Nanomechanics of Hemichannel Conformations CONNEXIN FLEXIBILITY UNDERLYING CHANNEL OPENING AND...

Nanomechanics of Hemichannel ConformationsCONNEXIN FLEXIBILITY UNDERLYING CHANNEL OPENING AND CLOSING*

Received for publication, May 25, 2006, and in revised form, June 9, 2006 Published, JBC Papers in Press, June 12, 2006, DOI 10.1074/jbc.M605048200

Fei Liu1, Fernando Teran Arce1, Srinivasan Ramachandran1, and Ratnesh Lal2

From the Neuroscience Research Institute, University of California, Santa Barbara, California 93106

Gap junctional hemichannelsmediate cell-extracellular com-munication. A hemichannel is made of six connexin (Cx) sub-units; each connexin has four transmembrane domains, twoextracellular loops, and cytoplasmic amino- and carboxyl-ter-minals (CTs). The extracellular domains are arranged differ-ently at non-junctional and junctional (gap junction) regions,although very little is known about their flexibility and confor-mational energetics. The cytoplasmic tail differs considerably inthe size and amino acid sequence for different connexins and ispredicted to be involved in the channel open and closed confor-mations. For large connexins, such as Cx43, the CTmakes largecytoplasmic fuzz visible under electron microscopy. If this CTdomain controls channel permeability by physical occlusion ofthe pore mouth, movement of this portion could open or closethe channel. We used atomic force microscopy-based singlemolecule spectroscopy with antibody-modified atomic forcemicroscopy tips and connexin mimetic peptide modified tips toexamine the flexibility of extracellular loop andCTdomains andto estimate the energetics of their movements. Antibody to theCTportion closer to themembrane stretches the tail to a shorterlength, and the antibody to CT tail stretches the tail to a longerlength. The stretch length and the energy required for stretch-ing the various portions of the carboxyl tail support the ball andchain model for hemichannel conformational changes.

Gap junctions are intercellular channels that directly coupletwo apposing cells. They allow diffusion-driven intercellulartransfer of ions and small molecules (�1000 Da), synchronizeelectrical activity, and regulate metabolic homeostasis, cellgrowth, and differentiation (1–3). Structurally, a gap junction ismade of two hemichannels (connexons) aligned head-to-headacross an extracellular “gap,” which is roughly cylindrical with acentral ion-permeable pore (4–9).Each connexon comprises six connexin proteins, and each

connexin has cytoplasmic amino and carboxyl termini, fourmembrane-spanning regions, two extracellular loops (ELs),3

and a single cytoplasmic loop. There is very limited variation inthe sequences of two disulfide-linked extracellular loops, whichare crucially positioned for the docking of the connexons toform gap junctions (3, 6). Docking between connexons involvesinteractions between the extracellular loops (7, 10). The detailsof how the extracellular loops are arranged and dock are cur-rently unresolved. Current models based on three-dimensionalstructural studies suggest that hemichannel docking involvesmultivalent interactions of subunits between hemichannels. Inaddition, hemichannels are also present in the non-junc-tional regions wherein their extracellular loops with manyhydrophobic amino acids would require a different conforma-tion to shield the hydrophobic amino acids from the aqueousenvironment (11).The opening and closing of hemichannels, in response to

both membrane potential and chemical signals, are beingexamined extensively, and several models, based on structuralinformation from x-ray diffraction and electron microscopy,have beenproposed for gap junction opening and closing. In theearly model by Unwin and Zampighi (12, 13), they proposedthat the channel is closed by differential rotations of apposinghemichannels thus narrowing the pore diameter. But, little isknown about non-junctional hemichannels present in the freemargins of the plasma membrane. Sosinsky and coworkershave reported calcium-induced extracellular conformationalchanges in Cx26 hemichannels using atomic force microscopy(AFM) (8). Recently, Thimm et al. (14) have proposed calcium-dependent folding/refolding of extracellular loops leading toaltered pore mouth opening or closing. They obtained three-dimensional conformations of the open and closed Cx43hemichannels reconstituted in lipid bilayer and have providedan estimate of conformational energetics (14). Such informa-tion is missing about the channel gating from the cytoplasmicside. For both hemichannel and gap junctional gating, espe-cially for connexins with a large cytoplasmic tail such as Cx43,Delmar and coworkers (15, 16) proposed the “particle-recep-tor” hypothesis similar to the “ball and chain” model of potas-sium channels. According to this, the long cytoplasmic tail“chain” folds into a “ball” (gating particle) and binds to the cyto-plasmic loop domain (a possible receptor) and leads to channelclosure (16, 17). However, little structural or mechanistic dataare available to support this model.AFM-based single molecule force spectroscopy is being used

as an effective approach to define protein folding and unfoldingand three-dimensional conformations. The binding of ligandsimmobilized on AFM tips to their receptors adsorbed on a sur-face is examined by applying a force to the receptor-ligandcomplex until the bond breaks at a measurable unbinding force

* The work was supported by the National Institutes of Health and the PhilipMorris External Research Program. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

1 These authors contributed equally to this work.2 To whom correspondence should be addressed: Neuroscience Research

Institute, University of California, 5126 BioSci II Bldg., Santa Barbara, CA93106. Tel.: 805-893-2350; Fax: 805-893-2005; E-mail: [email protected].

