1 Department of Physiology, Arizona Health Sciences Center, University of Arizona, Tucson, Arizona 85724; and 2 Cancer Research Center and Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813
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ABSTRACT |
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Connexin (Cx)43 gap junction channels are phosphorylated by numerous protein kinases, with the net effect typically being a reduction in gap junction communication (GJC). This reduction must result from a decrease in channel open probability, unitary conductance, or permselectivity, because previous results suggest that channel number is unaffected. Coexpression of v-Src with wild-type Cx43 (Cx43-wt) but not Cx43 with tyrosine to phenylalanine substitutions at 247 and 265 (Cx43-Y247,265F) resulted in reduced electrical and dye coupling but no change in single-channel amplitudes. EGF treatment of cells expressing Cx43-wt but not Cx43 with serine to alanine substitutions at 255, 279, and 282 (Cx43-S255,279,282A) resulted in reduced GJC, also with no change in single-channel amplitude. Dye coupling was reduced to a far greater extent than electrical coupling, suggesting that channel selectivity was also altered but with minimal effect on unitary conductance. The absence of Src- and MAPK-induced reductions in single-channel amplitude suggests that the decreases in GJC induced by these kinases result from reduced channel open probability and possibly altered selectivity.
connexins; growth factors; dye permeability; electrophysiology; epidermal growth factor
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INTRODUCTION |
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INTERCELLULAR GAP JUNCTIONS are created by the aggregation of channels formed by the docking of two hexameric hemichannels. The integral gap junction proteins (connexins, Cx) belong to a relatively large gene family composed of at least 20 members in the human species. Nearly all gap junction channels permit the passage of ions, and some (e.g., Cx43) permit the passage of metabolites, secondary messengers, and other molecules of <1,000 Da. It is well established that direct cell-to-cell communication plays an important role in the growth of multicellular organisms. Cell survival in these organisms requires that each cell type retain its individuality while at the same time coordinating its activities with other neighboring cells. Gap junctions facilitate this by allowing intercellular communication to take place in a cell-specific and coordinated fashion (5).
Loss of gap junction communication (GJC) contributes to the transformed phenotype of many cancerous and precancerous cells (34). Furthermore, intracellular signaling cascades activated by numerous growth factors are known to decrease GJC (25), with these decreases being strongly correlated with phosphorylation of the Cx protein (11, 33). Thus a major task in the gap junction research community is to identify the permeant molecules, determine their biological significance, and define the mechanisms for regulation of junctional permeability.
The mechanisms by which phosphorylation of Cx43 alter GJC are poorly
understood. Electrical coupling between cells can be expressed as
gj = N · Po · j,
where gj is macroscopic electrical conductance,
N is channel number, Po is the open
probability of the channels, and
j is their unitary
conductance. Dye coupling (believed to be indicative of metabolic
coupling) can be expressed in a similar manner as GJC = N · Po
· Perm, where Perm reflects the permeability of
the channel to the dye of interest, which can vary based on the dye's
size, charge, and chemical composition. (These variations are
indicative of channel selectivity.) Perm and
j both
reflect the ease with which molecules move through the channel's pore.
These equations predict that phosphorylation-induced decreases in
gj or GJC must result from decreases in one or
more of the product factors.
Over two decades ago it was discovered that GJC is reduced between cells that express an activated temperature-sensitive mutant of the avian sarcoma virus (ts-Src) (3). Since then, numerous studies have identified the COOH-terminal tail of Cx43 protein as a target for tyrosine phosphorylation by v-Src and related protein tyrosine kinases (10-12, 23, 30), with v-Src being shown to directly interact with Cx43 through SH-2 and SH-3 binding domains (16, 24). Two tyrosine residues in the COOH terminus of wild-type Cx43 (Cx43-wt) have been determined to be crucial for v-Src binding, phosphorylation, and inhibition of GJC (22, 30), although others have suggested that MAPK phosphorylation of serine residues may also be involved in v-Src-mediated decreases of GJC under acute treatment conditions (35).
The mechanism of GJC reduction in v-Src-stimulated, Cx43-expressing
cells has not been examined in detail. Atkinson et al. (2)
hypothesized that a decrease in channel N might account for
the reduced coupling resulting from ts-Src activation. Contrary to
expectation, their freeze-fracture analysis of gap junction plaques
demonstrated that acute changes in ts-Src activity had no effect on
junctional area at cell interfaces. Lin et al. (22) also
found no evidence for change in Cx43-wt expression or
immunofluorescence patterns in cells stably expressing v-Src. Together,
these studies indicate that acute and steady-state v-Src activation do
not influence channel N, and therefore must affect
j, permselectivity, or Po. Moreno
et al. (29) found a direct relationship between increased phosphorylation of Cx43-wt (by unknown kinases) and decreased
j. This finding was extended in studies of the PKC
pathway, in which PKC activation increased the frequency of smaller
subconductance events of Cx43-wt (19, 21, 28). In the
typical setting where multiple gap junction channels are active and
displaying multiple open states, Po is not
readily determined; therefore, to ascertain whether v-Src influences
Po,
j (or permselectivity) must
be eliminated as a factor under the influence of v-Src.
