Proc. Natl. Acad. Sci. USAVol. 93, pp. 5836-5841, June 1996Biophysics

Voltage gating and permeation in a gap junction hemichannel(connexins/intercellular communication/ion channels/permeability/rectification)

E. BRADY TREXLER, MICHAEL V. L. BENNETTr, THADDEUS A. BARGIELLO, AND VYTAS K. VERSELIS

Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461-1602

Contributed by Michael V. L. Bennett, February 27, 1996

ABSTRACT Gap junction channels are formed by mem-bers of the connexin gene family and mediate direct intercel-lular communication through linked hemichannels (connex-ons) from each oftwo adjacent cells. While for most connexins,the hemichannels appear to require an apposing hemichannelto open, macroscopic currents obtained fromXenopus oocytesexpressing rat Cx46 suggested that some hemichannels can bereadily opened by membrane depolarization [Paul, D. L.,Ebihara, L., Takemoto, L. J., Swenson, K. I. & Goodenough,D. A. (1991), J. Cell Biol. 115, 1077-1089]. Here we demon-strate by single channel recording that hemichannels com-prised of rat Cx46 exhibit complex voltage gating consistentwith there being two distinct gating mechanisms. One mech-anism partially closes Cx46 hemichannels from a fully openstate, Yopen, to a substate, Ysub, about one-third of the con-ductance of Yoen; these transitions occur when the cell isdepolarized to inside positive voltages, consistent with gatingby transjunctional voltage in Cx46 gap junctions. The othergating mechanism closes Cx46 hemichannels to a fully closedstate, Yclosed, on hyperpolarization to inside negative voltagesand has unusual characteristics; transitions between yVcosedand Yopen appear slow (10-20 ms), often involving severaltransient substates distinct from Ysub. The polarity of activa-tion and kinetics of this latter form of gating indicate that itis the mechanism by which these hemichannels open in the cellsurface membrane when unapposed by another hemichannel.Cx46 hemichannels display a substantial preference for cat-ions over anions, yet have a large unitary conductance (-300pS) and a relatively large pore as inferred from permeabilityto tetraethylammonium (-8.5 A diameter). These hemichan-nels open at physiological voltages and could induce substan-tial cation fluxes in cells expressing Cx46.

Gap junctions channels are constructed as two hemichannels(connexons) in series, one provided by each of the coupledcells. These channels provide a direct connection for passageof ions between the coupled cells that is insulated from theextracellular space. Their permeability to relatively large mol-ecules, up to -1 kDa in molecular mass, and presence ininexcitable cells suggest a role in transmission of chemical aswell as electrical signals. The constituent hemichannels arehexamers of protein subunits of the connexin gene family,which has at least 12 members in mammals (1). One issue ingap junction channel physiology is the functional significanceof this diversity, which requires characterization of the chan-nels in terms of gating and permeability. However, character-ization of gating and permeability of these channels is ham-pered by their inaccessibility to direct patch recording. Al-though the double whole cell patch technique has been usedextensively in single channel studies, junctional conductance,gj, between cell pairs is often too large to permit visualizationof unitary currents, which has necessitated the use of phar-macological agents to reduce the number of operational

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channels (2). Furthermore, membrane capacity decreases fre-quency response well below that possible with excised patches,and ionic substitution studies, which depend on dialysis frompatch electrodes, have been limited because of technicaldifficulties (3).Here we undertook the study of gap junction gating and

permeation by recording from unapposed hemichannels. Al-though it is generally believed that unapposed hemichannelsare closed in order to prevent loss of cytoplasmic solutes andentry of extracellular ions, exceptions include hemichannelsformed by Cx46 (4, 5), Cx56 (6), and an unidentified connexinin fish horizontal cells (7). We expressed high levels of Cx46 inXenopus oocytes and were able to record single hemichannelcurrents in both on-cell and excised, inside-out, and outside-out patch configurations. These hemichannels exhibit voltagegating consistent with the Vj dependence of hemichannelsincorporated into cell-cell channels and in addition exhibit anovel voltage-gating mechanism that allows Cx46 hemichan-nels to open in the surface membrane of single cells. Multiplesolution exchanges applied to the exposed face of excisedpatches indicated permeability to large ions and a significantpreference for cations. These data demonstrate that unap-posed hemichannels possess attributes characteristic of gapjunction channels and establish the utility of the excisedhemichannel preparation in probing mechanisms of gap junc-tion voltage gating and permeation.

