Proc. Nati. Acad. Sci. USAVol. 79, pp. 441-445, January 1982Cell Biology

Gap junctional conductance: Comparison of sensitivities toH and Ca ions

(electrotonic coupling/channel/blastomere/Fundulus/killifish)

D. C. SPRAY*t, J. H. STERNtt, A. L. HARRIS*t, AND M. V. L. BENNETT*t*Division of Cellular Neurobiology, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461; tGraduate Program in Biophysics,Brandeis University, Waltham, Massachusetts 02154; and the tMarine Biological Laboratory, Woods Hole, Massachusetts 02543

Contributed by Michael V. L. Bennett, September 17, 1981

ABSTRACT One cytoplasmic aspect of the junctional mem-brane between coupled pairs of Fundulus blastomeres was per-fused with solutions of known H and Ca ion concentrations. Con-ductance of junctional membrane was decreased by either ion.The sensitivity to H ions was about 10,000 times greater than thatto Ca ions. The results suggest that junctional conductance can bemodulated by changes in H ion concentration near physiologicalpH, but that unphysiologically high concentrations ofCa ion, suchas would be reached only on cell death, are required for compa-rable changes in junctional conductance.

The cells ofmany tissues are connected by arrays ofintercellularchannels identified morphologically as gap junctions (1). Gapjunctional channels permit electrolytes and small molecules toflow between coupled cells. Abundant circumstantial evidencesuggests that this form of intercellular communication is im-portant in development and in organized functioning of tissues(2). Factors that regulate the conductance of gap junctions maytherefore play a central role in these important cellularprocesses.On the basis of experiments in which cells were uncoupled

by treatments which presumably increased intracellular freecalcium ions (Cai) it was proposed that increased Cai reducesjunctional conductance (ga) (3). Subsequent experiments inwhich Cai was monitored by aequorin luminescence duringthese treatments supported this hypothesis (4). However, therelationship between Cai and gj has been evaluated only semi-quantitatively to date (5, 6).

Recently it was reported that acidification of the cytoplasmofelectrotonically coupled embryonic cells reversibly abolishedthe coupling (7-9). In experiments on isolated pairs of coupledcells in which the intracellular pH (pH,) and gj were directlymeasured, the relationship between pHi and g. was shown tobe a simple sigmoid curve (10). The relationship was well fit bya Hill plot with an apparent pKH of 7.3, only 0.4 pH unit belowthe normal pHi (pH 7.7) of these cells.

Because at least some intracellular buffering systems inter-change H and Ca ions (cf. ref. 11), an experimentally producedincrease in the concentration of either ion might cause a sig-nificant secondary increase of the other. Consequently, severalrecent studies measured the levels ofboth H and Ca ions duringexperimental treatments that uncoupled cells. Low pHi wasshown to decrease g without increasing Cai, as measured withaequorin in teleost {Fundulus) blastomeres (9) and with intra-cellular Ca-sensitive electrodes in amphibian (Xenopus) blas-tulae (12) and mammalian (sheep) cardiac muscle (13). Insect(Chironomus) salivary gland cells could be uncoupled by treat-ments that increased Cai without decrease in pHi. Conversely,

treatments that lowered pHi could uncouple these cells withoutincreased aequorin luminescence (14), supporting the indepen-dent action of pHi on g, observed in the other studies cited.

However, in this report the absence of increased aequorinluminescence during uncoupling by low pHi was ascribed to pHsensitivity of the aequorin-calcium interaction in spite of thefact that in standard saline solutions Ca-evoked aequorin lu-minescence is essentially insensitive to pH over the same range(15). Although these studies suggest that H and Ca ions can in-dependently close gap junction channels, the ionic activities atthe junctional membrane were not determined. Furthermore,the possibility remains that these ions affect gj via a secondarycytoplasmic intermediate.