3 The abbreviations used are: EL, extracellular loop; AFM, atomic force micros-copy; CT, carboxyl-terminal; PBS, phosphate-buffered saline; PEG, polyethyl-ene glycol; NHS, N-hydroxysuccinimide; N, newton(s); pN, piconewton(s); FJC,Freely-Joint-Chain; DOPC, 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 32, pp. 23207–23217, August 11, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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(18–20). Such an approach has been applied to investigate thebinding potentials of receptor-ligand pairs involved in celladhesion (21), polysaccharide elasticity (22), DNA mechanics(23), and the function of molecular motors (24).In the present study, using single molecule force spectros-

copy, we examined the interactions between individual anti-Cx43 antibody and the carboxyl-terminal (CT) region of full-length Cx43 hemichannels reconstituted in supported lipidbilayer as well as between connexin mimetic peptides to theCx43 extracellular loop and reconstituted Cx43 hemichannels.Antibodies to specific connexin epitopes have been used to pro-vide some qualitative details about the hemichannel structureand subunit interactions (14, 25). Previously, connexinmimeticpeptides, short synthetic peptides to specific sequences inconnexins EL and cytoplasmic loop domains, have been usedto block the functional gap junction coupling in a variety ofsystems (26, 27).Our results provide distinct signatures of the extracellular

and cytoplasmic sides of hemichannels reconstituted in bilayer:mimetic peptide recognizes the EL loops, whereas anti-CTantibodies recognize the CT domain. Antibody binding to theCT domain displays typical single interactions and molecularstretches. The extendedmolecule stretch is consistent with theelongated random coil nature of the Cx43 CT domain and issensitive to calcium. The extent of stretching the various por-tions of the carboxyl tail suggests the ball and chainmechanismfor hemichannel conformational changes.

EXPERIMENTAL PROCEDURES

Hemichannel Isolation and Purification—Wild-type BICR-M1Rk (Marshall) cell line expressing Cx43 was grown asdescribed previously (28). Connexons were isolated asdescribed (14, 29) with modification. The entire procedure wascarried out at 4 °C. Briefly, confluent cell cultures were har-vested and homogenized in Hanks’ buffer containing 20 mMHEPES (pH 7.4), 1 mM MgCl2, dithiothreitol, and proteaseinhibitor mixture I (Calbiochem). The lysate was centrifuged at1,000 � g for 5 min, and the supernatant was collected andcentrifuged at 100,000 � g for 1 h. The pellet was resuspendedin Hanks’ buffer with 0.25 M sucrose, loaded on a 1.2/2.0 Msucrose step gradient, and centrifuged at 100,000 � g for 2.5 h.The plasmamembrane fraction containing connexons between1.2 and 2.0 M sucrose gradients was collected and solubilizedwith buffer S (20 mM HEPES, 50 mM NaCl, 50 mM octylglu-coside, pH 7.4). The solution was centrifuged at 100,000� g for30min, and the supernatantwas incubatedwith anti-CT360–382polyclonal antibody (Chemicon, Temecula, CA)-coupledSepharose 4B beads for 2 h under gentle stirring. The beadswere then transferred onto a column and washed with 50 ml ofbuffer S containing 1 mg of phosphatidylcholine/ml. Connex-ons were eluted with buffer S containing 5 �M control peptideto anti-CT360–382 (Chemicon) and 1 mg of phosphatidylcho-line/ml. The purity of extracted connexons was evaluated bysilver staining andWestern blot following SDS-polyacrylamidegel electrophoresis as described before (30).Hemichannel Reconstitution into Supported Bilayers—1,2-

Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, AvantiPolar Lipids, Alabaster, AL) was dissolved in chloroform, vacu-

um-desiccated overnight, and then resuspended in phosphate-buffered saline (PBS, pH7.2–7.4) at a concentration of 1mg/ml.Liposomes were formed by extruding DOPC through a set of0.4-�m pore size filters using a LipoFast device from AvestinInc. (Ottawa, Canada). To reconstitute connexons into lipo-somemembrane, purified connexons were thenmixedwith theliposomes (�1:1000 w/w protein: lipid) and bath-sonicated for15 min. The liposomes were then deposited on freshly cleavedmica for 20 min, rinsed with PBS, and heated at 37–39 °C for2–3 min to fuse liposomes into planar bilayers (31, 32).Immunofluorescence Microscopy—1, 2-Dipalmitoyl-sn-glyc-

ero-3-phosphoethanolamine-N-(Lissamine Rhodamine B sul-fonyl) wasmixedwithDOPCat a ratio of 1:1000. Planar bilayerswith reconstituted hemichannels were prepared on glass cov-erslips using the procedure described above. The coverslipswere washed with PBS and placed in blocking solution (PBScontaining 3%bovine serumalbumin and 1% goat serum) for 30min at room temperature. Samples were then incubated withmouse anti-Cx43monoclonal antibody against the cytoplasmicdomain CT252–270 (Chemicon) in PBS (1�g/ml) for 2 h at roomtemperature. Samples were rinsed with PBS for at least fivetimes. They were then incubated with fluorescein isothiocya-nate-conjugated goat anti-mouse IgG (1 �g/ml, ZyMaxTM,Invitrogen) for 2 h at room temperature (33). Samples werethen washed again with PBS. Fluorescence images were cap-tured using an Olympus IX71 inverted microscope with 100�oil lens (numerical aperture, 1.45). Immunofluorescenceimages were collected with red channel filter set (Ex 482/Em536 nm) for bilayers and with green channel filter set (Ex525/Em 585 nm) for connexons. Bilayers reconstituted withconnexons but without any antibody incubation, bilayersreconstituted with connexons incubated with secondary anti-bodies directly, and bilayers without connexon reconstitutionbut incubated with both primary and secondary antibodieswere also imaged as controls.Preparation of Functionalized AFM Tips—Mouse anti-Cx43