MATERIALS AND METHODS

Expression of Cx46 mRNA in Xenopus Oocytes. Cx46 DNAwas cloned from rat genomic DNA using PCR amplificationwith primers corresponding to amino- and carboxyl-terminalsequences. Preparation of oocytes and synthesis of RNA havebeen described previously (8-10). Each oocyte was injectedwith 50 nl of an aqueous solution ofmRNA (2 mg/ml) togetherwith DNA antisense to the endogenous XenCx38 (8 pmol/,Il)(11). We use the phosphorothioate antisense oligo 5'-GCTTTA GTA ATT CCC ATC CTG CCA TGT TTC-3', which iscomplementary to XenCx38 commencing at nt -5.

Electrophysiological Recordings. Voltage clamp recordingsof macroscopic currents from single Xenopus oocytes wereobtained by utilizing a two-electrode voltage clamp. Bothvoltage-measuring and current-passing microelectrodes werefilled with 1 M KCl. The oocytes were bathed in a solutioncontaining 88 mM KC1, 2 mM MgCl2, 5 mM glucose, 10 mMHepes, pH 7.6. Nominal Ca2+ in this solution was 5-10 ,uM asdetermined by a Moller Ca2+ ion-selective electrode.

In all patch-clamp experiments, pipette solutions consistedof 100 mM KC1, 1 mM CaC12, 2 mM MgCl2, 5 mM EGTA, 10mM Hepes, pH 7.6. Oocytes were bathed in a solutioncontaining 88 mM NaCl, 1 mM KCl, 2 mM MgCl2, 5 mMglucose, 10 mM Hepes, pH 7.6. In experiments where relativepermeabilities were determined, a flow chamber was designedwith two separate compartments, one to hold the oocyte and

Abbreviations: TEA, tetraethylammonium; TMA, tetramethylammo-nium.

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one to perfuse solutions containing different salts; the com-partments were separated by a recessed septum. For eachexperiment, the chamber was first filled with the oocytebathing solution to a level such that the compartments were incommunication. After obtaining a cell-attached patch, thepatch was excised and moved over the septum to the perfusioncompartment. Then, the solution level was lowered so that theseptum separated the two compartments. The solution in theperfusion compartment was first replaced with the patchpipette filling solution to determine offsets due to electrodeasymmetries; these were usually small (1-2 mV) and werenulled prior to further solution exchange. Activity coefficientsfor salts were obtained from Robinson and Stokes (12) andwere used in the Goldman-Hodgkin-Katz equation for cal-culation of permeability ratios; for TEA and TMA, activitieswere estimated from Cl- activities of TEACI and TMAC1 saltsmeasured with a Cl--selective electrode (Orion Research,Cambridge, MA). Connection of the perfusion compartmentto ground consisted of a 3 M KCI agar bridge that led to aseparate chamber filled with the patch pipette filling solution,into which a Ag/AgCl wire was placed. Only patches in whichthe leak conductance was small (<10% of open channelconductance) were used for these studies. No differences insingle channel conductance or voltage dependence were seenbetween inside-out or outside-out patches. Data were filtered

at 1 kHz with a four-pole lowpass Bessel filter and digitized at5 kHz using an Axopatch 1-D patch clamp amplifier andDigidata 1200 interface (Axon Instruments, Foster City, CA).

RESULTS AND DISCUSSION

Macroscopic recordings from Xenopus oocytes expressing ratCx46 typically show large slowly activating currents upondepolarization from a holding potential of -70 mV (Fig. 1A).In K+ salt solutions with no added Ca2+, the currents reversenear 0 mV. With larger inside positive voltages (e.g. +65 mV)at relatively low levels of expression, currents show a peakfollowed by relaxation to a lower value. With higher levels ofexpression, the currents no longer show a peak followed byrelaxation (data not shown, but see ref. 5), which is attributableto increased voltage drop across the series access resistance(13). Patch recordings from oocytes expressing Cx46 showlarge conductance channels (-300 pS, Fig. 1B), which weascribe to functional Cx46 hemichannels. These large conduc-tance channels are not seen in oocytes injected only with DNAantisense to the endogenous oocyte connexin, XenCx38, or thisantisense together with Cx32 mRNA. Furthermore, currentsfrom multichannel patches from Cx46 expressing oocytes (Fig.1C) display properties consistent with those observed macro-scopically, i.e., slowly activating currents that reverse near 0