The present paper reports experiments in which junctionalconductance was monitored while one cytoplasmic aspect ofthejunctional membrane was perfused with solutions in which theconcentrations of H and Ca ions were controlled. This tech-nique involved replacing the bulk ofthe cytoplasm from one cellof a coupled pair with a well-defined solution that could be rap-idly changed while preserving and measuring junctional con-ductance (16). The technique of internal perfusion of cell so-mata, pioneered by Kostyuk et aL (17) and Lee et aL (18) andmodified for multiple solutions by Stern and Lisman (19), wasadapted to this problem (Fig. 1). H and Ca ions were found todecrease g, independently, H ions being effective at concen-trations three to four orders of magnitude lower than those re-quired for Ca. Because concentrations ofthese two ions are sim-ilar in the cytoplasm of "healthy" cells, these findings imply thatphysiological regulation of gj is more likely to be accomplishedby H ion changes, whereas the role ofCa in regulation ofgj maybe confined to traumatic or pathological conditions.

METHODSEmbryonic cells from Fundulus heteroclitus (killifish) wereused. Cleavage-stage blastomeres were mechanically disso-ciated into single cells and reassociated as pairs. The cells werebathed in either Leibowitz L-15 medium (GIBCO) or double-strength Holtfreter's solution (120 mM NaCl/1.3 mM KC1/1.8mM CaCl2, buffered to pH 7.6 with 4 mM Hepes) containing0.5% colchicine to inhibit mitosis. Cell pairs were used eitherwith the perfusion apparatus (Fig. 1) or penetrated with fourmicroelectrodes (filled with 3 M KCl; resistance 5-20 MI) forindependent current delivery and voltage measurement in eachcell. In the latter case, conductances of junctional and non-junctional membranes were calculated from input and transferresistances (20). In some experiments, solutions were pressure-

Abbreviations: Cai, intracellular free calcium ions; gj, junctional con-ductance; pHi, intracellular pH; NTA, nitrilotriacetic acid; g, conduc-tance of perfused membrane patch; gn, conductance of nonperfusedmembrane.

441

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injected into one cell of a pair through its current-passingmicroelectrode.The perfusion solutions contained 100 mM KC1, 5 mM

Hepes, and 10 mM EGTA or nitrilotriacetic acid (NTA). Forsolutions of each pH, appropriate amounts of CaCl2 and MgCl2were added to result in 1 mM free Mg and the desired free Caconcentration between 0.1 1LM and 1 mM (pCa 7 to 3). Con-centrations of H and Ca ions were calculated according to theequations of Raaflaub (21) and Wolf(22). H and Ca ion activitiesofthe perfusion solutions were measured with calibrated H andCa selective electrodes; values given in the text are measuredactivities.The perfusion technique is illustrated in Fig. 1 and described

in the legend. The measure ofjunctional conductance is takenas gp/gl in which gp is the conductance of the patch of mem-

4, ~~~Perfusion head

Pipettetip ~~~Breaking-@-~~~Pipette tip~~~~~electrode

Vb VocI

Vi

FIG. 1. Apparatus and equivalent circuit for perfusion experi-ments. The internal perfusion apparatus (19) was similar to systemsused by others for intracellular dialysis of neuron somata (17, 18). Theperfusion head (top) included four small glass tubes extending towithin 50 Am of the pipette tip through which different solutions couldbe passed [only one (labeled In) shown above]. This arrangement per-mitted the solutions at the tip of the pipette to be changed rapidly andcompletely. To mechanically and electrically isolate the junctionalmembrane, one cell of a coupled pair was sucked into a perfusion pi-pette with a 20-j.m orifice until the second cell sealed against the tip.The first cell ruptured while being drawn through the 20-gm orificeor was subsequently broken by an additional concentric electrode(Breaking electrode) that was independently movable. The cytoplasm,which is quite fluid in these cells, was washed away from the rupturedcell's interior. This procedure resulted in the isolation of one cyto-plasmic surface ofjunctional membrane in the orifice of the perfusionpipette without apparent intervening cytoplasm. The rapidity andthoroughness of solution changes at the tip were occasionally moni-tored visually with the addition of a nontoxic dye (fast green) to oneof the solutions. To evaluate the conductance of the patch of membraneinside the pipette tip, the intact cell was penetrated with a voltagemicroelectrode (Vi). Two calomel electrodes in the bath were used tomeasure voltage (Vb) and to deliver current (I), which was measuredinside the outlet tube with a virtual ground current probe. From theequivalent circuit for the apparatus (bottom) the ratio of conductanceof the patch (gp) to the conductance of the membrane of the intact celloutside the pipette (ge) is given by:

gp/g. = (Vb - Vi)/(Vi - V8), [1]

in which V, = Ig8 is the voltage drop across the series conductanceof the perfusion pipette (g8), which was directly measured without acell present in each experiment and was typically 2-5 uS. The ratiogdgn was not affected by the value of the leakage conductance(gl) between the pipette tip and the cell membrane [the value ofg1 + gpgJ/(gp + gn) was generally <0.2 uS]. This ratio is a valid mea-sure of changes in gp (the conductance of interest) when gn is constant(see text).