Mechanisms of growth factor-mediated reduction of Cx43-wt GJC are
greatly complicated by the numerous signaling cascades involved and
possible cross talk between these cascades. As a result, rigorous investigation of the mechanisms of phosphorylation-induced reduction of
GJC must be performed to delineate the effects of differing growth
factor cascades on gap junction function. By using cell lines
specifically engineered to explore the effects of v-Src and MAPK on
Cx43-wt phosphorylation, we have tested the mechanisms by which these
signaling cascades cause decreases in GJC. Unlike the PKC pathway,
phosphorylation of Cx43-wt by Src (at tyrosines 247 and 265) or MAPK
(at serines 255, 279, and 282) had no detectable effect on Cx43-wt
channel j, although the selectivity of these channels to
different dyes was affected. Our data indicate that the decrease in
electrical coupling induced by v-Src or MAPK was the result of channel
closure (reduced Po) whereas the decrease in GJC
also involved increased channel selectivity.
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MATERIALS AND METHODS |
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Cells and cell culture.
The Cx43 knockout mouse cell line, /
3 (26), expressing
exogenous Cx43-wt (wtC1 clonal line), Cx43-wt and v-Src (wtS1 and wtS3
clonal lines), or the double mutant Cx43-Y247,265F (encoding phenylalanine substitutions at Y247 and Y265) and v-Src (dbS2 clonal
line) (22) were used to assess mechanisms of
v-Src-mediated reductions of GJC. The same knockout mouse cell line
expressing the triple mutant Cx43-S255,279,282A (with serine to alanine
substitutions at sites 255, 279 and 282; G11 cell line)
(32) was used to assess mechanisms of MAPK-mediated
reductions in GJC.
Electrophysiology. The dual whole cell voltage-clamp technique was used to assess both macroscopic and single-channel conductance between pairs of cells in culture. Cells were grown to confluence in a 100-mm dish, lifted with 0.25% trypsin in Ca2+- and Mg2+-free buffer, and replated at low density on glass coverslips. Cells were allowed to adhere to the coverslips by incubating at 37°C for either 2-3 h, for single-channel recordings, or 24 h, for macroscopic conductance measurement.
Dual whole cell voltage-clamp experiments were carried out as previously reported (14, 17). Glass electrodes were filled with one of two internal solutions. Solution 1 (KCl) contained (in mM) 124 KCl, 14 CsCl, 9 HEPES, 9 EGTA, 0.5 CaCl2, 5 glucose, 9 TEA-Cl, 3 MgCl2, and 5 Na2ATP, and solution 2 (LKM) contained (in mM) 67.8 CsCl, 67.8 K-glutamate, 10 TEA-Cl, 0.5 CaCl2, 3 MgCl2, 5 glucose, 10 HEPES, 10 EGTA, and 5 Na2ATP. Solution 2 has lower bulk conductivity than solution 1, which results in lower unitary conductances for an equivalent channel population. The rationale for use of two different solutions was twofold: first, to mimic the previous electrophysiology performed on these same cells (21), and second, to increase the likelihood of a higher-resistance channel excluding or impeding the flow of larger ions between the cells, thereby enhancing the chances of observing conductance shifts in the presence of EGF. After the dual whole cell voltage-clamp configuration was achieved, both cells were held at 0 mV and then alternately stepped toDye coupling studies.
The extent of dye coupling was assessed using two separate dyes,
Lucifer yellow [mol wt 443, net negative charge of 2, 5% (wt/vol)]
and
[2-(4-nitro-2, 1,3-benzoxadiol-7-yl)aminoethyl]trimethylammonium (NBD-TMA; mol wt 280, net positive charge of 1, 5 mM; kindly provided by Dr. Stephen Wright, University of Arizona, Tucson, AZ; see Ref.