monoclonal antibody against the cytoplasmic domain CT252–270and mouse anti-Cx43 polyclonal antibody against CT360–382(Chemicon), connexin mimetic peptide GAP26 (VCYDKSF-PISHVR) and scrambled GAP26 (PSFDSRHCIVKYV) (Sigma-Genosys, Haverhill, UK) were used to functionalize the AFMtips as described (34) with slight modification. Briefly, standard200-�m-long Si3N4 tips (Veeco, Santa Barbara, CA) and 450-�m-long silicon tips (Applied NanoStructures, Santa Clara,CA) were cleaned with chloroform three times and dried withN2. Tips were then washed with ethanol twice and dried withN2. The tips were incubated overnight in 5.6 M ethanolamine-HCl dissolved in Me2SO over molecular sieve beads (pore size,0.33 nm) and washed three times with Me2SO followed by twotimes with ethanol, and then dried with N2. A synthesized het-ero-bifunctional polyethylene glycol (PEG) tether with alde-hyde and N-hydroxysuccinimide (NHS) reactive ends (alde-hyde-PEG-NHS)was attached to theAFM tip by incubating thetips for 2 h at room temperature in a solution containing 3.3mgof tether molecule, 0.5 ml of chloroform, and 10 �l of triethyl-amine. The functionalized tips were then washed twice withchloroform and dried with N2. Finally, the tips were incubatedin 200 �l of antibody or peptide (0.2 mg/ml in PBS) for 50 min

Intermolecular Force Spectroscopy of Connexin Hemichannels

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to link the free NH2- on the antibody or peptide to the func-tionalized tips. 2�l of 1MNaCNBH3 (32mgofNaCNBH3, 50�lof 100 mMNaOH in 450 �l of H2O) were added to the solutionat the very beginning of the reaction. At the end of the reaction,5 �l of 1 M ethanolamine (in 20% NaOH) was added to thesolution to passivate unreacted aldehyde groups. Functional-ized tips were washed and stored in PBS buffer at 4 °C until use.Cantilever Calibration—Cantilever spring constants were

measured using two methods. For cantilevers with a nominalspring constant of 0.06 newton (N)/m (Veeco), the spring con-stant of the functionalized tip was estimated by measuring thespring constants with standard force calibration cantilevers asdescribed previously (35). The spring constant of antibody- orpeptide-conjugated tips was measured to be k � 0.063 � 0.005N/m (n � 5) and is consistent with published results (36).Whenever feasible, we used the same conjugated tip for exper-iments involving internal control: for example, in mappinginteractions between antibody and receptor alone, adding on-line inhibitor of their interactions. For cantilevers with a nom-inal spring constant of 0.02 N/m (Applied Nanostructures), amodified (developed by Ben Ohler from Veeco) thermal noisemethod (37) was used to determine their spring constants.Briefly, the cantilever was withdrawn from the surface and wasallowed to oscillate freely at its natural frequency. An AFMimage (512 � 512 pixels) recording the cantilever deflection ofthese oscillations was obtained at a point sampling rate of�62.5 kHz (i.e. 61 Hz lines/s). A MatLab (MathWorks, Natick,MA) code was written to obtain the power spectrum density ofthese oscillations and by application of the equipartition theo-rem, the cantilever spring constant was calculated. The meas-ured spring constant was �0.014–0.016 N/m.AFM and Force Measurement—AFM images were collected

using a NanoScope III Multimode AFM (Digital Instruments).The AFM probes were oxide-sharpened silicon nitride (Si3N4)tips and were operated at frequencies of 5–35 kHz with freeoscillating amplitudes of �20–35 nm. Most of the AFM imag-ing was carried out in tapping mode with a scanning frequencyof 0.5–2 Hz that allowed simultaneous collection of the heightand amplitude data. All AFM imaging of lipid bilayers with orwithout reconstituted Cx43 connexons was conducted at roomtemp (22–24 °C) in PBS buffer (pH 7.2–7.4) as described (28).Force-volume maps were obtained to collect force curves

between antibody/mimetic peptide (conjugated to the AFMtip) and connexons reconstituted in the planar lipid bilayer.One complete force curve was recorded at each position whilethe AFM tip was scanned across the surface of the bilayer in64 � 64 pixels. The force curves were collected with a z-deflec-tion rate of 1–4 nm/ms, a z-scan size of 400 nm, and a maxi-mum cantilever deflection (relative trigger) of 20 nm.To determine the specificity of the antibody-Cx43 or

GAP26-Cx43 interaction, 100 �l of 50 �g/ml antibody or pep-tide (the same ligands conjugated on tips) was then added intothe imaging solution, and successive force-curves were col-lected again after 20 min of incubation with the blocking anti-body or peptide. As an additional control experiment, forcecurves were also recordedwith a scrambledGAP26-conjugatedtip in identical experimental conditions.

Data Analysis—A MatLab (MathWorks, Natick, MA) codewas used to analyze the force-distance curves obtained in force-volumemap as described (38). This program identifies unbind-ing events by searching for all significant local minima in aretraction curve. To differentiate unbinding events from noise,filters were introduced so that unbinding events with adhesionforces below a given threshold were not considered. A smallfraction of force-distance curves were identified as corruptcurves, e.g. due to excessive noise or ill-defined approachand/or retraction curves, and were excluded from the analysis.Each retraction curve was analyzed by first finding the horizon-tal region in the approach curve. The zero force line was deter-mined as the average value of points in the horizontal region.The intersection of this line with the retraction curve identi-fies the tip-surface contact point and defines the origin fromwhich the polymer extensions and the magnitude of the adhe-sion/unbinding forces aremeasured in the retraction curve. Foranunbinding event, the adhesion force corresponds to themag-nitude of the force in the retraction curve measured from thezero force line. The corresponding separation distance, d, rep-resents the distance traveled by the piezo from the origin of thecurve (corresponding to the initial horizontal cantilever posi-tion) to the unbinding event. The polymer extension length, L,is simply given by subtracting the cantilever deflection, �, fromthe piezo displacement, L � d � �. The unbinding force isdetermined from the difference between the forces corre-sponding to a local minimum and the next local maximum inthe direction of increasing values of the separation distance.The probability of finding either single or multiple events in aforce curve (determined by the number of significant localmin-imum) was calculated by dividing the number of curves in eachcategory by the total number of force curves acquired. Thesewere represented in a histogram displaying the probability forfinding zero events, one event or multiple events in a forcecurve.Force-extension curves were fitted with the extended FJC

model (39, 40),

Ex(F )