brane being perfused and g& is the conductance of the mem-brane not being perfused. Changes in g,/gn correspond tochanges in junctional conductance if g& remains constant andonly the conductance of junctional membrane in the patch isaffected. Because ofthe rapidity and reversibility ofthe changesobserved, the cytoplasm of the intact cell is unlikely to havebeen significantly altered by the perfusion solutions. Thus, g.would have remained constant. Further evidence against actionthrough changes in the cytoplasm of the intact cell is givenbelow.To evaluate effects of nonjunctional membrane in the patch,

the perfusion technique was applied to single isolated cells. Theperfusion solutions had no large or consistent effects on theexternal surfaces of membrane patches of isolated cells. It isunlikely that much of the intact cell of cell pairs was exposedto the perfusion solutions because bathing intact cell pairs inthe control solution [0.1 ,uM Ca (pCa 7.0), pH 7.8]-which doesnot affect gJ. when injected intracellularly in large volumes-rapidly uncouples the cells and results in loss of cell-to-celladhesion, which, at best, is slowly reversible. Actually, changesin gp/g. were usually smaller than expected for well-coupledcell pairs, and junctional conductance may have been reducedsomewhat by the action of low Ca on the extracellular aspectof the junctions. Effects of H and Ca ions on the cytoplasmicaspect of the nonjunctional membrane are unlikely to accountfor our results because this face of only one of the two mem-branes in series is exposed to perfusion solution, and in intactcells changes produced by comparable concentrations ofH andCa ions are quite small (10) (see Fig. 6). We conclude thatchanges in the conductance of the patch membrane during ex-posure to perfusion solutions were primarily an action on thecytoplasmic aspect ofjunctional membrane.

pH7.8 7.2 7.8 6.8 7.2 6.8 7.2 7.8

V P1P. Tt F P EV

I1TRI

l11 li

5z 1 1 I II/

o .--- ..

0 20 40 60 80Time. sec

FIG. 2. Effects on patch conductance of lowering the pH at 0.1 ,MCa. Current pulses (I) were passed between the bath and the interiorof the perfusion pipette, and voltage in the intact cell (Vi), bath voltage(Vb), and the difference between the two (Vb -Vi) were recorded, thelast at higher gain. The ratio gp/gn is plottedbeneath the chart records.The pH of the initial perfusion solution was 7.8 and the Ca was 0.1 AM(pCa 7.0). Between the arrows the solutions were changed to the in-dicated pH values with Ca held at 0.1 1M. Decreases in pH withoutchanges in Ca level caused repeatable and reversible decreases ingp/g., and the values at pH 7.2 were the same whether pH was fallingor rising.

442 Cell Biology: Spray et al.

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3.9 3.6 3.3_

pCa3.0 3.3 3.6 3.9

_ __~in~ ~ _mw o,C_

-p-I-I-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I-I'

Vb

-IT S-'75 i

).5

25

0p0 50 100 150 200 250

Time, sec

3 pH 6.8 pH 7.8 pH 7.8pCa 4.3 pCa4.3 pCa 7

l:E~~~~~~~~~~~W

jago~~~~~~~~~~~~~c

pH 7.EpCa 7

Vb - Vi

V1EC)

Vb0

FIG. 3. Effects of raising Ca levels atpH 7.8 on patch conductance.Recording and display as in Fig. 2. Perfusion of the membrane patchwith solutions increasing in Ca concentration from 0.13 to 1 mM (pCafrom 3.9 to 3.0) at constant pH 7.8 caused progressive reduction ing,,/g that reversed when calcium levels were restored.

