”) as a function of time. Various concentrations of CyRP-62 were introduced in the microfluidics system
at the time indicated by the upward arrow. Despite the small
size of the analyte (798 Da), which approached the detection
limits of the SPR instrument (⬇500 to 700 Da), a clear
change in resonance was recorded, thus indicating direct
binding of CyRP-62 to Cx43CT. Dissociation ensued on
washout (downward arrow). The rapid time course of association and dissociation precluded us from direct calculation
of the dissociation constants. Yet, the results show that
CyRP-62 binds Cx43CT. In previous studies, we demonstrated that peptide RXP-E prevents chemical gating of
Cx43.16 As a next step, we tested whether this property was
preserved in the CyRP peptides.
Patch-Clamp Results
Junctional conductance was measured in N2a cell pairs
expressing Cx43. Patch pipettes were filled with an internal
solution containing the test peptide. Time course and extent
of octanol-induced uncoupling was compared to that observed in the absence of the peptide. Figure 2B shows average
measurements of junctional conductance (Gj) recorded at
various times after the onset of octanol superfusion. Red
symbols and line correspond to data obtained when the
cyclized hexapeptide CyRP-62 was dissolved in the internal
pipette solution, and black symbols correspond to the average
time course of octanol-induced uncoupling in control conditions. The data show that, in the presence of the cyclized
peptide, the progression of octanol-induced uncoupling was
delayed, and 100% of cells remained coupled after 10
minutes after of octanol superfusion (see also Online Figure
I, A). The minimum Gj value recorded at the end of the
10-minute octanol exposure was significantly different from
that obtained in the absence of the peptide (P⬍0.01).
The results in Figure 2B suggested that CyRP-62 contains
an active core sequence capable of interfering with octanolinduced uncoupling. Other studies have demonstrated the
importance of aromatic side chains in the preservation of
pharmacophore activity.18 Consistent with this notion, substitution of Y5 with a larger aromatic residue (W) disrupted the
activity of the peptide (Figure 2C; also see the Table; peptide
CyRP-63). Indeed, only 1 of 6 pairs remained coupled at
the end of octanol exposure (Online Figure I, B), and the
average Gj value was not statistically different from 0
(P⬎0.05). The side chains of N and Q differ in having only
one and 2 methylene groups, respectively. Consistent with
this structural preservation, the activity of peptide CyRP-61
(N in position 6) was similar to that observed for CyRP-62
(Figure 2D; Online Figure I, C). These data show that CyRP
peptides represent a new class of core active molecules that
interfere with Cx43 chemical gating. Additional improvement
was achieved by increasing the balance of charge via addition
of an arginine residue in the cyclized sequence.
Increased Charge: Cyclized Heptapeptides
One of the characteristic features of RXP peptides was the
high balance of positive charge in their sequence (see16).
Thus, an alternative cyclized peptide (in this case, a heptapeptide) was generated by separating the RXP-4 sequence
between amino acids R6 and V7 (blue line in Figure 3A), then
forming a backbone-cyclic peptide using either asparagine or
glutamine to complete the structure (see also the Table;
peptides labeled CyRP-71 and CyRP-72, respectively). These
peptides, although somewhat larger, had an increased electrostatic balance (⫹3 instead of ⫹2). As shown in Figure 3B,
an overlay of CyRP-71 with CyRP-61 reveals that both
peptides share similar coordinates for the position of amino
acids R1, R2, and Y5, whereas CyRP-71 presents an additional arginine (R6). This simple modification had a significant impact on the binding Cx43CT, as demonstrated by the
Verma et al
A
Novel Pharmacophores With Cx43 Action
179
B
V
H-R
R
P-P
K
R B
A
Y5
R6
Y5*
L
R1*
F-NH2
Y
R1
R2*
P-P
R2
D
C
CyRP-71
Control
1.4
CyRP-71
1.0mM
500µM
250µM
125µM
62.5µM
31.25µM
15.63µM
7.8nM
Normalized Gj
Resonance Units
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1400
1200
1000
800
600
400
200
0
-200
-40 -20 0
1.2
1.0
0.8
0.6
0.4
0.2
Figure 3. Addition of an arginine in CyRP
sequence improved function. A, Diagram
indicating RXP-4 sequence. Here, the
peptide was separated between R6 and
V7. B, Cyclized heptapeptide (CyRP-71;
red) showed strong correlation in space
with analog CyRP-61 (green and asterisks), although an arginine was exposed
as potential site for additional interaction
with a ligand. C, SPR results demonstrated binding of CyRP-71 to a sensor
chip coated with recombinant Cx43CT. D,
Time course of octanol-induced uncoupling in Cx43-expressing N2a cells. Cell
pairs were recorded with pipettes containing CyRP-71 (100 ␮mol/L; red symbols) or
only the internal filling solution (black symbols). Difference between groups was
significant (P⬍0.001).
0.0
20
40
60
80 100 120
Time (sec)
0
100 200 300 400 500 600
Time (sec)
SPR experiment shown in Figure 3C (compare data with
those in Figure 2A; notice different scale in the ordinates).
