Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641
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ABSTRACT |
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Exposure of the urinary bladder epithelium of Necturus maculosus (NUB) to protease and collagenase yields ~50% isolated polarized cells. These cells express a membrane current slowly activated by depolarization or by removal of external divalent cations. The biophysical and pharmacological properties of the current are largely consistent with those of gap junctional hemichannels. After removal of divalent cations, the cells can also be loaded with 5(6)-carboxyfluorescein, a hydrophilic fluorescent anionic dye, and exposure to dye reduces the current in a manner dependent on membrane voltage and side of application. In contrast, Necturus gallbladder (NGB) cells exhibit no membrane conductance attributable to gap junctional hemichannels, although previous studies reveal the persistence of gap junction plaques on the plasma membrane. We conclude that functional gap junctional hemichannels can be expressed on the surface of certain isolated epithelial cells and that this is not a necessary consequence of the isolation procedure. These structures may contribute to cell damage under pathological conditions involving cell detachment.
gap junctions; epithelial cell injury; connexons; hemi-gap junctions
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INTRODUCTION |
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EPITHELIAL CELLS ARE COUPLED via gap junction channels that allow the intercellular exchange of ions and small molecules such as ATP and second messengers, thus making the epithelium a metabolic and functional syncytium. Gap junctions consist of two connexons in series. Each connexon is formed by six connexin molecules arranged concentrically to form a central cavity. Although it has been shown that gap junctional hemichannels can be expressed by native isolated cells (e.g., Ref. 3), their functional significance and detailed properties have not been ascertained. An experimental system permitting the study of a gap junctional hemichannel in its native cell is interesting for two reasons. First, it can shed light on the functional properties and regulation of connexons without the interference of a changed cellular environment; second, it can help us understand the potential physiological or pathophysiological roles of these structures. The latter point is especially pertinent in epithelia, in which damage may involve detachment of cells from the basal lamina and/or from neighboring cells (9).
In this communication we report that exposure of the urinary bladder of Necturus maculosus (NUB) to a mixture of protease and collagenase yields a population of isolated epithelial cells, of which about half remain polarized. The isolated cells exhibit membrane currents with properties consistent with gap junctional hemichannels and, in medium devoid of divalent cations, can be loaded with 5(6)-carboxyfluorescein (CF), a hydrophilic fluorescent dye. In contrast to the NUB cells, Necturus gallbladder (NGB) cells exhibit no membrane conductance attributable to hemichannels, although previous electron microscopic studies demonstrate that connexons remain present on the surface. We conclude that functional gap junctional hemichannels can be expressed on the surface of isolated epithelial cells and that this is not a consequence of the isolation procedure. Some of these results have been presented in preliminary form (22).
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METHODS |
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Cell isolation. NUB and NGB were excised, opened longitudinally, and pinned serosal side up on Sylgard-coated petri dishes. Epithelial cells were dissociated by a 1.5-h incubation at room temperature in physiological salt solution (PSS) containing collagenase type IV and protease type XIV (1 mg/ml each; Sigma Chemical, St. Louis, MO). The composition of the PSS was (in mM) 90 NaCl, 0.5 sodium phosphate, 2.5 KCl, 1.0 MgCl2, 1.8 CaCl2, 10 HEPES/NaOH, and 7.5 glucose, pH 7.60, osmolality ~195 mosmol/kgH2O. After the incubation, the tissue was washed with ice-cold, nominally Ca2+- and Mg2+-free PSS containing 0.5 mM EDTA. The cells were then resuspended in PSS and maintained in the refrigerator until used (up to 4 h later).
Wheat germ agglutinin labeling. Apical surfaces of stretched NUBs were labeled with wheat germ agglutinin conjugated with FITC (WGA-FITC; 25 µg/ml for 30 min; Sigma). After the extracellular dye was washed out, the epithelial cells were dissociated as described above and were observed with a confocal fluorescence microscope (Odyssey, Noran, Middletown, WI). The basolateral surface of the epithelium was never exposed to the dye.
CF uptake. Separate aliquots of dissociated cells were incubated at room temperature for 20 min in the presence of 0.5 mM CF in either PSS or in nominally Ca2+- and Mg2+-free PSS containing 0.5 mM EDTA. After removing the extracellular dye by centrifugation, the cells were resuspended in PSS and plated on Cell-Tak-coated glass coverslips. In experiments employing Gd3+, sodium phosphate and calcium chelators were deleted from the Gd3+-containing and control solutions. Intracellular fluorescence was monitored using confocal fluorescence microscopy. Uptake was quantified as the percentage of viable epithelial cells loaded. Viability was detected from cell morphology, based on control experiments comparing morphological features with trypan blue or propidium iodide exclusion. Individual cells with a homogeneous loading pattern and with a fluorescence level over a threshold established from the fluorescence intensity distribution were considered positive. All measurements were carried out with Metamorph V2 (Universal Imaging, West Chester, PA).