4). Cells were grown to confluence on 25-mm glass
coverslips. Injection electrodes (15-20 M) were filled with dye
by capillary action, backfilled with either 150 mM LiCl (for Lucifer
yellow dye) or 200 mM KCl (for NBD-TMA dye), and lowered onto the
surface of the cell. Cells were impaled by the overcompensation of the capacitance feature of the amplifier (A-M Systems) and withdrawn after
10 s, and the cells receiving dye were determined after 1 min.
Data analysis.
Single-channel records were analyzed with several strategies. The
amplitudes of single-channel event transitions were manually determined. The minimum detectable difference between amplitudes was
0.25 pA, which corresponds to 6.25 pS at 40 mV transjunctional driving
force. No binning of the data was performed. The frequency of events at
each distinguishable current level was calculated as a percentage of
the total number of events. The event data and event frequency data
were used to construct histograms for each cell pair, and the data were
fit (with Origin software) with one, two, or three Gaussian curves. The
model resulting in the best fit was determined with the following
criteria: 1) standard deviations for the peak center
parameters were <5% of the peak center value, and 2)
reduced 2 and R-squared values predicted significance at
the P < 0.05 level. For each experimental group, the
mean peak center values were compared by single-factor ANOVA or
t-test as appropriate. All-points histograms were also
generated from the digitized single-channel records with pCLAMP 8.1 (Axon Instruments) and Sigmaplot (SSPS). Event amplitudes determined
directly from single-channel records and their corresponding all-points
and event-frequency histograms were similar. Additionally, the
percentage of total events in the 30- to 70-pS (patch solution
1) or 25- to 60-pS (patch solution 2) range was
calculated for each experiment and treatment group and compared across
treatment groups with a single-factorial ANOVA to assess whether v-Src
or EGF treatment induced shifts of Cx43-wt channels to a lower
conductance state.
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RESULTS |
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Electrophysiology: junctional conductance.
Relative to Cx43-wt expressing cells (wtC1 clone), electrical coupling
was reduced in cells expressing v-Src with Cx43-wt (wtS1 and wtS3
clones) but not with Cx43-Y247,265F (dbS2 clone). Figure
1A presents uncorrected
gj and summed Rs values
for the four cell types. Figure 1B presents the corrected
[with the equations of van Rijen et al. (31)]
gj values. The wtC1 cells displayed a corrected
gj of 25.8 ± 3.7 nS (n = 9), whereas wtS1 and wtS3 cells had significantly less electrical
coupling at 6.4 ± 2.3 (n = 13) and 13.7 ± 3.5 (n = 12) nS, respectively (Fig. 1B). The extent of coupling in dbS2 cells (27.6 ± 7.6 nS;
n = 9) did not differ significantly from that in wtC1
cells, suggesting that the Cx43-Y247,265F mutations were sufficient to
prevent significant electrical uncoupling by v-Src in these cells.
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Electrophysiology: unitary conductance.
Single-channel records from Cx43-wt-expressing wtC1 cells revealed
predominantly 90- to 110-pS events (Fig.
2A), although smaller events
in the range of 30-70 pS were observed in most cell pairs.
Peak-to-peak separation on the corresponding all-points histogram
confirmed a main-state conductance of ~100 pS and a substate
conductance of ~60 pS. The amplitudes of 210 events from this cell
pair (86 s of record) were used to construct an event-frequency histogram that was best fit with two peaks (Fig. 2E) with
conductances corresponding to 93 ± 0.3 pS (SD) and 51 ± 1.2 pS (SD). Data from only three of six cell pairs were best fit with two
peaks; the remaining cell pairs were best fit with only one peak.
Across six cell pairs, the mean main-state conductance was 94 ± 1.6 pS (SE); for the three pairs whose data were best fit by two peaks the mean conductance of the substate was 47 ± 2.5 pS.
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GJC and junctional selectivity. The introduction of negatively charged inorganic phosphate moieties to the COOH terminus of Cx43 could shift gap junction channel selectivity toward preferential cation transfer. The dye Lucifer yellow has two negative charges at physiological pH; therefore, its transfer may not be favored through phosphorylated Cx43 gap junction channels. Here we used both Lucifer yellow and the novel cationic dye NBD-TMA (9) to assess the impact of Src and EGF on GJC and junctional selectivity. In addition, the v-Src inhibitor PP2 was used to ensure that the differences in dye coupling between cell types resulted from v-Src and not chance clonal differences between cell populations.