......... +50mV-'-- ..........- ....--- 35mV:-'- ' . .' +65mV

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FIG. 1. Comparison of macroscopic and single channel recordings from Xenopus oocytes expressing Cx46. (A) Example of macroscopic currentselicited in a two-electrode voltage clamp experiment from a single Xenopus oocyte 6 h after Cx46 mRNA injection. From a holding potential of-70 mV, voltage steps from -10 to +65 mV in increments of + 15 mV were applied to this cell. Currents activate with depolarization and reversenear 0 mV. At large inside positive voltages, conductance decreases as indicated by a reduced incremental change in current between +35 and+50 mV, and a pronounced current relaxation following a peak at +65 mV. Repolarization to -70mV produces inward tail currents. Time constantsof the tail currents are strongly voltage-dependent (data not shown). (B) Example of a cell-attached patch recording of a single Cx46 hemichannel.The currents shown were elicited at a net transmembrane voltage, V = -35 mV (V = Vrp - Vpipette). Vrp is the resting potential of the cell as measuredwith a separate intracellular microelectrode (for this cell, Vrp = -20 mV). These channels were found in >80% of the patches from this oocyte.(C) Example of an excised (inside-out) patch recording containing several Cx46 hemichannels from the same oocyte as in Fig. 1B. From a holdingpotential of -70 mV, steps to -10 mV and +20 mV elicit slowly activating inward and outward currents, respectively. Stepping to +50 mV elicitsa faster rising outward current (not resolvable at this time scale) that slowly decays to a lower value. Single channel currents are not resolvable duringany of the three depolarizing steps but are clearly visible in the tail currents upon repolarization back to -70 mV. Ensemble averaging of the tailcurrents and extrapolation to the peak indicated that there were seven hemichannels in the patch that were activated by depolarization.

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A+35 y"YCI,p·1H1i M Z-. . -.-d -d.. A-.. ;& Al

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FIG. 2. Cell-attached patch recordings from single Cx46 hemichannels in Xenopus oocytes. (A) Representative 5-sec segments of 60-secrecordings of single channel activity at positive and negative voltages. The hemichannel assumes three principal conductance states: 'open, the fullyopen state; Ysub, a subconductance state approximately one-third of the magnitude of y/op,en; and Yclosed, a fully closed state. All-points amplitudehistograms of the 60-sec recordings are shown to the right of each trace. Arrowheads indicate the histogram peak assigned to Yopen. At -55 mV,the hemichannel spends most of the time in yclosed, with occasional openings to 'open. With depolarization to -35 mV, openings to 'open increasein frequency and duration. At smaller voltages of either polarity, e.g., 15 mV, the hemichannel remains predominantly in yopen, with occasionalclosures to either Yclosed or Ysub. More positive voltages push the channel into longer residence times in ysub, and at +35 mV, closures to yclosedare rare. (B) Currents obtained with 16-sec voltage ramps from -70 mV to +70 mV applied to an on-cell patch containing a single hemichannelfrom a different cell than in A clearly illustrate the differential distribution of conductance states with voltage. Shown are three ramps applied insuccession. At large negative and positive voltages, the hemichannel closes to Yclosed and to Ysub, respectively, and remains predominantly in 'openat intermediate voltages of either polarity. The conductance level of yciosed corresponds to the leak conductance obtained by applying voltage rampsto the same patch after channel activity spontaneously disappeared (current through the leak conductance is superimposed on the three ramps).Leak conductances of patches containing no active hemichannels were of similar magnitude. (C) Examples of the complex gating transitions between'open and Yclosed. On the left are shown three consecutive 1-sec traces of an on-cell patch recording (V = -45 mV). Epochs in 7open are designated