Electrophysiological techniques.
Isolated cells were studied by the whole cell broken-patch
configuration of the patch-clamp technique (11). The pipette solution
contained (in mM) 90 KCl, 1.0 EGTA, 1.0 MgCl2, 10 HEPES/KOH, and 2 Na2ATP, pH 7.40, osmolality ~190
mosmol/kgH2O. The control bath
solution was PSS without phosphate. The reference electrode was an
Ag-AgCl wire in a bath solution/3% agar bridge. To increase the yield
of gigaohm seals, the isolated NGB cells were exposed for 5 min to
hyaluronidase (1 mg/ml, Sigma) immediately after completion of the
incubation in collagenase and pronase and then washed twice with PSS.
Cells were plated on dishes or coverslips coated with Cell-Tak. Whole
cell currents were measured with 3-5 M borosilicate glass
pipettes using an Axopatch 200B patch-clamp amplifier and pCLAMP6 (Axon
Instruments, Foster City, CA). All measurements were corrected for
liquid junction potentials (1). The hyaluronidase treatment had no
effect on the whole cell currents in either NGB or NUB cells. The
pharmacological agents used in the electrophysiological experiments
were halothane (2 mM, Halocarbons Labs., Hackensack, NJ),
n-octanol (0.5 mM, Sigma), and
18
-glycyrrhetinic acid (GA; 100 µM, Sigma); GA was dissolved in
DMSO to a final vehicle concentration of 0.1% vol/vol, which had no
electrophysiological effects by itself.
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RESULTS |
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Sizable fractions (40-60%) of cells isolated from NUB or NGB retain structural and functional polarity. Polarized cells have a characteristic "figure eight" shape, with two asymmetric domains corresponding to the apical (smaller) and basolateral (larger) regions. In the experiment shown in Fig. 1, the apical surface of the NUB epithelium was labeled with WGA-FITC before cell dissociation. In polarized (figure eight) cells, the fluorescent label stayed restricted to the apical membrane domain, whereas in nonpolarized (round) cells the label was distributed throughout the plasma membrane, although only the apical surface of the epithelium was exposed to WGA-FITC. This result indicates that apical membrane glycoproteins redistribute by lateral diffusion in cells that lose their polarity. In NGB polarized cells, apical and basolateral membrane domains have been shown to differ in detailed lipid and protein compositions, whereas nonpolarized cells are round and no plasma membrane domains exist (20).
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The expression of functional hemi-gap junction channels has been
suggested for isolated cultured cells (14) because exposure to
low-Ca2+ medium induces uptake of
small (<1 kDa) hydrophilic fluorescent dyes. We observed that, after
removal of external divalent cations, CF (mol wt 376.3, z = 2) was taken up by NUB but
not by NGB cells (Fig.
2A). In
NUB cells, the percentage of loaded cells increased significantly,
following removal of Ca2+ and
Mg2+, whereas in NGB cells
divalent cation removal had no effect (Fig. 2B). With each preparation there
were no appreciable differences between fluorophore loading in
polarized and nonpolarized cells. As shown in Fig.
2C, the percentage of loaded cells in
divalent-cation-free medium was significantly inhibited by 10 µM
GdCl3 and by 100 µM GA, both
blockers of gap junctional intercellular communication or gap
junctional hemichannels (2, 6). The loading of NUB cells with CF was
not altered by reducing the incubation temperature from ~23°C to
~4°C. At the same temperatures, there was no significant Ca2+-sensitive loading of 3-kDa
dextran labeled with fluorescein (0.5 mM). These results indicate that
the Ca2+-sensitive uptake of CF is
not due to endocytosis. Propidium iodide loading was assessed in the
presence of Ca2+ and
Mg2+, in cells preincubated in
either PSS or divalent-cation-free PSS. There was no difference in
loading, indicating that removal of divalent cations does not cause
irreversible membrane permeabilization.
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Isolated NUB cells (Fig. 3B), but not isolated NGB cells (Fig.
3A), display a slowly activating current in response to
membrane depolarization in control bath solution (Fig.