� Ns� Lp

e�G/kBT � 1�

Lh

e��G/kBT � 1��coth �FLK

kBT��kBT

FLK��

NsF

Ks

(Eq. 1)

where Ex(F) is themeasured extension length;�G� 3kBT is thedifference in Gibbs free energy between a planar and a helicalPEG subunit in the presence of a forceFwithLh� 0.287 nmandLp � 0.358 nm for their respective lengths (in the absence offorce). TheKuhn lengthLK� 0.7 nm, and the segment elasticityKS � 150 N/m are fitted to the experimental data.NS, the num-ber of monomers for each tether (polymer contour length) isthe only fitting parameter.

RESULTS AND DISCUSSION

Connexon Reconstitution in Lipid Bilayer—Hemichannels,when reconstituted in an artificial lipid bilayer, insert into themembrane with almost equal probability for both extracellularand cytoplasmic sides facing outside on a supported system.



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The differing lengths and amino acid sequences for the twosides, however, enable them to be distinguished with AFMimaging (14).As shown in Fig. 1, we designed our experimental system to

utilize the specific interactions either between antibody andCx43 CT domains or between mimetic peptides and Cx43extracellular domains in Cx43 hemichannels. Flexible polymertethers were used to attach antibodies or mimetic peptides tothe AFM tip, which spatially isolates nonspecific probe-sampleinteractions from the specific interactions of the tethered mol-ecules (Fig. 1B). Anti-CT252–270 antibody is used to target themiddle region of the Cx43 CT tail, whereas anti-CT360–382 isused to bind the end of the CT tail. Connexin mimetic peptideGap26 is used to probe the Cx43 extracellular domain EL163–75(Fig. 1C).The reconstitution of connexons in the supported bilayer

was confirmed by immunofluorescence labeling. Fig. 2A1showed a planar bilayer reconstitutedwith hemichannels (sam-ple) and exhibited immunofluorescence labeling (green) for

Cx43, whereas no fluorescence was detected in the samplewithout immunofluorescence labeling (Fig. 2A2). Insignificantnonspecific immunolabeling was observed when the samplewas incubated with the fluorescein isothiocyanate-conjugatedsecondary antibody directly (without primary anti-Cx43 anti-body) (Fig. 2A3), and no fluorescence was observed in bilayerswithout connexons after both primary and secondary antibodylabeling (Fig. 2A4). Fig. 2 (B1–B4) shows the correspondingfluorescence images of Lissamine Rhodamine B-labeled planarbilayers (red) on the coverslip. Some holes in the bilayer, whereliposomes did not fuse, are also seen (arrows).As shown in an AFM height image (Fig. 3A), the surface

topography of the bilayer without any reconstituted connexonsappears to be smooth and featureless with the height fluctua-tion of �0.3 nm (Fig. 3B). The thickness of the bilayer is �5.0nm, as determined from the cross-section along the unfusedbilayer holes, consistent with previous results (14). Connexonsreconstituted in bilayers appear as structures protruding outfrom the bilayer surface in a randomly distributed pattern.

FIGURE 1. A, schematic of force spectroscopy measurement showing binding of antibody (anti-CT252–270 or anti-CT360 –382) or connexin mimetic peptide(GAP26) connected to the AFM tip with flexible PEG spacers to Cx43 hemichannels reconstituted in lipid bilayer. B, schematic of the linkage of antibody (orpeptide) to the AFM tip via a PEG spacer molecule synthesized according to the procedure described in a previous study (34). C, schematic of Cx43 depictinglocations of two extracellular loops (EL1 and EL2), one cytoplasmic loop (CL), and the cytoplasmic carboxyl-terminal domain (CT) and locations of antigenicbinding sites for anti-CT252–270 (blue) and anti-CT360 –382 (red) as well as the peptide segment corresponding to GAP26 (EL163–75, pink). (Adapted from Ref. 41.)



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These connexons appear to fall into two groups with distinctextramembranous protrusion heights of �0.5–1.0 nm (red cir-cles) and 2.0 nm (white circles) (Fig. 3C). Fig. 3D shows cross-sections along lines in Fig. 3C: open arrows represent the firstgroup of low protrusion heights (red circles), whereas solidarrowheads represent the second group of high protrusionheights (white circles). The height difference is due to the larger

cytoplasmic CT domain protrudingfrom the membrane. Thus, a cleardistinction of the sidedness ofimaged connexons is achieved thatis consistent with previous bio-chemical and structural studies ofCx43 connexons (14, 30, 41).Anti-CT Antibody and Mimetic

