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Selective transfer of endogenous metabolites through gap junctions composed of different connexins

Gary S. Goldberg*†‡, Paul D. Lampe§ and Bruce J. Nicholson**Biological Sciences, Cooke Hall, State University of New York at Buffalo, Buffalo, New York 14260, USA

§Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA†Present address: Experimental Pathology and Chemotherapy, National Cancer Center Research Institute, 5-1-1 Tsukiji Chuo-ku, Tokyo 104, Japan

‡e-mail: [email protected].

ap junctions, the only channels that allow direct exchange ofsmall metabolites between cells, are composed of a family ofintegral membrane proteins, called connexins in vertebrates.

Different connexins have been implicated in a diverse array of bio-logical processes and diseases1, indicating that gap-junction proper-ties may vary with connexin composition. For example, channelscomposed of different connexins have different conductances2 andpermeabilities to ions3 and fluorescent dyes4,5. However, althoughthe permeability of gap junctions to some nucleotides6, cyclicAMP7, calcium8 and possibly inositol-1,4,5-trisphosphate8 has beendocumented, the comparative selectivities of different connexinsfor biologically significant molecules remain an enigma. Here,using new techniques, we show that the rate of permeation ofmetabolites through gap junctions differs according to connexincomposition.

One demonstration of the biological consequences of expressionof different connexin isotypes is their variable effectiveness in sup-pressing growth of tumour cells9. Although transfection of C6 gli-oma cells with connexin-43 (Cx43) suppressed their in vitro growthpotential10, Cx32 did not11. Paradoxically, however, Cx32 transfect-ants are far better coupled to each other, as assayed by calcein-dyetransfer (Table 1; for methods see ref. 12). A diffusion constant foreach cell interface was obtained from the time course of calceintransfer in two C6 cell transfectants12, using quantitative modellingof dye diffusion through cell monolayers5. Independently, we deter-mined the ratio of calcein permeabilities of Cx43 and Cx32 chan-nels to be 1.08 from quantitative measurements of calcein diffusionbetween Xenopus oocyte pairs expressing defined numbers of Cx43or Cx32 channels, calculated from the junctional and single-chan-nel conductance of each connexin (P.A. Weber, Y.I. Chen, J.Nitsche and B.J.N., unpublished observations). Applying the rela-tive calcein permeability of the two connexins to the diffusion con-stants calculated above for the transfected C6 cell lines produced anestimate of the relative number of channels in the Cx32 versus theCx43 transfectants of 16:1. Hence, the differential effects of thesetwo connexins on cell growth were related not to their expressionlevel but rather to some specific property of the channels, such asselective permeabilities for endogenous signals.

To address this possibility, we have developed techniques withwhich to ‘capture’, identify and quantify the junctional transfer ofendogenous metabolites between cells transfected with Cx43 or Cx32(ref. 13). ‘Donor’ cells were metabolically labelled overnight with[14C]glucose, fluorescently stained with DiI, and plated with unla-belled ‘receiver’ cells at a 1:6.25 ratio to yield confluent monolayers.After allowing 2 h for plating and establishment of cell contact andcommunication, we separated donors and receivers from each otherby fluorescence-activated cell sorting (FACS). Potential transjunc-tional molecules, which travelled from the donors to the receivers,were then resolved from larger components of the cellular lysates byfiltration (selecting material of relative molecular mass <3, 000)13.

Several controls indicated that nearly all of this transfer occurredthrough gap junctions. Analysis of the medium revealed no signifi-cant leakage of radioactivity from the cells. C6 cells transfected withthe empty vector (LTR) and connexin transfectants incubated inthe presence of the junctional blocker α-carbenoxolone (ACO)served as negative controls in each experiment. Low levels of trans-fer of metabolites were detected in LTR cells because of the presenceof low endogenous levels of Cx43 (ref. 12).