RESULTSEffect of Low pH on Junctional Membrane. Patches in-

cluding junctional membrane were perfused with solutions atpH values of 7.8, 7.2, and 6.8 containing 0.1 1uM free Ca (pCa7.0, Fig. 2). The ratio gp/g decreased reproducibly and re-

(14)(5)

pCa

(7)(3) i

=1-------.0

G

0.5 Ox6.8 7.8

pH

5.0

FIG1-4. Dependence of patch conductance on pCa and pH. Thelarger graph shows the relation between gg,/ and Ca concentrationexpressed as pCa at pH 7.8. Data points from pCa 4.0 to 6.0 are meanvalues normalized with respect to the mean value at pCa 6.0. Eachpoint is the mean value of trials performed on each of three or morecell pairs; bars represent standard errors and the numbers of trials areindicated. For data points from pCa 3.0 to 3.9 the mean at pCa 3.9 wasassumed to be equal to the mean at 4.0 and was similarly normalized.The curves are plots of the Hill equation for normalized patch con-ductance, gp/g, = K^/(K' + [Ca]'), with values of K, the equilibriumconstant, and n, the Hill coefficient, chosen by eye. The solid curve isbased on the assumption that the minimal value of g/gn was almostreached at pCa 3.0. The dashed curve is based on the assumption thatgpl/gn approached zero asymptotically. The Inset summarizes the nor-malized data from seven experiments (see text) with pH values of 6.8,7.2, and 7.8 with pCa 7.0 (mean and SEM at pH 7.2). The dotted linein the Inset is a plot of the Hill equation for normalized conductanceG with pKH 7.3 and n = 4.5 previously shown to describe the pH1-gjrelation in intact cell pairs (10).

I

0.5,, _0 20 40 60 80 100

Time, sec

FIG. 5. Effects of change in pH and pCa in the same preparation.Change from a solution at pH 7.8, pCa 7.0 to a solution at pH 6.8, pCa4.3 resulted in a profound decrease in g?,/lg When the patch was per-fused with a solution at pH 7.8, pCa 4.3 (50 ,uM Ca) gplgn completelyrecovered. Decreasing pCa from 4.3 (50 ,uM) to 7.0 (0.1 jLM) had no fur-ther effect on gp/gn. The overshoot seen upon recovery at normal pHpresumably reflects a commonly observed progressive change ing,,/g that is ascribable to increase in the area of membrane suckedinto the pipette.

versibly with decreased pH. The changes were rapid and theironsets coincided with arrival of new solution at the pipette tipas monitored visually with dye-containing solutions.

These data were normalized for comparison with previousexperiments in which the cytoplasm ofintact cells was acidifiedby superfusion with saline containing weak acid (10). The nor-malization procedure is based on the pH sensitivity of gj in in-tact cells, where junctional conductance is close to maximal atpH 7.8 and minimal at pH 6.8. If the same holds for pH sen-sitivity of gp/g& in the perfused preparation, the fraction of thetotal changes in g/gn at pH 7.2 is predicted by the curve re-lating gjand pHi in intact cells (see Fig. 4 Inset). The agreementat pH 7.2 indicates that H ions affect gj similarly in the perfusedand intact preparations.

Effect ofCa Ion on Junctional Membrane. Patches includingjunctional membrane were perfused with solutions containingfree Ca concentrations ranging between 0.1 AM and 1 mM (pCa7.0 to 3.0) at pH 7.8. Solutions containing 0.1 mM free Ca orless (pCa 2 4.0) had no significant effect on gp/&, whereas so-lutions containing 0.3-1.0 mM free Ca (pCa 3.6 to 3.0) de-creased gp/gn (Figs. 3-5). The actions of Ca ions were revers-ible, and intermediate values ofg&/g, obtained at intermediateCa concentrations were similar whether the preceding Ca con-centration was higher or lower.

In Fig. 4, values of gp/g, for pCa 6.0 to 4.0 were normalizedwith respect to the value at pCa 6.0. In the separate series ofexperiments for pCa 3.9 to 3.0, the value of gp/g, at pCa 3.9was assumed to be equal to the mean value at pCa 4.0. Becausegp/g. may not have reached its minimum even at the highestcalcium concentration used (1 mM, pCa 3.0) the maximal effectofCa is uncertain. However, the minimal value ofg/gn at highcalcium levels must lie between zero and 0.25 (its value at 1 mMCa). By fitting Hill plots to the data for each of these lower lim-its, we estimate that the apparent pKCa falls betwen 3.25 and3.45. The corresponding range for the apparent dissociationconstant (Kc) is 0.6 to 0.4 mM.