Peptide Recognitions of Reconsti-tuted Cx43—Identification of theEL- versus CT-sided hemichannelswas defined by the specific interac-tions of either the mimetic peptideto EL domain or antibodies to CTdomain for a given hemichannel inthe sample. A typical force-exten-sion plot obtained from the meas-urements of anti-CT252–270 anti-body-Cx43 interaction force isshown in Fig. 4A. As the cantileverwas pulled away from the surface,the probe left the tip-sample repul-sive contact region (I) and enteredthe attractive interaction region (II)that is distinguished by a character-istic spacer stretching shape andcorresponds to a specific interactionof the tethered antibody with Cx43.The non-linear parabolic increasein the interaction force prior to anti-body-peptide unbinding providesthe viscoelastic properties of thespacer-antibody-Cx43 connections.This interaction resembles theshape described by the extendedLangevin function (red line) forcharacteristic polymer stretchingfollowing the Freely-Joint-Chain(FJC) model (39). In region III, thecantilever separates completelyfrom the sample surface and returnsto its equilibrium deflection. A typ-ical force of �100 pN and extensionof 30–50 nm was detected fromforce curves with single unbindingevents.Force curves with similar shape

and unbinding force were alsoobtained for the anti-CT360–382-Cx43 intermolecular interactionforcemeasurements (Fig. 4B). How-

ever, there was a significant difference with respect to theanti-CT252–270-Cx43 interaction: an extra extension of �105nmwas detected in the anti-CT360–382-Cx43 interaction thatis attributed to the stretching of Cx43 CT tail as discussedlater. Moreover, as described later, these extensions werebeyond the expected stretching of the antibody (if any) andthe linkers.

FIGURE 2. Fluorescence imaging. A1, planar bilayer reconstituted with hemichannels exhibiting immunoflu-orescence labeling for Cx43 after both primary and secondary antibody labeling; A2, without any antibodylabeling, no fluorescence was observed; A3, no fluorescence was observed when sample was incubated withthe fluorescein isothiocyanate-conjugated secondary antibody directly without primary anti-Cx43 antibodyincubation; A4, there was also no fluorescence observed after both primary and secondary antibody labeling inbilayers reconstituted without connexons. B1–B4, fluorescence images of corresponding planar bilayerslabeled with Lissamine Rhodamine B. Unfused holes were observed as indicated by arrows. Scale bar: 5 �m.



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The force-extension curve between the connexin mimeticpeptide GAP26 and the extracellular domains of reconstitutedCx43hemichannels showed a smaller unbinding force (Fig. 4C).This is in contrast to the large unbinding force observed for theanti-CT antibody and Cx43 interactions.The specificity of the antibody-Cx43 interactions was con-

firmedwhen excess free anti-Cx43 antibody was added into theimaging medium to block the antigenic sites of the Cx43hemichannels. After addition of excess antibodies, the compli-cated rupture structure was abolished and no specific ruptureevent was observed (Fig. 4,D and E, respectively). The specific-ity of GAP26-Cx43 interactionwas also verified by the blockingassay: excess free GAP26 was added into the imaging mediumthat abolished unbinding events, most likely by blocking thepeptide binding sites on the ELs (Fig. 4F). Further evidence ofthe specificity of the interactions was also provided from theforce measurement between scrambled GAP26 and recon-

stituted Cx43 hemichannels (Fig.4G). Thus, the specific ruptureevents were abolished by on-lineaddition of excess free GAP26 andwhen using the scrambled peptidebound to the AFM tip approaches.The sidedness (EL versus CT)

identification and the specificity ofthe antibody/peptide interactionsto the EL and CT sides were evalu-ated carefully. In the reconsti-tuted bilayer, both EL-side-up andCT-side-up Cx43 hemichannels arepresent (Fig. 3 in Thimm et al. (14)).In our study, using AFM tip conju-gated with mimetic peptide GAP26showed a weak force of unbindingand a short extension (�30 nm).Moreover, on-line addition ofanti-CT antibodies did not affectsuch unbinding force or extension.On the other hand, using AFM tipconjugated with anti-CT antibodiesshowed strong forces of unbindingand large (30 nm) stretching. Fur-thermore, these interactions werenot affected by on-line addition ofGAP26 but by the free anti-CT anti-body. Also, in the AFM imaging andforce map, we clearly observed thebimodal but random distribution ofbumpswith heights representing ELand CT sides up.Our preliminary results also indi-

cate that no apparent specific inter-action is detected when the sameforce measurement was performedusing the anti-CT antibody-conju-gated AFM tip to scan the carboxyl-terminal truncated mutant of ratheart Cx43 gap junction plaques.4

These isolated Cx43 gap junction plaques that were obtainedfrom Dr. Mark Yeager at The Scripps Research Institute weretruncated after Pro263, therefore, the antigenic binding site foranti-CT antibody was unavailable (42).As shown in the peptide-hemichannel unbinding event

histogram (Fig. 5A), �29% of all the force curves for anti-CT252–270-Cx43 interactions were identified as single ruptureevents. About 60% of these single rupture events occurred withmuch higher forces of�200–250 pN (Fig. 5B). Considering therelatively constant tether stretching distance in all single rup-ture curves, single unbinding events with higher forces wouldprobably be attributed to ruptures of multiple parallel anti-body-antigen bonds (43). In addition to the single ruptureevents as described above, ruptures with multiple unbinding

4 F. Liu, F. Teran Arce, S. Ramachandran, and R. Lal, unpublished data.

FIGURE 3. A, an AFM height image of planar lipid bilayer without any reconstituted connexons. The bilayersurface appears to be smooth and featureless. Scale bar: 200 nm. B, height profile cross-section along the line inA shows the thickness of the bilayer (�5 nm). C, an AFM height image of connexons reconstituted in planar lipidbilayer with the extramembranous protrusions of both the cytoplasmic (white circles) and extracellular (redcircles) sides. Scale bar: 100 nm. D, height profile cross-sections along the lines in C show the thickness of thebilayer and two connexon populations that protrude �0.5–1.0 nm (open arrows) and �2.0 –3.0 nm (arrow-heads) from the membrane plane. These two populations represent the extracellular and the cytoplasmic facesof connexons, respectively.