-15Ci1d-15 y,,~V ¥sui^pc

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Hemichannel Gating

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FIG. 3. Schematic diagram of voltage gating of hemichannels when unapposed and when incorporated into cell-cell channels. Hemichannelsare composed of six connexin subunits. The accepted topology of a subunit includes four transmembrane domains (TM1-4) with two extracellularloops (El and E2) and the N-terminus (NT), cytoplasmic loop (CL), and carboxyl terminus (CT) located intracellularly (illustrated at bottom right)(14). Hemichannels in the surface membrane of cells are generally closed and require docking with another hemichannel to open. Docking can

be viewed as a form of chemical gating, analogous to ligand gating, mediated by El and E2. In cell-cell channels, each hemichannel contains a

separate Vj sensor/gate, and Vj gating can be mediated by either one. Cx46 hemichannels are closed by Vj, which is relatively positive on theircytoplasmic side (+ and - in the diagram indicate voltage of one cell relative to the other). Since Vj gating shows little sensitivity to the voltagebetween inside and outside of the cells and amino acid residues located at the beginning of the NT of Cx26 and Cx32 are integral components ofthe Vj sensor (15), we depict Vj gating as occurring at the cytoplasmic end of each hemichannel. For Cx46 hemichannels unapposed by anotherhemichannel, closure is favored at inside negative voltages, state [1], but the conformational energy associated with closure can be surmountedby moderate depolarization, allowing hemichannels to be open in the surface membrane, state [2]. We ascribe this form of voltage gating to theextracellular loops. Further polarization to inside positive voltages close the Vj gate of the hemichannel, state [3]. Thus, the loop and Vj gates are

in series and have opposite polarities of closure, inside negative and positive, respectively. The large difference in the voltage at which these twomechanisms operate, together with long dwell times in the open state near 0 mV (Fig. 2), indicates that simultaneous closure of both gates wouldseldom occur, state [4].

mV and that peak and relax to lower values at higher insidepositive voltages.To illustrate the steady-state voltage gating properties of

Cx46 hemichannels, recordings from a cell-attached patchcontaining a single Cx46 hemichannel are shown in Fig. 2A. Atlarge inside negative voltages, the hemichannel remains pre-dominantly in Yclosed, with occasional openings to open. Thereare frequent lower amplitude noisy bursts that are not resolv-

able as discrete events and may be incomplete hemichannelopenings. With depolarization, dwell times in Yopen markedlyincrease. From open there are brief sojourns to several sub-states including Ysub, a conductance state about one-third ofthat of Yopen. At voltages near 0 mV of either polarity, thehemichannel remains predominantly in yopen with continuedbrief sojourns to substates and Yclosed. At larger inside positivevoltages, dwell times in s,ub become strikingly longer. This

by solid bars labeled a, b, and c. At the onset and end of open epochs, the hemichannel transitions between Yclosed and yopen often involveat least two identifiable transient substates distinct from Ysub. Epoch b, shown in an expanded time scale, exhibits an initial opening to thefirst identifiable transient substate (S1) lasting approximately 15 msec, followed by an opening to the second transient substate (S2), beforefinally reaching Yopen. Within this open epoch, a sojourn to Ysub is evident, along with brief sojourns to S2 (asterisks). At the end of this epoch,conductance rapidly decreases from Yopen to S1, and then slowly decays to Yciosed over approximately 20 msec. Periods in yclosed are interruptedby a number of short noisy bursts (arrows) that may represent incomplete openings. Illustrated on the right side is another epoch in Yopen fromthe same patch at the same voltage. Segments representing the initial opening (d) and final closing (e) are shown below at an expanded timescale. Numbered arrows mark specific events. An incomplete opening [1] falls back to S1 and then transits to S2 [2] before fully opening [3].Closure from yopen appears slow and continuous [4] and may be due to fast unresolved transitions among the identified transient substatesand/or other unresolved transient substates; with a -3 dB cutoff at 1 kHz, the transitions would have to be <400 ,usec to go unresolved. Ashort, noisy burst [5] yields to Yclosed.

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complex gating behavior is clearly evident in I-V curvesobtained by applying slow voltage ramps between -70 and+ 70 mV to patches containing a single Cx46 hemichannel (Fig.2B). The negative quadrant is dominated by transitions be-tween Yclosed and Yopen, whereas the positive quadrant exhibitstransitions between yopen and 7sub. Residence in Yopen domi-nates at intermediate voltages. Yopen shows marked rectifica-tion; the chord conductance changes as a smooth monotonicfunction over the entire voltage range and in Fig. 2B is 300 pSat -50 mV and 135 pS at +50 mV (also see Fig. 4A).