Vb - Vi

vi

0.R! o

ho

bD0.1

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An example of the insensitivity of junctional conductance tosolutions of 50 ,M Ca (pCa 4.3) is shown in Fig. 5, which alsoillustrates the capability of the technique to make simultaneousand separate changes of different ions. In this experiment con-

ductance of a patch initially exposed to a solution at pH 7.8 and0.1,M Ca (pCa 7.0) was decreased by exposure to a solutionat pH 6.8 and 50,M Ca (pCa 4.3). The patch completely re-

covered its conductance when exposed to a solution at pH 7.8that still contained 50 AuM Ca (pCa 4.3). Subsequent reductionof Ca to 0.1,M (pCa 7.0) at pH 7.8 had no additional effect on

gp/gn.Injection of Calcium Buffers into Cell Pairs. The low sen-

sitivity of junctional conductance to intracellular Ca indicatedby the above perfusion experiments is consistent with the ef-fects ofintracellular pressure injection ofthe perfusion solutionsinto one of a pair of intact coupled cells. In the experiment ofFig. 6 Left a small injection (about 20% of the cell volume) ofthe 0.5 mM Ca solution (pCa 3.3) rapidly decreased the transferresistance and increased the input resistance of both cells. Cal-culations from these data showed that the junctional conduc-tance decreased profoundly, recovered, and then increased be-yond its initial value. (Such overshoots did not always occur.)The nonjunctional conductance of the injected cell transientlyincreased and then recovered, and the nonjunctional conduc-tance of the uninjected cell was unchanged. The brief increasein nonjunctional conductance ofthe injected cell may have been

V 'T !7aM,.r,uvirr...... iI[!IIIg![Ia I | @ p

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6 -pCa 3.9

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FIG. 6. Changes in conductances of intact cell pairs

jection of Ca buffers at 0.5mM and 0.13mM free Ca

The three traces show voltage in the injected cell

second cell NVO), and the current pulses of approximately

plitude applied alternately in the two cells (I). The

corresponding calculated values of g. (e) and nonjunctionaltances (20) (', cell 1;*, cell 2). (Left) 6a buffer at

3.3) in amount estimated visually as 20% of the

jected (arrows). Over a period of about 20 sec the

of each cell decreased as indicated by increase

deflection in each cell due to the current applied

dicated on the graph below). The transfer conductances

indicated by the fall in the smaller potential in

current applied in the other cell. Recovery to initial

about 150 sec. The graph shows that gj decreased 0.1

MiS, recovered, and then increased to about 3 MS. Nonjunctionalductances were unaffected except for a brief increase

probably due to electrode movement. (Right) A

injected with solution buffered to pCa 3.9. A much

given, which approximately doubled the cell volume.

a brief decrease in gj from about 4.8 to 3.2 AS and

junctional conductances. These sensitivites to injected

rable to those observed in the perfused patch membrane

due to trauma that accompanied the injection. In contrast, a

large injection (100% increase in cell volume) of the 0.13 mMCa (pCa 3.9) solution in another cell pair produced a brief mod-est decrease in gj with little change in the nonjunctional con-

ductances (Fig. 6 Right). Smaller injections of this solution werewithout effect.

The injection experiments allow unambiguous determinationof junctional and nonjunctional conductances, but they do notpermit precise determination of Ca,. Total Ca concentration,bound and free, in the injection solutions was 2-10 mM, butfree Ca concentration in the cells after injection would dependon the amount of Ca and buffer injected, the intracellularpH,and the capacity of intracellular Ca buffering systems. Althoughrecovery of gj after the injections indicates that intracellularbuffering was only transiently overcome, the relatively smallduration and magnitude of the effects of large volumes of the0.13 mM Ca (pCa 3.9) perfusion solution are in agreement withthe perfusion experiments.