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steps were also detected for the anti-CT252–270-Cx43 interac-tions. Only a few multiple rupture events, however, wereobserved for anti-CT360–382-Cx43 or Gap26-Cx43 interactions(Fig. 5A). The total specific rupture events (including single aswell as multiple unbinding) in Gap26-Cx43 interactions weresignificantly reduced as well. The binding probability as well asthe rupture forces in the Gap26-Cx43 map was significantlylowered, as compared with antibody-Cx43 map.The anti-CT252–270 monoclonal antibody and the anti-

CT360–382 polyclonal antibody used in this study are madeagainst synthetic peptides corresponding to positions 252–270(GDLSPSKDCGSPKYAYFNG) and 360–382 (DQRPSSRASS-RASSRPRPDDLEI) of the native sequence within the cytoplas-micCTdomainof rat cardiacCx43, respectively (44–47).As indi-cated in Fig. 1, the antigenic sites for both anti-CT252–270 and anti-CT360–382 antibodies are locatedat theCTdomain.Therefore, the

force curves obtained in the force maps shown in Fig. 5 (B and C)recognize the CT-side-up reconstituted Cx43 hemichannels.The connexin mimetic peptide GAP26 used in this study

corresponds to positions 63–75 of the native sequence withinthe extracellular loop 1 (EL1) of Cx43. The force curves

FIGURE 4. A–C, represent force-extension curves showing single ruptureevents between anti-CT252–270, anti-CT360 –382, GAP26, and reconstitutedCx43, respectively. The parabolic trace of region II reflects the extension of thedistensible PEG-antibody (or peptide)-Cx43 connection. Red lines: fittings tothe extended FJC model. The specificity of anti-CT252–270-Cx43 (D), anti-CT360 –382-Cx43 (E), and GAP26-Cx43 (F) is demonstrated by competitiveblocking assay by on-line addition of respective antibodies/peptides to blockthe binding sites on the reconstituted connexons. G, representative force-extension curve showing no specific interaction between scrambled-GAP26and reconstituted Cx43.

FIGURE 5. A, distribution of specific unbinding events. B–D, force-volumeinteraction maps of anti-CT252–270-Cx43, anti-CT360 –382-Cx43, and GAP26-Cx43. The rupture forces are scaled in each pixel with color values (0 –250 pN).Representative force curves corresponding to each individual pixel as indi-cated by arrows are shown on the right side.



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observed (Fig. 4C) may occur between GAP26 and its comple-mentary sequence in the extracellular loops of the Cx43hemichannels as proposed previously (10, 48). Significantlyfewermultiple unbinding eventswere detected inGAP26-Cx43force measurements. Characteristic tether stretching curvesand blocking competition assay as well as scrambled peptideunbinding observed in the present study suggest that theobserved interactions are specific interactions between GAP26and Cx43. Therefore, the force map adhesions in Fig. 5D rec-ognize the EL-side-up Cx43 hemichannels.Connexin mimetic peptides have been used to block the

functional gap junction coupling in a variety of systems, includ-ing electrophysiological, biochemical, and immunochemicalassays (26, 27, 49, 50). No structural data is yet available for thebinding of peptides to connexins. The present study providesan approach for further examination of connexin sequences

recognized by inhibitory peptides and thus could provideinsights intowhich amino acid sequences from the extracellularloops are involved in channel gating or interact to form wholegap junction channels.Mimetic Peptide-Cx43 and Antibody-Cx43 Rupture Forces

and Molecule Stretching—The Cx43-mimetic peptide andCx43-antibody unbinding force analysis is complicated if thespecific interactions arise from a number of different interact-ing molecules or variable strength of bonds. Previous studieshave revealed a typical unbinding force of �100 pN for singleantibody-antigen pairs (19, 43, 51), although the unbindingforces vary considerably (49–244 pN) for different systems(52). The unbinding force histogram reveals frequent occur-rence of unbinding force with distinct values for different bind-ing domains. Unbinding forces of 63� 11, 116� 22, and 222�16 pN were observed for the anti-CT252–270-Cx43 interactions(Fig. 6A). The anti-CT360–382-Cx43 interactions show an aver-age unbinding force of 125 � 18 pN (Fig. 6B). The ruptureforces for GAP26-Cx43 interactions occur at 52 � 10 and 96 �14 pN. Few unbinding forces higher than 200 pNwere observedfor the mimetic peptide-connexon interactions (Fig. 6C).Both the anti-CT antibodies and mimetic peptides were

covalently linked to an AFM tip via the distensible aldehyde-PEG-NHS spacers tominimize the impact of nonspecific inter-actions. The spacers spatially isolate nonspecific probe-sampleinteractions from the specific interactions of the tethered mol-ecules and allowed us to discriminate between rupture eventsusing both the unbinding force values and rupture location. Anavidin-biotin unbinding system (34) was used to estimate thePEG spacer’s average length: avidinmolecules were attached tothe AFM tip via the aldehyde-PEG-NHS spacer with the samemethod used for antibody, whereas biotinylated-bovine serumalbumin was attached on the mica surface. Force curves wererecorded under the same condition as in the antibodymeasure-ments. As shown in Fig. 6 (D and E), our results are similar tothe previously reported force curves for biotin-avidin unbind-ing events (34) and typical unbinding forces�40 pN.The force-extension curve was fitted to the extended FJC model asdescribed under “Experimental Procedures.” The perfect fittingin Fig. 6D indicates that the observed extension�28 nm resultsmainly from the spacer stretching, even considering the�5 nmbiotin-avidin complex. Fitting parameters obtained from theextended FJC fitting aswell asmeasured extension lengths fromdifferent interactions are listed in Table 1. The fitted contourlength shows large deviations from the measured extensionlength in anti-CT252–270-Cx43 and anti-CT360–382-Cx43interactions.As shown in Fig. 6F and Table 1, an average extension of�31

nm was observed in the GAP26-Cx43 interactions, which isvery close to the PEG spacer length measured in our biotin-