3). Consistent with the cell-loading
results, these currents were observed in both polarized and
nonpolarized cells but were ~40% smaller in nonpolarized cells (data
not shown). In NUB cells clamped at depolarized voltages, returning to
the holding potential (60 mV) caused distinct tail currents. The
observation that a pulse to 0 mV elicited no appreciable current, but
resulted in a small inward tail current on returning to the holding
potential (
60 mV), suggests that the reversal potential is near
0 mV. Hence the underlying channels are either anion selective [the
Cl
equilibrium potential
(ECl)
0] or poorly selective between Na+ and
K+. These features are strikingly
similar to those of the currents obtained after expression of Cx46 or
Cx38 in Xenopus oocytes (4, 6, 17). In
contrast, in NGB cells a fast-activating current (<100 ms) was
observed at positive voltages; after returning to the holding
potential, no tail currents were observed (Fig.
3A). The current in NGB cells is
insensitive to removal of external divalent cations and is mediated by
apical membrane maxi-K+ channels
(19). In NUB cells held at a negative holding potential (
60 mV),
fast superfusion with a solution free of divalent cations elicited a
reversible increase of the (inward) holding current (Fig.
3C). The time course of this
response was similar to that of the current sensitive to external
Ca2+
(Ic)
observed in oocytes (24), which has been attributed to functional
endogenous Cx38 hemichannels. In the absence of external Ca2+ and
Mg2+, the magnitude of the
depolarization-activated currents and the rate of activation were
increased, and the time constant for decay of the tail currents was
prolonged. A small instantaneous and linear component of the whole cell
current appeared under these conditions. In contrast to the
Ic observed in
oocytes, the whole cell current activated by removal of external
Ca2+ and
Mg2+ is slightly anion selective,
as demonstrated by the change in reversal potential elicited by
dilution of bath salt (Fig. 3D). Equimolar reduction (
= 50%) of external
K+ and
Cl
(isosmotic sucrose
replacement) shifted the reversal (zero-current) potential by 7.4 ± 0.8 mV (n = 9), a value consistent
with the permeability ratio of
Cl
to
K+
(PCl/PK)
2.4. When the current was activated by membrane depolarization, the
reversal potential yielded a similar anion/cation permeability ratio
(data not shown). When external and internal
Cl
were partially replaced
with cyclamate, equiosmolar replacement of 50% of the potassium
cyclamate with sucrose shifted the reversal potential by 1.1 ± 1.8 mV (n = 5). This observation suggests
that Pcyc/PK
1.
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The pharmacological properties of the whole cell current are similar to
those of the dye uptake and to those of hemichannels expressed in
Xenopus oocytes.
GdCl3, GA, and halothane reduced reversibly the whole cell current (Fig. 4,
A and
B) at concentrations that inhibited
CF uptake. The blocks were observed both in the presence and in the
absence of extracellular divalent cations. Octanol (0.5 mM) exerted a
small but significant inhibition of the current activated by removal of
divalent cations. Higher octanol concentrations produced irreversible
cell permeabilization. As shown in Fig.
4A, GA blocked the
depolarization-activated current. Similar results were obtained with
the other blockers (summarized in Fig.
4B).
GdCl3 (1 µM) inhibited the
current by ~35% (n = 4; not shown).
Like GA, both halothane and octanol have been used as blockers of gap
junctional communication (16). As shown in Fig.
4C, addition of CF to the pipette
solution reduced the inward current activated by removal of external
Ca2+ and
Mg2+. Addition of the dye to the
bath solution reduced the outward current activated by divalent cation
removal (Fig. 4D). These results are
consistent with channel block by the voltage-dependent entry of CF
(z = 2) in the permeation
pathway.
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DISCUSSION |
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Most NGB epithelial cells dissociated with collagenase and pronase retain viability, and a significant fraction remains structurally and functionally polarized (20). With this preparation both membrane domains are accessible to patch-clamp recordings. Because the NGB epithelium is a leaky tissue (i.e., high transcellular conductance relative to the paracellular conductance, see Ref. 8), it was of interest to test whether similar isolated-polarized cells could be obtained from a tight epithelium (higher paracellular conductance). The dissociation of NUB epithelium (12) also yielded polarized cells (Fig. 1). Unexpectedly, we found whole cell currents with features similar to those of the currents elicited by heterologous expression of connexins. Because these currents are not observed in cells isolated from NGB with the same enzymatic procedure, we conclude that their presence in the NUB cells is not a necessary consequence of the enzymatic treatment.