In addition to gating between different conductance levelsat inside negative and positive voltages, i.e. yopen > y/closed andYopen > 7/sub, respectively, the transitions between those levelshave markedly different kinetics (Fig. 2C). Transitions be-tween Yopen and 'closed are slow and usually involve severaltransient subconductance states distinct from ysub (Fig. 2C,epoch b). In some cases, these transitions appear to becontinuous, lasting 10-20 ms (Fig. 2C, segment e). Unlikegating observed in most ion channels, where transition timestypically are unresolved, the apparently slow transitions be-tween Yclosed and Yopen may result from rapid gating among theidentified transient substates and, perhaps, other unresolvedsubstates. Transitions between Yopen and Ysub are rapid and donot appear to involve any other substates (Fig. 2A, +35 mV).We attribute the voltage-dependent differences in occupa-

tion of hemichannel states and kinetics of transitions betweenthem to the actions of two distinct voltage gates as diagrammedin Fig. 3. Hemichannels typically are closed when unapposedby another hemichannel to prevent loss of cytoplasmic solutesand entry of extracellular ions. After contact with anotherhemichannel to create a cell-cell channel (i.e., docking) thehemichannels open to create a continuous pore connecting thecytoplasms of the two apposed cells. This process can beviewed as comparable with ligand-receptor binding, withhemichannels acting in both roles. Once formed, the cell-cellchannel can gate in response to Vj by closing Vj gates locatedin each of the hemichannels. In the case of Cx46, the hemichan-nel can be opened by moderate depolarization in the absenceof contact with an apposed hemichannel. This property isconferred by a gating mechanism characterized by transitionsbetween Yclosed and Yopen involving multiple, short-lived sub-states. The features of complete closure at inside negative

voltages and opening on depolarization are consistent with theproperties inferred from macroscopic currents observed insingle Cx46-expressingXenopus oocytes (Fig. 1A). We ascribethis gating mechanism to the extracellular loops of the con-nexin subunits (Fig. 3, inset) because of the resemblance ofthese transitions to "docking" currents observed during cell-cell channel formation (16). We provisionally term this gatingmechanism "loop" gating, and the multiple conformations ofextracellular loops that would be possible in a hexamerichemichannel may account for the apparently gradual gatingtransitions. At large inside positive voltages, when the "loop"gate is open, the hemichannel can close by a gating mechanismcharacterized by transitions between Yopen and Ysub (see Fig.2A, +35 mV). It has recently been shown for homotypic gapjunction channels formed of several different connexins thatgating by Vj is characterized by transitions between a fully openstate and a substate, which explains the residual conductanceobserved in these cell-cell channels at large Vj values (2, 16,17). Thus, the incomplete closure of the Cx46 hemichannel(i.e., gating to a substate) at inside positive voltages is consis-tent with gating by Vj. That the substate is favored at insidepositive voltages is consistent with the polarity of Vj gating ofCx46 in cell-cell channels as inferred from heterotypic Cx46/Cx32 and Cx46/Cx26 junctions (18). These data indicate thatCx46 hemichannels in both the unapposed and cell-cell chan-nel configuration have the same Vj gating polarity instead ofthe opposite Vj gating polarity as suggested by White et al. (18).The polarity of hemichannel opening in single cells corre-sponds to loop gating, not Vj gating.Cx46 hemichannels can be opened by moderate depolariza-

tion, which may be important physiologically. To help under-stand the potential physiological roles of Cx46 hemichannels,we determined their permeability characteristics (Fig. 4).Single salt gradients of KCI and TEAC1 shift the reversalpotential (Erev) negative on the side with the higher concen-tration of cations to a degree indicating a substantial prefer-ence for cations. Erev was -25.6 ± 0.6 and -14.0 ± 0.8 mV(n = 4) for a 4.2-fold activity gradient of KCl and TEAC1,respectively, compared with -36.3 mV for ideal cation selec-tivity (Fig. 4). Calculated PK/PC1 was 10.3:1 and PTEA/PCI was2.8:1. In symmetrical 100 mM KCl, the channels rectified (Fig.4A) as noted above (Fig. 2A). In a 500:100 KCl gradient (Fig.