When normalized by molecular weight, the amplitude of the
SPR response elicited by CyRP-62 (125 ␮mol/L) was 3.71 (in
arbitrary units); in comparison, the response elicited by
CyRP-71 was 9 times higher (33.92 arbitrary units; immediately consecutive experiments conducted on the same sensor
chip). In fact, the molecular mass–normalized response from
CyRP-71 binding was larger than that obtained from RXP-E
on the same sensor chip (9.91 arbitrary units), although there
was a noticeable difference in the time course of the reaction
(data not shown). Finally, patch-clamp results showed that
CyRP-71 also prevented octanol-induced uncoupling (Figure
3D). In fact, there was an improvement in the ability of this
core active component to prevent uncoupling after insertion
of the basic residue, further underlying the importance of the
balance of charge in the activity of these compounds (see also
Online Figure I, D). Overall, this is the first demonstration of
small cyclized peptides that can interfere with the octanolinduced closure of Cx43 channels.
Structural Correlation Between RXP-E
and CyRP-71
In previous studies, we reported that RXP-E prevents chemically induced uncoupling of Cx43 channels, and action
potential propagation block.16,17 Here, we tested whether
structural features thought to be of relevance in the cyclized
peptides were also present in RXP-E. Figure 4 (top) shows
the entire RXP-E sequence (in the one letter amino acid
code), divided into 3 regions: the first RXP-containing
domain (the amino side; colored red), the linker (green), and
the second RXP-containing domain (the carboxyl side; colored blue). Figure 4A and 4C shows the overlay of peptide
CyRP-71 (green) with either the carboxyl terminus (amino
acids 29 to 34) or the amino terminus (amino acids 1 to 11)
of RXP-E (red), respectively. Clearly, the spatial coordinates
of the basic residues R1, R2, and R6 of the cyclic peptide
match with those of residues K29, R31 and R34 of RXP-E.
The similar dimensions, spatial distribution and electric
charge of the core residues make it likely for the carboxyl
terminal region of RXP-E and the cyclic peptides to share a
common binding motif. The opposite conclusion can be
drawn from the comparison of the cyclic peptide with the
amino end (N-terminal) of RXP-E; in that case, the space
occupied by R6 in the cyclized peptide would correspond to
the location of 2 acidic residues (D2, D3) in RXPE. If binding
between Cx43CT and the peptide is mediated, at least in part,
by electrostatic forces, the presence of negative charges at a
relevant position in space would prevent binding. Overall,
these modeling results led us to predict that binding of RXP-E
to Cx43CT occurs via the carboxyl end of RXP-E. The SPR
results shown in Figure 4B and 4D were consistent with this
hypothesis. Duplicates of either the carboxyl side, or the
amino side of RXPE, separated by the linker region, were
used (see sequences at the top of Figure 4B and 4D). As
shown in Figure 4B, a significant change in the angle of
incidence for resonance was detected when a peptide containing the carboxyl end of RXPE was presented to Cx43CT
(upward and downward arrows represent the addition of the
peptide and its washout, respectively). In contrast, no binding
was detected for the peptide formed by the amino end of
RXP-E and its linker (Figure 4D). Overall, these results show
a convergence between modeling predictions and experimental results and suggest defined structural constraints for the
binding of RXP-derived peptides to Cx43CT. This structural
information led us to the prediction of a minimum Cx43CT
binding motif.
Identification of a Minimally Active Motif: RRNY
Cyclized compounds show longer bioavailability given their
limited degradation in the intracellular space. An alternative
path toward pharmacological development is the use of
180
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July 17, 2009
RXPE: SDDLRSPQLHNGGGSAVPFYSHSHMVRRKPRNPR
A
B
HMVRRKPRNPRGGGSAVPFYSHSHMVRRKPRNPR
1400
Resonance Units
K29
R6
R31
R1
R34
R2
D2 R6
200
0
-200
-20
0
20
40
60
800
100
1.0mM
250µM
25µM
600
400
200
0
-200
-40
-20
0
20
A
40
60
B
N
100
whether RRNY-containing peptides could bind Cx43CT.