Our main observations are that isolated NUB epithelial cells express
poorly selective whole cell currents activated by depolarization or
removal of divalent cations from the external medium. These currents
are inhibited by several blockers of gap junctional communication, by
Gd3+ and by CF. The gating and
kinetic properties are similar to those found in oocytes after
expression of connexins (Cx) -3 (rat Cx46, bovine Cx44, and chicken
Cx56; Refs. 6, 10, 17, 18),
-2
(Xenopus Cx38; Ref. 4) or
-8 (rat
Cx50; G. A. Zampighi, personal communication). The
properties of such currents appear to result from the presence of
hemi-gap junction channels (connexons) in the plasma membrane of these
cells (G. A. Zampighi, personal communication), and many properties of
these hemichannels are consistent with the properties of assembled gap
junctions (5). However, the currents described here are predominantly
anionic, an unusual observation with gap junctions (23).
The expression of hemichannels in the plasma membrane of isolated cells has been associated with electrophysiological effects, changes in net solute permeability (e.g., oocyte osmotic fragility), and dye loading of Novikoff cells (endogenous hemichannels) or HeLa cells (transfected with connexin cDNAs) (14). After removal of external divalent cations, we observed four kinds of internally consistent results in NUB cells: first, uptake of CF, a gap junction permeable hydrophilic dye; second, activation of whole cell currents; third, inhibition of dye uptake and whole cell currents by agents known to inhibit gap junction intercellular communication in tissues or hemichannels expressed in Xenopus oocytes; and fourth, inhibition of the whole cell current by a gap junction-permeant anionic hydrophilic dye, in a manner consistent with permeation of the same pathway. Taken together, these results support the hypothesis that current and dye uptake are due to the expression of functional native gap junctional hemichannels. In NGB cells neither hemichannel-like currents nor dye uptake is observed, supporting the hypothesis that both phenotypes are correlated. Freeze-fracture electron microscopy suggests that gap junctions (or hemi-gap junctions) are expressed on the plasma membrane of both NGB and NUB cells (Ref. 20 and data not shown). However, the functional consequences differ in the two cell types, suggesting that they either express different connexin isoforms or they express the same isoform, but its regulation differs.
Conductances activated by removal of external Ca2+ have been observed in many preparations, including the Xenopus oocyte, in which an endogenous current has been correlated with the expression of Cx38 (4). Oocytes form gap junctions with granulosa cells, and the gap junctions are disrupted by the preparation of oocytes for electrophysiological recordings. Functional gap junctional hemichannels have also been proposed to exist in cultured cells that express Ca2+-sensitive dye uptake (14). Currents similar to the ones described here and expressed in oocytes have also been observed in isolated horizontal cells from catfish retina (3) and, more recently, in a preparation of isolated rat lens fibers, which express endogenous Cx46 and Cx50 (7).
The role of functional hemichannels is not clear. Conventional thinking is that connexins are incorporated in the plasma membrane after the formation of hexamers (connexons) and then dock to the homologous structure present in the neighboring cell. If connexons exist in isolation, they are supposed to be impermeable (closed) under normal conditions (cell negative membrane voltage, millimolar levels of external Ca2+). The high permeability of the hemichannels would cause cell damage if they remained open. Hence we speculate that the hemichannels may play a role in cell damage during pathological conditions such as ischemic, toxic, or inflammatory epithelial injury, in which partial cell separation is known to occur (9). For instance, membrane depolarization of a cell subjected to ischemia and ATP depletion may activate the hemichannels. Because of their high permeability, this could result in Na+ loading (reducing further cell ATP levels by stimulating the Na+-K+-ATPase), Ca2+ loading, and loss of important intracellular solutes (ATP, cAMP, inositol trisphosphate). All of these phenomena can have deleterious effects. Primary ATP depletion (15) and elevation of intracellular Ca2+ (21) by themselves can cause cell death.
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ACKNOWLEDGEMENTS |
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We thank Drs. Malcolm S. Brodwick and Simon A. Lewis for comments on the manuscript, Kelli Spilker for technical assistance, and Deborah Robb for secretarial help.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38734 and a minority supplement to C. G. Vanoye.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: L. Reuss, Dept. of Physiology and Biophysics, The Univ. of Texas Medical Branch, Galveston, TX 77555-0641.
Received 3 September 1998; accepted in final form 1 October 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barry, P. H.
JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements.
J. Neurosci. Methods
51:
107-116,
1994[Medline].
2.
Davidson, J. S.,
and
I. M. Baumgarten.
Glycyrrhetinic acid derivatives: a novel class of inhibitors of gap-junctional intercellular communication. Structure-activity relationships.
J. Pharmacol. Exp. Ther.
246:
1104-1107,
1988[Abstract].
3.
DeVries, S. H.,
and
E. A. Schwartz.
Hemi-gap-junction channels in solitary horizontal cells of the catfish retina.