Despite the fact that the number of Cx32 channels was 16-foldhigher than the number of Cx43 channels, the transfer of endog-enous metabolites between cells expressing Cx32 or cells expressingCx43 was similar (Table 1). This supported the contention thattransfer of calcein did not accurately reflect the transfer of biologi-cally relevant molecules between these cells. The ACO block pro-vided a more dynamic view of transfer rates by allowing us toobserve the capture of metabolites following a limited period afterACO washout13. These results showed that the transfer of radiola-belled metabolites through Cx43 channels approached steady-statelevels within 20 min of communication. In contrast, calcein transferthrough either channel, or metabolite transfer through Cx32 chan-nels, reached no more than 50% of steady-state levels. Correctingfor the expression level of the two connexins described above, theseresults indicated that Cx43 channels were up to 50 times more per-meable to these metabolites than were Cx32 channels.

We subjected cell lysates to successive C18 reverse-phase andAminex HPX-87H ion-exchange high-performance liquid chroma-tography (HPLC) to identify specific metabolites and compare theirtransfer rates through the two channel types. Fractions from thesecond column were resolved by thin-layer chromatography (TLC),with internal standards being loaded in each lane, and capturedmetabolites were detected by exposure to storage phosphor screens.All radioactive measurements were normalized for the number of

G

Table 1 Dye and metabolite coupling of C6 cell transfectantsCell type Calcein coupling (total

number of receiver cells)*

*Number of receiver cells filled with dye per donor cell preloaded with calcein.

14C metabolites(percentage of donor cell)†

†14C metabolites (relative molecular mass <3,000) captured per receiver cell. Expressed aspercentage of similar labelled material in each donor cell (plating ratio of donors:receivers is1:6.25).

Steady state‡

‡Data taken 2 h after plating of donors and receivers.

ACO washout§

§Donors and receivers were plated in the presence of ACO. Data were taken 20 min after ACOwashout, or with no washout, and the difference is shown.All data are shown as means ± s.e.m. (with number of experiments in parentheses).

Steady state‡ ACO washout§

Control (LTR)

6.0 ± 0.7 (27) 0.6 ± 0.3 (16) 2.7 ± 0.8 (2) 0.8 ± 0.3 (2)

Cx32 106 ± 10 (8) 44.5 ± 5.9 (10) 6.4 ± 1.3 (2) 1.8 ± 0.4 (2)Cx43 14.1 ± 1.0 (45) 7.4 ± 0.6 (25) 8.0 ± 1.5 (2) 6.0 ± 0.5 (2)

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cells loaded on the columns. As shown previously13, a late fractionfrom the Aminex column contained labelled ADP only (Fig. 1a).Minimal transfer was seen in both LTR and ACO controls, whiletransfer through Cx32 transfectants was significantly less thanbetween the Cx43 transfectants (Fig. 1a).

Four more components in fractions eluting earlier from theAminex column (glucose (fraction 14), glutamate (fractions 13,14), glutathione (fractions 13–18), and ATP (fractions 14, 15)) weretentatively identified (Fig. 1b, c) by co-migration with unlabelledinternal standards in both columns and TLC plates. The identitiesof these components were verified by either hydrolysis with 6 MHCl at 100 °C for 1 h or treatment with bacterial alkaline phos-phatase overnight. Treatment of fraction 13 with alkaline phos-phatase caused the predicted shift of ATP to adenosine, withoutaffecting other components of the fraction (Fig. 1d). Acid hydroly-sis had predictably broader effects, causing a general reduction inTLC mobility of all components and cleavage of acid labile peptideand phosphodiester bonds (see, for example, fraction 15). Glutath-ione was cleaved into its constituent amino acids, but only gluta-mate was detected, because cold cysteine and glycine were presentin excess in the medium. ATP was reduced to adenosine and severalmodified variants (marked by asterisks).

Glucose transfer was detected in the Cx43 transfectants (Fig. 1b)

but not in Cx32 transfectants or control transfected cells. Glucosewas also not evident in these donors, possibly reflecting a higherglucose usage in the control and Cx32 transfectants compared withthe growth-inhibited Cx43-transfected cells. In addition to theseidentified compounds, one strongly labelled band that did not co-migrate with any internal standard was detected in fraction 18 (Fig.1b). This compound was notable in that it transferred efficientlybetween Cx43 transfectants but appeared to be effectively excludedby the Cx32 channels.