Assessment of binding was carried out by SPR. Recombinant
Cx43CT was used as ligand, covalently linked to the sensor
chip, and peptides were presented as analytes. The mass of
the analyte was increased by concatenating 2 “RRNY” motifs
(ie, RRNYRRNY). An example of sensograms obtained on
introduction of this peptide into the chip containing Cx43CT,
is presented in Figure 5C. Clearly, angle of incidence for
resonance changed rapidly on onset of superfusion (time
“zero”) and returned to baseline on washout (see downward
arrow). The rapid transitions signaled a fast on– off ligand–
analyte interaction and prevented us from determining dissociation constants for these peptides. However, the data
showed that RRNYRRNY was capable of interacting with
Cx43CT in a concentration-dependent manner. Similar re-
Y
Y
R
R
P
R
R
P
C
D
RRNYRRNY
RRPPYN
Control
1 mM
500µM
250µM
100µM
25µM
500
400
300
200
100
0
Normalized Gj
1.0
600
0.8
0.6
0.4
0.2
0.0
-100
0
80
Time (sec)
synthetic scaffolds that mimic peptide structure. Toward that
aim, we sought to minimize the active sequence of the
cyclized compounds by deleting the proline residues in
CyRP-61 (Figure 5A). Indeed, in an ␣-helix, the proline–
proline component would retain R1, R2 and Y5 at the
appropriate spatial coordinates (see also Figure 1C). We
therefore speculated that, because this spatial conformation
was achieved by introduction of N6, the prolines would no
longer be essential to hold the relative distances. Molecular
modeling confirmed this expectation, as shown by the overlay
of the side chains for R1, R2, and Y4 in a short tetrapeptide
RRNY (green) with R1, R2, and Y5 of the cyclized CyRP-61
hexa-peptide (red; Figure 5B). Functional and biochemical
assays were therefore designed to assess the prediction that
RRNY is a Cx43CT binding motif. As a first step, we tested
-20
80
SDDLRSPQLHNGGGSAVPFYSHSSDDLRSPQLHN
1000
R2
Resonance Units
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H10
R1
400
Resonance Units
D3
R5
1000
800
600
Time (sec)
D
C
40µM
20µM
10µM
5µM
2.5µM
1.25µM
0.625µM
0.313µM
1200
Figure 4. Structure–function comparison
of CyRP-71 with RXP-E. Top, Amino acid
sequence (1-letter code) of RXP-E (see
also elsewhere16). A, Molecular overlay of
predicted spatial position for amino acids
R1, R2, and R6 of CyRP-71 (green) with
positively charged amino acids K29, R31,
and R34 in the carboxyl end of RXP-E
(red). L spatial correlation was consistent
with the ability of peptide to bind Cx43CT
by SPR (B; peptide sequence on top;
peptide concentrations as noted). C, At
the amino end of RXP-E, overlay of positively charged residues R5 and H10 of
RXP-E (red) R1–R2 of CyRP-71 (green)
predicts that 2 acidic residues (D2 and
D3) occupy the position held by a basic
amino acid (R6) in CyRP-71. The latter
would be inconsistent with occupation of
the same binding pocket. D, As predicted,
a peptide of the amino end of RXP-E
(sequence at top) failed to bind Cx43CT
by SPR.
20
40
60
Time (sec)
80 100
0
100 200 300 400 500 600
Time (sec)
Figure 5. Strategy for identification of sequence
RRNY as potential core active molecule. A, Proline
residues of hexapeptide CyRP-61 were removed,
under the assumption that the structure formed by
residues R1, R2, and Y5 represented the core
Cx43-binding element. B, Overlay of predicted
positions of amino acids R1, R2, and Y5 of
CyRP-61 with those of RRNY. C, SPR traces
obtained by presenting peptide RRNYRRNY to
Cx43CT. Peptide concentrations as noted. D, Peptide RRNYRRNY in the pipette (red symbols) prevented octanol-induced uncoupling of Cx43
(P⫽0.005). Octanol uncoupling proceeded as control (black symbols) in the presence of a linearized
version of CyRP-61 (RRPPYN; green symbols;
P⫽0.63).
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Normalized Gj
A 1.0
CyRP-71
Control
0.8
0.6
0.4
0.2
0.0
0
B
0.8
0.6
0.4
0.2
0.0
0
100 200 300 400 500 600
CyRP-71
Control
1.0
0.8
0.6
0.4
0.2
0.0
0
200 400 600 800 1000 1200
Time (sec)
100 200 300 400 500 600
Time (sec)
D
Normalized Gj
C
181
CyRP-71
Control
1.0
Time (sec)
Normalized Gj
sults were obtained in 3 separate runs. Additional studies
showed that RRNY-containing peptides can pull down Cx43
from an adult rat heart lysate preparation (Online Figure II).
The binding results led us to assess whether the motif
RRNY would be sufficient for preventing octanol-induced
uncoupling. Cx43-expressing N2a cells were dialyzed with an
internal pipette solution containing peptide RRNYRRNY.
Time course of octanol-induced uncoupling is shown in
Figure 5D. In the presence of the peptide, average Gj recorded
10 minutes after the onset of octanol was significantly
different from that recorded in control at the same time point
(P⬍0.05) and coincided with the preservation of electric
coupling in 4 of the 6 cells studied (see also Online Figure III,
A). These results indicate that RRNY is a minimum sequence
capable of interfering with the chemical regulation of gap
junctions by octanol. Moreover, because amino acids RRNY
are contiguous in the cyclized CyRP-61 peptide (see Figure
1D and the Table), we speculated that a linearized (noncyclic)
RRPPYN peptide would fail to affect uncoupling, given the
loss of continuity of the RRNY motif. As expected, the time
course and extent of octanol-induced uncoupling in the
presence of the linear RRPPYN peptide was not different
from control (Figure 5D; trace in green; also Online Figure
III, B), thus supporting the notion that pharmacophore activity is related not only to the presence of specific amino acids,
but to the preservation of their molecular conformation in
space.
Novel Pharmacophores With Cx43 Action
Normalized Gj
Verma et al
CyRP-71
RRPPYR
Control
1.0
0.8
0.6
0.4
0.2
0.0
0
200
400
600
800
Time (sec)
Figure 6. CyRP-71 did not prevent octanol-induced uncoupling
in N2a cells expressing Cx43 mutant M257 (A) or an alternative
connexin isotype, Cx40 (B). CyRP-71 prevented acidificationinduced uncoupling of Cx43-expressing N2a cells (C). CyRP-71
(0.1 mmol/L) was diluted in internal pipette solution buffered to
pH 6.2. In the absence of peptide, Gj decreased to 17.3⫾1.7%
of initial value. In the presence of CyRP-71, Gj decreased only
to 49.0⫾5.9% of maximum (P⫽0.002). D, CyRP-71 prevented
acidification-induced uncoupling of neonatal rat ventricular gap
junctions (pH 6.2). Fifteen minutes after patch break, Gj in control and in the presence of linear peptide RRPPYR decreased to
8.4⫾3.2% and 16.6⫾6.5% of initial value, respectively (probability value RRPPYR, P⫽0.48 when compared to control). In the
presence of CyRP-71 (0.1 mmol/L), Gj decreased only to
47.0⫾9.5% of maximum (P⫽0.006).