DISCUSSION

The present study indicates that H and Ca ions can indepen-dently reduce junctional conductance. However, we must con-

sider the possibility that these ions affect the junctional con-

ductance by changing the level of a cytoplasmic intermediatein the intact cell. Tracer studies with other small ions and muchlarger molecules suggest that on the basis of size and charge bothH and Ca ions should permeate gap junctions (23) althoughChironomus junctions have been reported to be impermeableto Ca ions (6).

In intact cells injection of Ca ions produces only transientchanges ingj, indicating that the cell can rapidly restore its freeCa concentration. Therefore, in the intact cell of the perfusedpreparation, Ca concentrations would be expected to be muchlower than in the perfusion solution. The similar sensitivity ofgj to Ca ions when injected or when perfused implies that theCa ions directly affect junctional membrane rather than actingby crossing the junctions. In perfusion experiments with H ionsthe pHi in the intact cell would be higher than in the perfusateif the cytoplasmic buffers were not overloaded. As with Ca, thesimilar sensitivity ofgj in perfusion experiments and in intactcells would imply a direct action on the junctional membrane.Conceivably in perfusion experiments H ions crossing the junc-tions might overcome the intact cell's buffers so that pH wouldapproach the same value on both sides of the junction. In intactcells injection of strong acids results in stable decreases ing

(reversible by bathing with weak base; unpublished observa-tions). In the perfused preparation decrease in patch conduc-tance caused by H ions crossing the junctions should trap H ionsin the intact cell once the junctions were largely closed andpatch conductance should then recover only slowly. In fact,even large decreases in patch conductance caused by H ionswere rapidly reversible, and we conclude that H ions act di-rectly on the junctional membrane rather than through a cy-

toplasmic intermediate in the intact cell.In the perfused preparation g was very sensitive to changes

in pH, near the normal resting level. This sensitivity is also seen

in cell pairs ofAmbystoma and Fundulus (10), in intact Xenopusblastulae (8), in Chironomus salivary gland (14), and in crayfishseptate axon (24). In sheep Purkinje fibers, in which pHi is nor-

mally 7.2-7.3, sensitivity to hydrogen ions can be inferred fromthe opposite actions of externally applied CO2 and NH3 on gj(13).The low sensitivity of the junctional conductance to Ca ions

observed here is in only moderate disagreement with the re-

ported effectiveness of Ca in uncoupling cells of Chironomus

V,

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0

[..!7..PIT"'. I.IIII MMMRIOM,IRIIJOWN

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Proc. Natl. Acad. Sci. USA 79 (1982) 445

salivary gland. Oliveira-Castro and Loewenstein (5) reportedthat when a hole was made in one of a chain of coupled cells,"uncoupling" was observed when the bath concentration of Cawas 0.08 mM (pCa 4.1) or above, but not when it was 0.04 mM(pCa 4.4). The degree of uncoupling was not specified nor wasjunctional conductance determined. The Ca levels in the bath-ing solutions were not buffered or measured, and actual valuesmay have been higher due to contamination.We found that there was little effect of Ca levels of 0.13 mM

or below and that a lower limit for maximal reduction of gj wasabout 1.0 mM. In cardiac muscle, uncoupling by dihydro-oua-bain and dinitrophenol causes Cai to increase to only 4-40 AM(pCa 5.4-4.4) as measured with Ca-sensitive microelectrodes,but possible changes in pHi were not determined (25). Sup-porting the insensitivity ofgap junctions to Ca is the low affinityof the major Ca-binding component found in junctions isolatedfrom ox and guinea pig ventricles (26). The observed Kd ofabout0.5 mM (pCa 3.3) and maximal slope of 1.77 are both within therange of our estimates from dependence of g. on Ca levels.

In our experiments, near their pKs H and Ca ions affectedgj with slopes differing by a factor ofaround 2. Conceivably, twoH ions or a single Ca ion could bind at the same site, althoughwith much different affinities.

The experiments described above show that (i) H and Ca ionscan act independently to decrease junctional conductance, and(ii) H ions are effective at levels 1/10,000th of those requiredfor Ca (pKH = 7.3, pKC, = 3.3). In the present study, in contrastto previous studies of intercellular coupling, the concentrationsofthe two ions ofinterest close to thejunctional membrane wereknown and could be independently altered. These features re-duce the quantitative ambiguities ofprevious studies, includingthe problem of spatial resolution of ion activity.