Quantitative comparisons of metabolite transfer through thegap-junction channels are presented in Fig. 2 as the percentage ofradioactive compound in a donor cell that was captured in (that is,transferred into) a receiver cell. Transfer of each compoundbetween the connexin transfectants exceeded that between LTRcontrols, and was reversibly reduced by ACO. The incomplete blockof metabolite transfer by ACO was consistent with earlier electricalcoupling that indicated that ACO did not mediate complete closureof the channels14. In stark contrast to calcein, which passed mostefficiently between Cx32 transfectants, glutathione and/or gluta-mate passed between the two transfectants to a similar degree,whereas ADP and/or ATP passed more efficiently through Cx43.No significant differences were evident in the transfer of ATPbetween Cx32 transfectants in the presence, absence and after wash-

Figure 2 Cx43 mediates the transfer of ADP and ATP between C6 cells better than does Cx32, but neither shows selectivity for glutamate and glutathione. Transfer of ADP, ATP, glutamate and glutathione between cells transfected with Cx32, Cx43 or empty vector (LTR) was calculated as the percentage of the compound present in each donor cell that was captured by an individual receiver cell (means ± s.e.m., n = 2). Given that the ratio of donors to receivers was 1:6.25, transfer to equilibrium would result in the capture of ~14% of the original material in the donor cell by the receiver cell. Values were quantified from TLC plates and include results from transfer in the absence (no ACO) and presence (ACO) of gap-junction blocker, and after a 20-min washout of ACO. Values for LTR cells were not determined in the cases of ACO treatment and ACO washout. The 20-min ACO-washout studies provided a better estimate of the rates of transfer of metabolites than did the steady-state measurements taken after plating in the absence of ACO. However, the actual rates of transfer in the Cx43 cultures were likely to be higher than estimated here, as metabolite distribution had already approached equilibrium within the 20-min capture period. Statistical analysis: transfer of all four of the individual compounds between both transfected cell lines exceeded that between LTR controls, and could be reversibly reduced by ACO. The probability that this pattern could occur by any chance means other than through gap-junctional coupling is <0.015 (sign test). Application of multivariant analysis (by analysis of variance, ANOVA) to the transfer of all four metabolites in the two connexin transfectants under each condition showed that the connexin type significantly influenced the pattern of transfer in the absence of ACO, or after ACO washout (P<0.002 for both). As expected, the effect of connexin type was abolished (P>0.7) in the presence of ACO, when junctional coupling is significantly inhibited.

Steady state (no ACO)

ACO block

20 min. ACO washout

Per

cent

of d

onor

s

15

10

5

0LTR 32 43 LTR 32 43 LTR 32 43 LTR 32 43

ADP ATP Glutathione Glutamate

Figure 1 ADP, ATP, glutamate and glutathione constitute the majority of transjunctional material derived from glucose. Fractions containing transjunctional metabolites resolved on an Aminex column were analysed by TLC to identify transjunctional components. Material eluting between 30 and 35 min was pooled and shown in a; fractions eluting between 13 and 18 min are shown in b, c. Data from Cx32, Cx43 and control (LTR) transfectants are shown. a, Material eluting at 30–35 min co-migrated with an ADP standard, and appeared in all donors (0D) and in Cx43 receivers in the absence (–R) or after washout (WR) of the gap-junction blocker ACO. Minimal levels of radioactive ADP were found in Cx32 or LTR receivers, or Cx43 receivers in the presence of ACO (+R). b, c, Material present in the 13–18-min fractions from donors and receivers. Identities of the labelled metabolites were determined by co-migration with internal standards in each fraction as indicated on the left and/or right of each panel (Glu, glutamate; GSSG, glutathione). One band that was not identified is indicated by ‘unknown’. Significant metabolite transfer was not evident between the control transfectants in c, whereas differential metabolite transfer was seen in comparisons of the Cx32 and Cx43 transfectants in b (see text). d, Further characterization of the metabolites shown in b, c. Treatment of fraction 13 with bacterial alkaline phosphatase (BAP) specifically converted ATP to adenosine (A). Acid hydrolysis (HCl) of fraction 15 reduced ATP to adenosine, as well as to some modified variants (A*), and glutathione to glutamate. Acid treatment also caused a shift in the mobility of all components. N, native material.