J. Physiol. (Lond.)
445:
201-230,
1992[Abstract].
4.
Ebihara, L.
Xenopus connexin38 forms hemi-gap-junctional channels in the nonjunctional plasma membrane of Xenopus oocytes.
Biophys. J.
71:
742-748,
1996[Abstract].
5.
Ebihara, L.,
V. M. Berthoud,
and
E. C. Beyer.
Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels.
Biophys. J.
68:
1796-1803,
1995[Abstract].
6.
Ebihara, L.,
and
E. Steiner.
Properties of a nonjunctional current expressed from a rat connexin46 cDNA in Xenopus oocytes.
J. Gen. Physiol.
102:
59-74,
1993[Abstract].
7.
Eckert, R.,
P. Donaldson,
K. Goldie,
and
J. Kistler.
A distinct membrane current in rat lens fiber cells isolated under calcium-free conditions.
Invest. Ophthalmol. Vis. Sci.
39:
1280-1285,
1998[Abstract].
8.
Frömter, E.,
and
J. Diamond.
Route of passive ion permeation in epithelia.
Nat. New Biol.
235:
9-13,
1972[Medline].
9.
Gailit, J.,
D. Colflesh,
I. Rabiner,
J. Simone,
and
M. S. Goligorsky.
Redistribution and dysfunction of integrins in cultured renal epithelial cells exposed to oxidative stress.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F149-F157,
1993
10.
Gupta, V. K.,
V. M. Berthoud,
N. Atal,
J. A. Jarillo,
L. C. Barrio,
and
E. C. Beyer.
Bovine connexin44, a lens gap junction protein: molecular cloning, immunologic characterization, and functional expression.
Invest. Ophthalmol. Vis. Sci.
35:
3747-3758,
1994[Abstract].
11.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
12.
Higgins, J. T.,
L. Cesaro,
B. Gebler,
and
E. Frömter.
Electrical properties of amphibian urinary bladder epithelia.
Pflügers Arch.
358:
41-56,
1975[Medline].
13.
Karnovsky, M. J.
A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy (Abstract).
J. Cell Biol.
27:
137A,
1965.
14.
Li, H.,
T. F. Liu,
A. Lazrak,
C. Peracchia,
G. S. Goldberg,
P. D. Lampe,
and
R. G. Johnson.
Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells.
J. Cell Biol.
134:
1019-1030,
1996[Abstract].
15.
Lieberthal, W.,
S. A. Menza,
and
J. S. Levine.
Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells.
Am. J. Physiol.
274 (Renal Physiol. 43):
F315-F327,
1998
16.
Niggli, E.,
A. Rüdisüli,
P. Maurer,
and
R. Weingart.
Effects of general anesthetics on current flow across membranes in guinea pig myocytes.
Am. J. Physiol.
256 (Cell Physiol. 25):
C273-C281,
1989
17.
Paul, D. L.,
L. Ebihara,
L. J. Takemoto,
K. I. Swenson,
and
D. A. Goodenough.
Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes.
J. Cell Biol.
115:
1077-1089,
1991[Abstract].
18.
Rup, D. M.,
R. D. Veenstra,
H. Z. Wang,
P. R. Brink,
and
E. C. Beyer.
Chick connexin-56, a novel lens gap junction protein. Molecular cloning and functional expression.
J. Biol. Chem.
268:
706-712,
1993
19.
Segal, Y.,
and
L. Reuss.
Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium.
J. Gen. Physiol.
95:
791-818,
1990[Abstract].
20.
Torres, R. J.,
G. A. Altenberg,
J. A. Copello,
G. Zampighi,
and
L. Reuss.
Preservation of structural and functional polarity in isolated epithelial cells.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1864-C1874,
1996
21.
Trump, B. F.,
and
I. K. Berezesky.
The role of cytosolic Ca2+ in cell injury, necrosis and apoptosis.
Curr. Opin. Cell Biol.
4:
227-232,
1992[Medline].
22.
Vanoye, C. G.,
L. A. Vergara,
G. A. Zampighi,
and
L. Reuss.
Differences in expression of functional hemi-gap junctions in isolated epithelial cells (Abstract).
FASEB J.
12:
A377,
1998.
23.
Veenstra, R. D.
Size and selectivity of gap junction channels formed from different connexins.
J. Bioenerg. Biomembr.
28:
327-337,
1996[Medline].
24.
Zhang, Y.,
D. W. McBride,
and
O. P. Hamill.
The ion selectivity of a membrane conductance inactivated by extracellular calcium in Xenopus oocytes.
J. Physiol. (Lond.)
508:
763-776,
1998