100:100 KCl(in)/KCl(out)

I (pA)40-

20

i -20-

-40-

-80 -40 0 40 80V (mV)

B 500:100 KCl(in)/KCl(out)

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FIG. 4. Cx46 hemichannels are cation selective. Erev for single Cx46 hemichannel currents in excised (inside-out) patches was measured as thevoltage at zero current during applied voltage ramps. Erev differs in (A) symmetric 100:100 KCl, (B) 500:100 (in/out) KC1 gradient, and (C) 100:100TMACl(in)/KCl(out) biionic conditions (numbers are concentrations in mM). A and B are from the same patch; C is from a different patch. Insymmetric KCl (A) the open channel I-V curve displays inward rectification with conductance changing from 340 pS at -70 mV to 160 pS at +70mV. Replacement of 100 mM KCl on the inside with 500 mM KCI (B) shifts Erev to -24 mV, indicating a substantial preference for cation overanions. PK/PCI was found to be 10.3 (n = 4 patches) when calculated using the Goldman-Hodgkin-Katz voltage equation (19). The I-V curve

appears nearly linear, which is attributable to the higher concentration of K+ on the inside face of the membrane. (C) Replacement of 100 mMKCl on the inside with 100 mM TMAC1 shifts Erev to +24 mV. PK/PTMA was calculated to be 2.9 (n = 4 patches) when using PK/PCI = 10.3 andapplying the Goldman-Hodgkin-Katz voltage equation. The increased rectification is consistent with the reduced current flow from the TMA sideat inside positive voltages.

A

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4B), the rectification was reduced, which is attributable to thehigher concentration of K+ on the inside face of the mem-brane. Using the PK/PCI ratio, permeability ratios for a seriesof cations and anions were obtained under biionic conditions.The cation selectivity sequence is Cs+ > K+ > Na+ > Li+ >TMA+ > TEA+ with permeability ratios relative to K+ of1.19:1.00:0.80:0.64:0.34:0.20. As expected for a large aqueouschannel, the alkali cation permeability sequence is in order oftheir aqueous mobilities. The permeabilities of the organiccations TMA and TEA are diminished more than expectedbased on aqueous mobility, perhaps due to interactions withthe channel wall. Anion permeabilities are low, and littlediscrimination could be detected among the anions Cl-, Br-,NO3 and acetate-, perhaps due to their small contribution toErev (data not shown).A substantial preference for cations as shown by Cx46

hemichannels is a feature of some gap junction channels (3,20). Rectification similar to that shown by Cx46 hemichannelswas recently observed in heterotypic Cx32/Cx26 channels (21).Our data indicate that rectification can be a property of thehemichannel. Linking identical hemichannels in series to formhomotypic channels can give rise to symmetry about Vj = 0,and possibly linearity in the single channel I-V relation,whereas linking different hemichannels to form heterotypicchannels can give rise to various degrees of rectificationdepending on the intrinsic properties of the hemichannels(unpublished observations).

Since unapposed Cx46 hemichannels can open in a physio-logical range of membrane potential, they may play a signifi-cant role in tissue function as hemichannels, as well as cell-cellchannels. Other connexins may have similar properties. Thusfar, Cx46 has been shown to be expressed in the lens and inproliferating Schwann cells (22, 23). Teleost and elasmobranchretinal horizontal cells appear to have gap junction-likehemichannels that can be opened by depolarization in lowextracellular Ca2+ or by pharmacological agents, although theputative connexin(s) involved are unidentified (7, 24). Thestudy presented here represents a step toward understandingthe physiological roles of both hemichannels and cell-cellchannels, and the extent to which cell-cell channel propertiesare derived from properties intrinsic to the hemichannel. Theapplicability of hemichannel recording to other connexinsremains to be determined. Replacing individual domains ofother connexins with those of Cx46 (8, 9) may convert theminto functional hemichannels, which could be valuable indetermining the characteristics of the unmodified part of themolecule.

We thank Seung Hoon Oh for cloning and sequencing rat Cx46;Christopher Ginter for technical assistance; and Elliot Hertzberg,

Alan Finkelstein, Steve Slatin, Paul Kienker, Paul Huynh, and RhodriWalters for helpful comments on the manuscript. This work wassupported by National Institutes of Health Grants GM46889 andNS07412. M.V.L.B. is the Sylvia and Robert S. Olnick Professor ofNeuroscience.

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1796-1803.7. Malchow, R. P., Qian, H. & Ripps, H. (1993)J. Neurosci. Res. 35,

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