CyRP-71 and the Integrity of the Cx43CT Domain
Figures 1 through 5 describe the various steps taken to
identify potential leading compounds. Additional experiments focused on CyRP-71, given its binding and functional
efficacy, and its potential as a more biologically stable
compound. We hypothesized that binding of CyRP-71 to
Cx43CT is linked to the ability of the peptide to prevent
uncoupling. As shown in Figure 6A, this hypothesis was
supported by experiments demonstrating that CyRP-71 failed
to prevent octanol-induced uncoupling in Cx43 channels
lacking the C-terminal domain (mutant M25716). In addition,
this peptide did not modify the time course of octanolinduced uncoupling in N2a cells expressing a different
connexin isoform, Cx40 (Figure 6B). Consistent with this
observation, CyRP-71 caused only a minor SPR deflection
when interacting with recombinant Cx40CT (Online Figure IV). The overall data suggest that there is a degree of
structural specificity to the effect of CyRP-71 and that the
Cx43CT domain is an essential component for peptide
action.
CyRP-71 and Acidification-Induced Uncoupling
Octanol served as a screening tool to identify peptide activity.
However, a more biologically relevant question is whether, as
in the case of RXP-E, the candidate compound can prevent
low pH-induced block.16 Experiments were conducted both in
Cx43-expressing N2a cells (Figure 6C) and in pairs of
neonatal cardiac myocytes (Figure 6D). Patch pipettes were
filled with an internal solution buffered to pH 6.2. In the
absence of peptide (black symbols and traces) Gj decreased
progressively, reaching minima of 17.3⫾1.7% in N2a cells
(Figure 6C) and 8.4⫾3.2% (Figure 6D) in cardiac myocytes.
In contrast, cells exposed to CyRP-71 remained coupled
throughout the same time course, Gj decreasing only to
49.0⫾5.9% (6C) and 47.0⫾9.5% (6D) of control (red symbols). The green symbols in Figure 6D depict data obtained
with a linearized peptide RRPPYR. Results were not different
from control, indicating that the structure of CyRP-71, rather
than only the net balance of charge, was important for its
functional effect.
CyRP-71 and Cx43CT Interaction Resolved
by NMR
The SPR results demonstrated direct interaction between
CyRP-71 and Cx43CT. Yet, further refinement of CyRP-71
will require a better understanding of the structural constraints imposed by its binding site in Cx43CT. As an initial
step, we identified the amino acids in Cx43CT whose position
in space change when presented to the peptide. Resonance
assignments for Cx43CT have been published before (X).
Figure 7A and 7B shows an 15N-HSQC spectrum for Cx43CT
alone (black) which has been overlaid with spectra obtained
in the presence of CyRP71 (red). A summary of the Cx43CT
residues affected by CyRP-71 is presented in Figure 7C. The
predominant regions of resonance shifts were: (1) near or
within the proline-rich region of Cx43CT (K258-A276); (2)
in the extreme end of the C-terminal domain (I382); and (3)
182
A
Circulation Research
July 17, 2009
108
B 118.8
G270
C271
G285
Black: Cx43CT Control
Red: Cx43CT/CyRP71
G261
Y267
112
119.8
T275
116
N15
N294
S273
F335
Q308
N302,Q304
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R293
L356
124
120.8
C271 Y267
N300
Y286
N15
S314
S272
120
Y265
Q308
K264
N269
V289
R299
Y265
Y286
F337
F268
121.8
K287
A305
A266
A315
F268
C
255
128
8.8
A276
8.6
8.4
8.2
H1
8.0
7.8
8.27
Cx43CT
8.17
H1
8.07
7.97
382
SPSKDCGSPKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNAKKVAAGHELQPLAIVDQRPSSRASSRASSRPRPDDLEI
strongly affected CT residues
weakly affected CT residues
Figure 7. NMR analysis of structural modification of recombinant Cx43CT when in the presence of CyRP71. A, Cx43CT was titrated
with CyRP71 to 1:500 and 1:900 molar ratio. 15N-HSQC spectrum for Cx43CT alone (black) was overlaid with spectra obtained in the
presence of CyRP71 (1:500, red; 1:900, green). B, Close-up of resonance peaks from box in A. C, Summary of Cx43CT residues
affected by CyRP71.
in the region contained within amino acids G285 to E316 of
Cx43. These results confirm the binding of Cx43CT to
CyRP-71 and provide a first lead as to the amino acids in the
C-terminal domain that are involved in the interaction. Future
studies will be needed to determine which residues form the
actual binding site and which shifts may result from secondary changes, perhaps some consequent changes in Cx43CT
dimerization.21
Discussion
We have previously demonstrated that RXP-E binds to
Cx43CT and prevents chemically induced gap junction closure.16 As such, RXP-E represented a proof of principle, and
the RXP series a starting point to develop a pharmacophore
model for new compounds capable of modifying gap junctions. Structure–function analysis of the RXP series led us to
a new ensemble of pharmacophores that bind Cx43CT and
prevent chemically induced uncoupling. To our knowledge,
the 2 core active structures hereby reported (one cyclized; one
linearized) represent the smallest known Cx43CT-binding
molecules with regulatory activity over gap junctions. More-
over, CyRPs are the first known cyclized molecules capable
of binding Cx43CT. Thus, our results represent a potential
foundation for development of target-based gap junction
pharmacology.