FIGURE 6. Probability histograms of the rupture forces of the measured anti-CT252–270-Cx43 (A), anti-CT360 –382-Cx43 (B), and GAP26-Cx43 interactions (C).The histogram is fitted with a Gaussian curve, and the corresponding mean �S.D. is indicated in the text. D, a representative force-extension curve showingspecific avidin-biotin interaction in the PEG spacer extension test system:avidin molecules were attached to the AFM tip via the aldehyde-PEG-NHSspacer with the same method used for antibody; biotinylated-bovine serumalbumin was attached on the mica surface; force curves were recorded underthe same conditions as in the antibody measurements and fitted to theextended FJC model (red line). E, the average extension of PEG spacer stretch-ing, �28 nm, is estimated from the extension of the testing system. F, histo-grams of measured tether extensions in anti-CT252–270-Cx43, anti-CT360 –382-Cx43, and GAP26-Cx43, respectively.

TABLE 1Parameters obtained from the extended FJC fittingNS , the number of monomers for each tether, is the only fitting parameter. LC is the corresponding polymer contour length (LC � NSLp/(e�GBT � 1) � Lh/(e��GBT � 1)�.Lx is the measured extension of the anti-CT(or peptide)-PEG-Cx43 complex.

Force curves Anti-CT252–270-Cx43 Anti-CT360–382-Cx43 GAP26-Cx43 Avidin-biotin/bovine serum albuminNS 94 � 24 266 � 18 125 � 29 136 � 33LC (nm) 26.7 � 6.8 75.5 � 5.1 35.5 � 8.2 38.6 � 9.4LX (nm) 56.8 � 14.1 104.9 � 20.9 31.1 � 4.3 27.6 � 8.1



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avidin testing system (Fig. 6E). This suggests that the extensionin the EL experiments results only from the spacer stretching,andmost likely there was very little, if any,molecular stretchingof the Cx43 extracellular loop or the GAP26 peptide during theunbinding event.A significantly larger extension of �105 nm was observed in

the anti-CT360–382-Cx43 interactions (Fig. 6F). In this situa-tion, the antibody binds to the CT positions 360–382, close tothe end of the CT domain (Fig. 1C). The extra extensionobserved for this situationmay reflect the total stretching of theseveral associated molecules, including the IgG, the PEGspacer, and the CT domains. In comparison to the mimeticpeptides against the ELs, the antibody for Cx43 CT epitopeswas relatively larger (�150 kDa) and this larger size may add tosome additional extension. Stretching lengths of antibodiesfrom 5 up to 18 nm have been reported (53, 54). However, it isunlikely that the antibody three-dimensional structure canbe unfolded significantly because of the external force duringthe unbinding events, and thereby provide the additional exten-sion of more than 20 nm, because the intramolecular forcesresponsible for stabilizing antibody conformations are signifi-cantly larger than the intermolecular antibody-antigen bindingforce (19).Recent NMR studies indicate that the overall structure of

Cx43 CT domain is primarily random coiled with two shorthelical regions (17, 55). Such an elongated random coil struc-ture might be highly flexible and easy to stretch. The additionalextension length of C-tail proteins from AFM force measure-ment has been previously reported (56). Cx43 CT domain con-tains �148 amino acid residues, and the contour length of thepolypeptide chain in its fully extended conformation mayextend up to 53 nm (1 amino acid �0.36 nm) (57) and evenlonger if the applied external force would permit the peptidebackbone to stretch (39).Consistent with the above analysis, a shorter extension of

�57 nm was detected in the anti-CT252–270-Cx43 interac-tions, where the antibody binding site is located in the mid-dle of the flexible Cx43 CT domain (Fig. 1C). Only a smallportion of the CT coil can be stretched (amino acid residues234–252). The good fitting of measured force curve to theextended FJC model (red line in Fig. 4A) suggested thatthe PEG spacer stretching (�30 nm) still contributes to themajor part of the overall extension.Flexibility of Cx43 CT Domain: Role in Hemichannel Gating—

Structural flexibility of the Cx43 CT domain is the basis of theparticle-receptor hypothesis for Cx43 (58).Muller and cowork-ers (8) reported a compressing energy of 11.9 kJ/mol requiredfor the conformational change in the CT domain of Cx26; sig-nificantly, though, the CT portion of Cx26 is much smallercompared with the CT portion of Cx43. The energy required tostretch Cx43 CT domain can be calculated by integrating theforce-extension function. The area under the force-extensioncurve from anti-CT360–382 interaction (Fig. 4B) corresponds tothe mechanical energy required to stretch both the PEG spacerand CT domain, whereas the area under the force curve fromanti-CT252–270 interaction (Fig. 4A) corresponds approxi-mately to the energy required to stretch only the PEG spacer;the mechanical energy for the anti-CT360–382 interaction and

for the anti-CT252–270 interaction are �375 kJ/mol and �130kJ/mol, respectively. The difference between these two valuesof�245 kJ/mol is themechanical energy required to stretch theCx43 CT domain. This value is similar to the previouslyreported mechanical energy required to stretch protein mole-cule complex (P-selectin/PSGL-1) (53).Electrophysiological studies have shown intracellular Ca2�-

dependent gating of Cx43 hemichannels as well as other type

FIGURE 7. A, representative force-extension curves showing specific anti-CT360 –382-Cx43 interactions at nominally Ca2�-free PBS buffer (pH 7. 4). B,with the same tip and same sample, specific anti-CT360 –382-Cx43 interactionswere diminished when 1.8 mM Ca2� was added on-line to the medium. C, theinteractions were restored when the imaging solution was changed back tocalcium-free PBS buffer.