ADP

ADP

0D WR-R 0D -R +R WR+R0D -RLTR

LTR

Cx32

Cx32

Cx43

Cx43Donors Receivers

Donors Receivers

ReceiversDonors

GluGlucose

UnknownGSSG

ADP

Glu

Glucose

Glu

Glu

AA*A*

A

Glucose

UnknownGSSG ADP

GSSG

ATPGSSG

ADP

GluGlucoseUnknown

GSSG

13 14 15 16 17 18 13 14 15 16 17 18

13 14 15 16 17 18 13 14 15 13 13 15 1516 17 18

13 14 15 16 17 18 13 14 15 16 17 18

N BAP HCIN

a

dc

b

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out of ACO, indicating that ATP passage through Cx32 channelswas at or below the measurable limits of this assay.

Given the ratio of donors to receivers of 1:6.25, diffusion toequilibrium would have resulted in the capture by a receiver cell ofabout 14% of the material in an original donor cell. By this crite-rion, transfer of the compounds between the Cx43 transfectantsapproached 50% (glutamate and glutathione) to 80% (ADP andATP) of equilibrium within 20 min after ACO washout. In contrast,transfer of neither ADP nor ATP between the Cx32 transfectantsapproached these levels. The relative permeabilities of these twoconnexins, on a per-channel basis, were obtained by normalizingthe data in Fig. 2 to the relative channel numbers expressed by theCx32 and Cx43 transfectants (16:1). The data, after subtraction ofnegative controls, indicate that Cx43 channels were as much as120–160-fold more permeable to ADP and/or ATP than were Cx32channels, and 30–45-fold more permeable to glutathione and/orglutamate, although both channels were similarly permeable to cal-cein (Table 2).

The preferred passage of endogenous metabolites such as ADPand ATP through Cx43 as opposed to Cx32 channels may underliethe selective ability of Cx43 to inhibit growth of C6 cells in vitro.Diffusion of these high-energy metabolites from metabolicallyactive cells to less active neighbours may dilute their mitogenic

potential, resulting in suppression of cell growth. Although wecompared the capture of ADP, ATP, glutamate and glutathione asindividual species, metabolic conversions were likely to haveoccurred in the receivers. Hence, a significant portion of labelledADP and glutathione in the receiver cells may have resulted fromthe transfer of ATP and glutamate, respectively. In the cases shownhere, other conversions were unlikely given the high concentrationsof the species detected in the cells compared with their immediateprecursors. Nonetheless, our data show for the first time, to ourknowledge, dramatic differences in the permeability of differentconnexins to endogenous metabolites in cells. The identification ofdifferential permeabilities of connexins for specific metabolites willultimately be essential in understanding the specialized roles ofconnexins in vivo, as illustrated by their genetic linkage to severaldiseases and a growing number of knockout studies that show thatone connexin can not always substitute for another1. h

RECEIVED 23 MARCH 1999; REVISED 8 JUNE 1999; ACCEPTED 30 SEPTEMBER 1999; PUBLISHED 18 OCTOBER 1999.

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ACKNOWLEDGEMENTS

We thank P. Weber and J. Nitsche for development of the dye-diffusion model and permeability estimates that allowed quantification of channel number in the transfectants; D. Sheedy and C. Stewart for cell sorting; I. Silver and G. Koudelka for helpful discussions; Whatman and Molecular Dynamics for technical assistance with TLC; and C. Naus for continued help and interest. This work was supported by grants from PachTech and the Wendy Will Case Cancer Research Fund (to G.S.G.), the Whittaker Foundation (to B.J.N. and J. Nitsche), and the NIH (to B.J.N., grants CA480490 and GM48773, and P.D.L., grant GM55632).Correspondence and requests for materials should be addressed to G.S.G.

Table 2 Metabolite transfer between Cx43- and Cx32-transfected C6 cellsLabelled metabolite Ratio of counts captured Cx43:Cx32 transfectants

(normalized for expressed channel number)*

*Relative expression levels of Cx32 and Cx43 in the C6 transfectants studied were establishedto be 16:1 from quantitative dye transfer and channel permeability estimates (see text).

Steady state†

†Difference in transfer seen between connexin and control transfectants 2 h after plating.

ACO washout‡

‡Difference in transfer seen in the continuous presence of the junctional blocker ACO, andfollowing a 20-min washout.

ATP 54 160ADP 36 122Glutathione 13 44Glutamate 18 29

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