Our compounds represent a new a pharmacophore model
for Cx43 regulation. Yet, major milestones need to be
reached before considering them of practical application.
First, our studies demonstrate that selected peptides prevent
closure of Cx43 channels induced by octanol superfusion.
CyRP-71 also prevented pH-induced uncoupling. Yet, other
assays will be needed to obtain a wider view of the overall
functional effects of this peptide on Cx43. Second, we have
shown that our candidate compounds interact with Cx43, but
a remaining question is whether these peptides bind other
cellular components (including other ion channel proteins)
and alter their function and/or regulation. Additional studies
will be required to characterize the selectivity and specificity
of these compounds and their range of pharmacological
applications. Yet, it is important to keep in mind that RXP-E,
a leading molecule from the RXP series, prevents action
potential propagation block without modifying other non-
Verma et al
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junctional membrane channels,17 and our studies show structural similarities between the pharmacophores and the active
domain of RXP-E. A lack of increase of Gj during application
of both RXPE17 and CyRP71 may also indicate that these
pharmacophores act not by recruiting new channels but by
stabilizing the open state of those at the membrane. Third, we
chose to explore the effect of CyRP-71 at one peptide
concentration. Thus, we know that 100 ␮mol/L CyRP-71
delivered through the patch pipette is enough to prevent
octanol, and low pH–induced uncoupling (Figure 6). Yet,
further structure-based refinement of the pharmacophore will
be needed to improve its affinity for the target. Fourth, the
linkage between Cx43CT binding in SPR and the patchclamp data may still seem correlative, although the hypothesis of a causative association is supported by results obtained
with the mutant lacking Cx43CT (Figure 6A). These caveats
notwithstanding, an important goal has been achieved: to find
a compound of low molecular mass (⬍1 kDa) that binds the
regulatory domain of Cx43, affects function, and shows
selectivity for Cx43 over Cx40. Improving the affinity and
selectivity of these compounds will make them valuable as
potential scaffolds to act as carriers for cargoes with biological activity.
Our functional studies are complemented with NMR analysis of Cx43CT in the presence of CyRP-71. The data provide
us with initial identification of those amino acids whose
position in space is affected by the peptide (Figure 7).
Differences were found with the resonance shifts caused by
RXP-E.16 Of note, resonance shifts detected by HSQC may
be consequent to direct binding, distant changes in conformation, and/or modifications of the dimerized state of
Cx43CT.21 The larger, more complex structure of RXP-E
(several “RXP” domains; likely more than one binding site)
may cause a wider range of modifications, direct and indirect,
that overlap (and may obscure) those occurring within the
binding pocket. The cyclized peptide, on the other hand, may
be less restricted for secondary interactions and therefore
yield a different resonance shift map. Future identification of
the actual binding pocket will be carried out by nuclear
Overhauser effect spectroscopy (NOESY). This analysis will
be an important step toward optimizing pharmacophore
binding and selectivity. Overall, these peptides represent a
potential imprint of a region of Cx43CT that is amenable for
binding to exogenous molecules that will affect the function
of the Cx43 channel as a whole. Combined structural and
biological studies may lead to a future generation of molecules of higher efficacy, selectivity and biological stability,
capable of crossing the cell membrane barrier to reach their
target in a living cell. Although the latter is only a goal, our
data suggest that we are heading in the right direction.
Identification of the CyRP group of compounds bears relevance, because cyclic peptides are more stable within the
intracellular space and, as such, have more potential for
future pharmacological applications.
Peptide RRNYRRNY failed to prevent uncoupling in
⬇40% of the cell pairs studied. From that standpoint, its
efficacy was less than that previously described for RXP-E.
On the other hand, CyRP-71 showed an effect similar to that
Novel Pharmacophores With Cx43 Action
183
of RXP-E. Interestingly, CyRP-71 also shows homology with
the Cx43CT-binding element of RXP-E. This cyclized molecule offers itself as an excellent platform for the next
generation of compounds, using peptide–mimetic substitutions on the core structure to minimize the size and maximize
the activity, stability, and bioavailability while preserving
pharmacological effect.
In summary, we have described a new generation of
Cx43-binding peptides. Our efforts focused on a series of in
silico modeling steps, combined with biochemical and cellular experiments, to identify the core active structure of the
RXP series. We have identified new candidate molecules
capable of binding Cx43CT and preventing chemically induced uncoupling of Cx43 channels. This is the first demonstration of a small, cyclic core active structure that chemically
and functionally interacts with Cx43 to prevent gap junction
closure. Our data opens a new line of investigation for
development of target-based gap junction pharmacology.
Sources of Funding
Supported by NIH grants HL39707, HL087226, GM57691, and
GM072631 (to P.L.S.) and a grant from the Carol M. Baldwin
Research Fund (to S.M.T.).
Disclosures
B.D.L. holds modest ownership interest in the compounds detailed in
this article, which were provided by Zealand Pharma.