Free cytoplasmic Ca is normally <0.1 ,M (pCa 7. 0) in manycells, including amphibian blastomeres (12), sheep Purkinje fi-bers (13), and squid axons (27, 28). Because pCai is normally wellbelow the levels required to have effects on junctional conduc-tance, it seems unlikely that Ca ions regulate junctional con-ductance under physiological conditions. However, cytoplas-mic Ca concentrations may approach millimolar levels underpathological circumstances such as cell death or membrane dis-ruption. Under these conditions Ca ions may provide the mech-anism by which a severely diseased or damaged cell may un-couple from its neighbors to preserve tissue integrity (5, 29, 30).

Cytoplasmic H ion activities, on the other hand, are normallyat levels appropriate for sensitive control of junctional conduc-tance. The pHi for Fundulus and amphibian embryonic cells isabout 7.7 and is several tenths of a pH unit lower in other celltypes (13, 31). Small deviations in pHi from its normal levelwould therefore modulate junctional conductance. Further-more, pHi may be important in pathological as well as physio-logical changes in coupling. For example, ischemia of the heartcauses uncoupling of cardiac cells (32) and a decrease in pH,(33, 34).

We gratefully acknowledge the technical assistance of C. LoBue,helpful discussions with J. Lisman, S. Levy, and S. Smith, and con-struction of the perfusion apparatus by D. Eaton of the Brandeis Uni-

versity Machine Shop. This research was supported by Grants HD-02428, NS-12627, and NS-07512 from the National Institutes of Health.A. L.H. was the recipient of National Research Service Award Post-doctoral Fellowship NS-06342.

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2. Bennett, M. V. L., Spray, D. C. & Harris, A. L. (1981) Am. Zool21, 407-422.

3. Loewenstein, W. R. (1975) Cold Spring Harbor Symp. Quant.Biol. 40, 49-63.

4. Rose, B. & Loewenstein, W. R. (1975) Nature (London) 254,250-252.

5. Oliveira-Castro, G. M. & Loewenstein, W. R. (1971)J. Membr.Biol. 5, 51-77.

6. Rose, B. & Loewenstein, W. R. (1976) J. Membr. Biol 28,87-119.

7. Turin, L. & Warner, A. (1977) Nature (London) 270, 56-57.8. Turin, L. & Warner, A. (1980) J. Physiol (London) 300, 489-504.9. Bennett, M. V. L., Brown, J. E., Harris, A. L. & Spray, D. C.

(1978) Biol Bull 155, 428.10. Spray, D. C., Harris, A. L. & Bennett, M. V. L. (1981) Science

211, 712-715.11. Meech, R. W. & Thomas, R. C. (1977) J. Physiol (London) 265,

867-879.12. Rink, T. J., Tsien, R. Y. & Warner, A. E. (1980) Nature (London)

283, 658-660.13. Weingart, R., Hess, P. & Reber, W. R., in International Sym-

posium on Normal and Abnormal Conduction in the Heart, eds.Hoffman, B., Lieberman, M. & Paes de Carvalho, A. (Elsevier,Amsterdam), in press.

14. Rose, B. & Rick, R. (1978)J. Membr. Biol. 44, 377-415.15. Shimomura, 0. & Johnson, R. H. (1973) Biochem. Biophys. Res.

Commun. 53, 490-494.16. Stern, J. H., Spray, D. C., Harris, A. L. & Bennett, M. V. L.

(1980) Biol. Bull. 159, 493.17. Kostyuk, P. G., Krishtal, 0. A. & Pidoplichka, V. I. (1977) Na-

ture (London) 267, 70-72.18. Lee, K. S., Akaike, N. & Brown, A. M. (1978)J. Gen. Physiol. 71,

489-507.19. Stern, J. H. & Lisman, J. E. (1980) Fed. Proc. Fed. Am. Soc. Exp.

Biol 39, 2137.20. Bennett, M. V. L. (1966) Ann. N.Y. Acad. Sci. 137, 509-539.21. Raaflaub, J. (1956) in Methods of Biochemical Analysis, ed.

Glick, D. (Interscience, New York), Vol. 3, pp. 301-318.22. Wolf, H. U. (1973) Experientia 15, 241-249.23. Bennett, M. V. L. (1978) in IntercellularJunctions and Synapses,

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