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hemichannels (Cx32, Cx46, and Cx50) (59–67). To furtherexamine the role of the Cx43 CT domain in the Ca2�-depend-ent hemichannel gating process, PBS buffer containing 1.8 mMCa2� was added into the anti-CT360–382-Cx43 force measure-ment medium. The specific rupture events frequently detectedunder 0 mM Ca2� condition (Fig. 7A) were significantly dimin-ished in the presence of 1.8 mM Ca2� (Fig. 7B). Significantly,this loss of rupture events in the presence of 1.8 mM Ca2� isreversible. When the same sample was washed with normallyCa2� free PBS medium (pH 7.4), the rupture events withcharacteristic rupture force and extension were detectedagain (Fig. 7C).The lack of antibody-hemichannel interactions in the pres-

ence of 1.8 mM calcium could result from the change in theantibody conformation such that its binding ability becomesunavailable.We believe that such a possibility is highly unlikely.In our previous study on mapping vascular epidermal growthfactor receptors in endothelial cell plasma membranes usinganti-vascular epidermal growth factor receptor-antibody-con-jugated AFM tips, we had reported the lack of any such effects,i.e. the binding/unbinding was not affected by the nature of theimaging media for both in vitro (isolated receptor-antibodyinteractions) and in vivo (receptors in the live cell plasmamem-brane and antibody on the AFM tip) (68). On the other hand,hemichannel closing under 1.8mMCa2� introduces conforma-tional changes in the Cx43 CT domain, which may result in apossible steric effect preventing the binding of anti-CT360–382to theCT tail. Sosinsky and colleagues (8) have reported that 0.5mM Ca2� changes the cytoplasmic conformation of Cx26 gapjunction. Similar conformational change may also occur inCx43 hemichannel with 1.8 mM Ca2�. Because the antigenicbinding site of anti-CT360–382 is located at the end of CTdomain, such a conformational change in the presence of cal-ciummay cause the binding site to become inaccessible to anti-CT360–382 antibody, probably due to the refolding of CTdomain according to particle-receptormodel (16, 69), thus pre-venting the binding of the antibody to the hemichannel. Thesignal transduction resulting from calcium binding/unbindingto the CT domain that results in the channel closing/openingwas not examined and is beyond the scope of this study.

We provide a simple model forchannel opening and closing that isconsistent with the earlier hypothe-sis byDelmar and colleagues (16). Intheir model, the CT blocks thechannel pore mouth physically byclumping as a ball (Fig. 8). Ourresults provide an estimate of theintrinsic distensibility of the flexibleCx43 CT domain, which is higherthan the relatively inelastic ELdomains. In brief, (a) higher disten-sibility of CT domain and (b) pres-ence of antibody-CT domain inter-action at 0 mM Ca2� and the lack ofit with 1.8 mM Ca2�, strongly sup-port calcium-dependent conforma-tion change of CT domain and the

ball and chainmodel of hemichannel gating. However, whetherthis estimated distensibility force translates into the requiredmechanical energy to move the ball from the pore mouthrequires further careful investigations. The mechanical move-ment of the CT domain in our study would probably overesti-mate the amount of energy needed to remove the ball in theactual cell interior wherein several complementary physico-chemical factors would modulate the overall connexinflexibility.In summary, we examined the interactions between individ-

ual antibodies and non-crystalline, non-truncated Cx43hemichannels reconstituted in supported lipid bilayers as wellas between connexinmimetic peptides andCx43 hemichannelsusing single molecule force spectroscopy. Our results providedistinct signatures of the extracellular and cytoplasmic sides ofhemichannels reconstituted in bilayer. Cx-mimetic peptidesrecognize only the extracellular side, whereas the anti-CT anti-bodies recognize only the cytoplasmic side. We provided aunique system to investigate not only the intermolecular inter-actions (antibody-antigen) but also the intramolecular interac-tions (CT stretching) of the reconstituted hemichannels. Suchan approach of force microscopy with a connexin mimeticpeptide-functionalized probe could be used for examiningthe biophysical basis of gap junction formation and analyz-ing hemichannel structure-function in vitro as well as in cellmodels.

Acknowledgments—We acknowledge helpful advice and criticalreading of the manuscript from Dr. Arjan Quist and Dr. Scott John.We thank Dr. Peter Hinterdorfer for providing PEG tethers used toconjugate mimetic peptides and anti-Cx43 antibody.

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FIGURE 8. Diagram illustrating the concept of a “particle and receptor” model for Ca2� regulation ofCx43 hemichannel closing. The flexible Cx43 CT domain acts as a gating particle. Under normal conditions,the gate particle is away from the hemichannel pore and the channel is open. The anti-CT360 –382 binding site (atthe end of CT domain) is accessible to the antibody. Upon Ca2� binding or other chemical stimuli (pH, etc.), thegate would swing toward the mouth of the channel, bind to the “receptor” affiliated with the pore (cytoplasmicloop domain), and close the channel. In this case, the anti-CT360 –382 antibody could not bind to the CT domain.



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