References
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2. Dhein S. Cardiac ischemia and uncoupling: gap junctions in ischemia and
infarction. Adv Cardiol. 2006;42:198 –212.
3. Srinivas M, Duffy HS, Delmar M, Spray DC. Prospects for pharmacological targeting of gap junction channels. In: Zipes DP, Jalife J, eds.
Cardiac Electrophysiology: From Cell to Bedside. 4th ed. Philadelphia,
Pa: Saunders; 2004:158 –167.
4. Eloff BC, Gilat E, Wan X, Rosenbaum DS. Pharmacological modulation
of cardiac gap junctions to enhance cardiac conduction: evidence supporting a novel target for antiarrhythmic therapy. Circulation. 2003;108:
3157–3163.
5. Haugan K, Petersen JS. Gap junction modifying antiarrhythmic peptides:
therapeutic potential in atrial fibrillation. Drugs Future. 2007;32:
245–260.
6. Lewandowski R, Petersen JS, Delmar M. Connexins as potential targets
for cardiovascular pharmacology. In: Zipes DP and Jalife J, eds. Cardiac
Electrophysiology: From Cell to Bedside. 5th ed. Philadelphia, Pa:
Saunders. 2009;205–213
7. Müller A, Schaefer T, Linke W, Tudyka T, Gottwald M, Klaus W, Dhein
S. Actions of the antiarrhythmic peptide AAP10 on intercellular coupling
.Naunyn Schmiedebergs Arch Pharmacol. 1997;356:76 – 82.
8. Kjolbye AL, Haugan K, Hennan JK, Petersen JS. Pharmacological modulation of gap junction function with the novel compound rotigaptide: a
promising new principle for prevention of arrhythmias. Basic Clin
Pharmacol Toxicol. 2007;101:215–230.
9. Rossman EI, Liu K, Morgan GA, Swillo RE, Krueger JA, Butera J,
Gruver M, Kantrowitz J, Feldman HS, Petersen JS, Haugan K, Gardell SJ,
Hennan JK. The gap junction modifier, GAP-134, improves conduction
and reduces atrial fibrillation/flutter in the canine sterile pericarditis
model. J Pharmacol Exp Ther. 2009;329:1127–1133.
10. Axelsen LN, Haugan K, Stahlhut M, Kjølbye AL, Hennan JK, HolsteinRathlou NH, Petersen JS, Nielsen MS. Increasing gap junctional coupling: a tool for dissecting the role of gap junctions. J Membr Biol.
2007;1:23–35.
11. Dhein S, Polontchouk L, Salameh A, Haefliger JA. Pharmacological
modulation and differential regulation of the cardiac gap junction proteins
connexin 43 and connexin 40. Biol Cell. 2002;94:409 – 422.
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12. Delmar M, Coombs W, Sorgen P, Duffy HS, Taffet SM. Structural basis
for the chemical regulation of connexin43 channels. Cardiovasc Res.
2004 62:268 –275.
13. Morley GE, Taffet SM, Delmar M. Intramolecular interactions
mediate pH regulation of connexin43 channels. Biophys J. 1996;70:
1294 –1302.
14. Ek-Vitorin JF, Calero G, Morley GE, Coombs W, Taffet SM, Delmar M.
pH regulation of connexin43: molecular analysis of the gating particle.
Biophys J. 1996;71:1273–1284.
15. Seki A, Coombs W, Taffet SM, Delmar M. Loss of electrical communication, but not plaque formation, after mutations in the cytoplasmic loop
of connexin43. Heart Rhythm. 2004;1:227–233.
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Taffet SM, Delmar M. Identification of a novel peptide that interferes
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Novel Pharmacophores of Connexin43 Based on the ''RXP'' Series of Cx43-Binding
Peptides
Vandana Verma, Bjarne Due Larsen, Wanda Coombs, Xianming Lin, Gaelle Spagnol, Paul L.
Sorgen, Steven M. Taffet and Mario Delmar
Circ Res. 2009;105:176-184; originally published online June 25, 2009;
doi: 10.1161/CIRCRESAHA.109.200576
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2009 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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World Wide Web at:
http://circres.ahajournals.org/content/105/2/176
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2009/06/25/CIRCRESAHA.109.200576.DC1.html
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ONLINE SUPPLEMENT. MATERIAL AND METHODS
Molecular modeling
The HyperChem (version 7.5, Hyper Cub. Inc.) program was used for visualization of
spatial structures, overlays and conformations. Force-field analysis was conducted
using Amber94, and for optimization, the Polak-Ribiere algorithm (conjugate gradient)
was applied.
Electrophysiological experiments
Patch clamp experiments were conducted in N2a cells transiently transfected with either
human Cx43, rat Cx40 or with a Cx43CT-truncated mutant coding for amino acids 1-257
of rat Cx431,2. In all cases, the dual-whole-cell voltage clamp technique was used to
record gap junction currents. Specifics for cell culture, transfection and recordings are
described elsewhere.1 Briefly, N2a cells were obtained from American Type Culture
Collection; Manassas, VA). Cells were grown in DMEM (Gibco, Invitrogen, Grand Island,
NY) and supplemented with 10% fetal bovine serum, 5000U/L penicillin and 5mg/ml
streptomycin (Mediatech, Herndon, VA). Cells were plated at 35-40% and transiently
transfected with the above-mentioned plasmids. Transfections were carried out using
between 0.25-0.5µg of plasmid DNA and Effectene (Qiagen, CA) according to the
manufacturer’s instructions. Transfection efficiency ranged between 70 and 80%. In all
cases, the dual-whole-cell voltage clamp technique was used to record gap junction
currents. Both cells in the pair (cell 1 and cell 2) were independently voltage clamped at
the same holding potential (-40 mV). The prejunctional cell (cell 1) was stepped to +20
mV, creating a potential difference across the junction (Vj) of + 60 mV during repetitive
10-30 sec steps. The current injected by the amplifier in cell 2 to maintain the holding
potential of that cell (-40 mV) during the voltage step in cell 1 was considered to be
equal and opposite to the current flowing through the gap junctions (Ij)
Junctional
conductance (Gj) was calculated from Ohm’s law (Gj = Ij/Vj).
Octanol superfusion was initiated 5 minutes after patch break and continued for 10
minutes. Concentration of octanol was 1.5 mM in all experiments. For experiments
assessing acidification-induced uncoupling patch pipettes were filled with a 2(Nmorpholino)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. Peptides were diluted in the internal pipette solution to a final
concentration of 100μM. A total of 20 experiments were carried out where octanolinduced uncoupling was tested in the absence of peptides. These experiments were
averaged, and data used as control for comparison with those series where a given
peptide was assessed.
Cell dissociation and culture of rat neonatal ventricular myocytes (NRVMs)
All experiments involving animals conformed to the protocols in the Guide for the Care
and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996). Primary
cultures of NRVMs for patch clamping, immunofluorescence, and monolayers were
obtained using established procedures3,4. Ventricular myocytes from neonatal SpragueDawley rats (Charles River, Mass) were isolated and cultured according to Rohr et al.
5,6
Briefly, the hearts from 1 and 2 day old rats were aseptically removed and collected in
calcium- and magnesium-free Hanks’ Balanced Salt Solution (HBSS; without Ca2+ and
Mg2+; Sigma). The ventricles were minced and incubated in a solution containing 0.125%
trypsin (Roche Applied Science) and 0.15% pancreatin (Sigma). Digestion took place at
36°C in consecutive steps. Two hour periods of differential preplating were used to reduce
the presence of noncardiomyocytes. Cells were then suspended in medium M199
(Cambrex) containing 10% fetal bovine serum (FBS) (Mediatech, Herndon, VA), 20 U/mL
penicillin, 20 mg/mL streptomycin, and 100 μmol/L bromodeoxyuridine (Sigma) to inhibit
fibroblast proliferation. Cells were plated on 35-mm tissue culture dishes at a low density
or in 22-mm coverslips for patch clamp experiments.
GST-Pulldown assay
Bound GST fusion proteins were incubated with approximately 15 mg of pre-cleared rat
or mouse heart lysate in 1ml of lysis buffer for 90 minutes, rocking at 4ºC. A separate
sample was incubated with lysis buffer only as a control. Unbound proteins were
removed by washing 2 times with lysis buffer. The final pellet was resuspended in
Laemmli sample buffer and probed by western blotting as described below.
Western blots for Cx43
The pulldown pellets from the GST-fusion protein assay described above were kept at
55°C in Laemmli buffer (10μl Laemmli buffer per 50μl of sample) for 10 minutes.
Samples were run on an 8-16% tris-glycine gradient gel, transferred to nitrocellulose
membranes and blocked for 1 hour at room temperature (blocking buffer consisted of
1% non-fat milk and 0.05% Tween in PBS). Membranes were then incubated in primary
o
antibodies overnight at 4 C followed by incubation in secondary antibody (anti-mouse
HRP, Sigma) for 45 minutes at room temperature. Signal was detected by
chemiluminescence (Pierce, SuperSignal West Pico, Chemiluminescent Substrate). The
primary antibody used was a monoclonal mouse Cx43 (diluted 1:100 in 0.05% Tween in
PBS) that recognizes the amino terminal (NT) domain of Cx43 (Fred Hutchinson Cancer
Research Center). Bands were analyzed by densitometry using Adobe Photoshop 7.0
(2002, Adobe Systems Inc., San Jose, CA) and background subtracted for each sample
Surface Plasmon Resonance (SPR)
SPR is a spectroscopic method to determine binding amplitude and kinetics in real time7.
Experimental details were as described previously1. Briefly, recombinant rat Cx43CT
was covalently bound to a carboxylmethyl dextran matrix and used as ligand. Peptides
were presented to the bound ligand, and the amplitude of the response was subtracted
from that obtained from a control chamber with no ligand7. No change in angle of
resonance was taken as an indication of absence of binding. The amplitude of the
change in angle of resonance (expressed as “response units”) is a function of the mass
of the analyte7 and therefore, for comparison, response units were calibrated by the
molecular weight of the tested compound. Data obtained for various concentrations,
tested sequentially on the same chip by serial 50:50 dilutions, are presented. For all
peptides presented in the paper, three separate runs were conducted for at least one
concentration.
Nuclear Magnetic Resonance (NMR)
All NMR data were acquired at 7°C using a 600 MHz Varian INOVA NMR spectrometer
outfitted with a cryo-probe. Gradient-enhanced two-dimensional
were used to observe all backbone amide resonances in
15
N-HSQC experiments
15
N-labeled Cx43CT in
presence or absence of unlabelled CyRP71. Data were acquired with 1024 complex
points in the direct dimension and 512 complex points in the indirect dimension. Sweep
widths were 10,000 Hz in the proton dimension and 2,500 Hz in the nitrogen dimension.
Statistical analysis
When appropriate, data were statistically compared by Student’s t test. Comparisons
were made utilizing the average asymptotic Gj value of either control, or peptide-treated
cells. Only one comparison (against control) was allowed for each data set. Differences
yielding p values <0.05 were regarded as significant. Average data are presented as
mean +/- standard error of the mean.
ONLINE RESULTS
Online Figure I
Kaplan-Meier plot showing percentage of cell pairs that remained coupled (i.e., Gj>0) at
the end of each minute after onset of octanol. Time zero corresponds to the onset of
octanol (1.5 mmol/L) superfusion (Black line absence of peptide; red line presence of
peptide under test). A: Effect of CyRP-62 on octanol-induced uncoupling in Cx43expressing N2a cells. In the absence of CyRP-62, 20 cell pairs were completely
uncoupled (Gj=0) whereas octanol failed to uncouple cell pairs when CyRP-62 was
present in the pipette solution (0.1 mmol/L; N=6). B: Effect of CyRP-63 on octanolinduced uncoupling in N2a cells; CyRP-63 treated cells showed delayed uncoupling,
only one out of five cell pairs (20%) remained coupled after 10 minutes of octanol
treatment. C: Effect of CyRP-61 on octanol-induced uncoupling in Cx43- expressing N2a
cells. In the absence of CyRP-61, all 20 cell pairs were completely uncoupled (Gj=0)
whereas octanol failed to uncouple 60% of cell pairs when CyRP-61 was present in the
pipette solution (0.1 mmol/L; N=20 in control; N=5 in the presence of CyRP-61). D:
Effect of CyRP-71 octanol-induced uncoupling in Cx43-expressing N2a cells. In the
absence of CyRP-71 20 cell pairs were completely uncoupled (Gj=0) whereas octanol
failed to uncouple cells when CyRP-71 was present in the pipette solution (0.1 mmol/L;
N=6).
Online Figure II
Western blot of Cx43 from samples obtained from a GST-pulldown assay. Symbols “+“
and “-” represent a sample obtained from tubes where the glutathione bead-bound
recombinant protein was presented (“+”) or not (“-“) to the heart lysate. Glutathione
beads were bound to either GST alone (first two lanes), RXP-E (lanes 3 and 4) of ZP71.
The latter is a protein of the sequence:RRNYGGGSAVPFYSHSRRNYGGGSAVPFYSHSRRNY i.e., three RRNY domains,
bound by the same linker sequence as in RXP-E.
Online Figure III
Kaplan-Meier plot showing percentage of cell pairs that remained coupled (i.e., Gj>0)
after the perfusion of octanol. Time zero corresponds to the onset of octanol superfusion
(Black line - absence of peptide; red line - presence of peptide under test). A: Effect of
RRNYRRNY on octanol-induced uncoupling in Cx43- expressing N2a cells. In the
absence of RRNYRRNY, all 20 cell pairs were completely uncoupled within 600 seconds
(Gj=0) whereas octanol (1.5mmol/L) failed to uncouple 60% of cell pairs when
RRNYRRNY was present in the pipette solution (0.1 mmol/L; N=5). B: Effect of
RRPPYN on octanol-induced uncoupling in N2a cells (N=20 in control; N=5 in the
presence of RRPPYN). All cells treated with peptide were uncoupled by 220 seconds of
octanol treatment.
Online Figure IV
Surface plasmon resonance trace obtained from presenting peptide CyRP-71 to
recombinant
Cx40CT,
covalently
bound
to
a
carboxylmethyldextran
surface.
Concentration of the peptide was 125 µM. Notice that the amplitude of the sensogram is
only a small fraction of that obtained from a chip seeded with Cx43CT (see Figure 3 in
the manuscript). Together with patch clamp data in Figure 6B, these data indicate that
CyRP-71 has selectivity for Cx43 over the related isotype, Cx40.
References
1.Shibayama J, Lewandowski R, Kieken F, Coombs W, Shah S, Sorgen PL, Taffet SM
and Delmar M.
Identification of a novel peptide that interferes with the chemical
regulation of connexin 43. Circ Res. 2006; 98:1365-72.
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B 100
100
% of coupled cells
% of coupled cells
A
CyRP-62
80
60
40
20
0
0
CyRP-63
80
60
40
20
0
100 200 300 400 500 600
0
Time (sec)
Time (sec)
D
100
CyRP-61
80
% of coupled cells
% of coupled cells
C
60
40
20
0
0
100
CyRP-71
80
60
40
20
0
100 200 300 400 500 600
Time (sec)
100 200 300 400 500 600
Online Figure I
0
100 200 300 400 500 600
Time (sec)
-
+
GST
-
+
RXPE
Online Figure II
-
+
ZP 71
A
B
RRNYRRNY
% of coupled cells
% of coupled cells
100
80
60
40
20
0
0
100 200 300 400 500 600
RRPPYN
100
80
60
40
20
0
0
100 200 300 400 500 600
Time (sec)
Time (sec)
Online Figure III
Resonance Units
350
300
250
200
150
100
50
0
-50
-50
0
50
100
150
200
Time (sec)
Online Figure IV