GJC was evaluated as extent of coupling, calculated as the total number of cells to which dye diffused within 1 min after injection. In wtC1 cells, NBD-TMA coupling was greater than Lucifer yellow coupling (13.7 ± 1.4 vs. 7.2 ± 0.8; Fig. 5A) and was significantly increased after PP2 treatment (18.6 ± 1.9 vs. 13.7 ± 1.4), perhaps reflecting the effects of basal c-Src activity. Lucifer yellow coupling was nearly absent, and NBD-TMA coupling was reduced from 13.7 cells to 0.2 ± 0.09 and 0.65 ± 0.28 cells in the wtS1 and wtS3 cells, respectively. After PP2 treatment, NBD-TMA coupling in wtS1 and wtS3 cells was significantly improved although still not equal to that of wtC1 cells. Extent of coupling in the Cx43-Y247,265F-expressing dbS2 cells was comparable for Lucifer yellow (5 ± 1 cells) and NBD-TMA (with PP2 pretreatment: 5 ± 1 cells; without PP2 pretreatment: 6 ± 1 cells) and well below that observed for the wtC1 cells. The lower levels of coupling in dbS2 compared with wtC1 cells most likely reflected differing growth morphologies of the dbS2 cells
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DISCUSSION |
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A decrease in GJC or electrical coupling must involve a decrease
in channel N, Po, j,
permselectivity, or a combination of these factors. We have
demonstrated that the previously reported v-Src- and MAPK-induced
decreases in Cx43-mediated electrical coupling occur independent of a
decrease in the unitary conductance of the channel. Because Cx43
expression and localization are also not affected by v-Src (2,
22) or MAPK (32) activation, the observed decreases
in electrical coupling must reflect decreased channel
Po. We have further demonstrated that v-Src and
MAPK activation reduced GJC and have shown that the magnitudes of the
decreases for the two dyes were not predicted well by the observed
decreases in electrical coupling. These results suggest that Src and
MAPK alter channel selectivity. These findings contrast with findings for the PKC pathway, in which PKC-mediated phosphorylation induces a
decrease in junctional conductance that can be at least partially attributed to a decrease in channel unitary conductance
(21).
The absence of a reliable measure for Po in cell
pairs coupled by multiple gap junction channels limits our analysis of
gating mechanisms. The presence in most junctions of several channels, each with multiple possible conductance states, complicates any analysis of Po. A simultaneous decrease in
main-state Po and increase in substate
Po, in combination with possible changes in
channel N, make Po-based
interpretations of changes in gj challenging at
best. An example of this is described by Kwak et al. (19), who found that gj increased despite decreased
predominance of large j events. Apparently, the
increased predominance of the substate conductance was accompanied by
either an increased Po of that channel state or
an increased channel N.
In the absence of Po data, it is necessary to eliminate the other factors that contribute to GJC as potential candidates for v-Src- and MAPK-mediated reductions in GJC. Early studies of v-Src effects on GJC used a temperature-sensitive v-Src mutant that was activated by shifting cells in culture from a nonpermissive temperature of 40°C to a permissive temperature of 35°C (2, 3). These experiments permitted the measurement of GJC and gap junction size, an indicator of gap junction channel N, after chronic (>12 h) or acute (<60 min) activation of v-Src. Chronic growth of cells at the permissive temperature resulted in a loss of GJC compared with cells grown at the nonpermissive temperature. Similarly, acutely shifting cells to and from the permissive temperature resulted in loss and gain of GJC, respectively. Measurements of gap junction area per interface demonstrated that chronic growth of cells at the permissive temperature resulted in a decreased gap junction area. However, this effect could not be attributed to the action of v-Src, because nontransfected (with vSrc) cells grown at 35°C also had a decreased plaque area compared with those grown at 40°C. In addition, acute shifts between the nonpermissive and permissive temperatures had no effect on gap junction area despite changes in GJC. These data indicate that the Src-induced decrease in GJC did not result from decreased channel N. Using Western blots, Lin et al. (22) found comparable Cx43 expression levels in wtC1, wtS1, and dbS2 cells, with no apparent differences in Cx43 localization observed after immunofluorescent labeling, lending further support to the conclusion that v-Src can cause decreased GJC without reducing channel N. Neither measure of channel N, morphological or biochemical, provides information on the functionality of the channels. Regardless of whether channel N differs between the wtC1 and wtS1 or wtS3 cell lines, our PP2 and EGF data, which explore the acute effects of v-Src inhibition and MAPK activation, strongly suggest that these kinases modify channel gating. These observations, combined with the absence of differing expression levels and localization of Cx43 between cell types (22) and the absence of change in gap junction area in v-Src-treated cells (2), argue strongly that the loss of GJC measured in our v-Src-transfected cells are not the result of decreased channel N.
Cx43 characteristically has three open conformations: the main state with a typical conductance of ~100 pS (dependent on pipette solutions used), a substate with a conductance of ~60 pS, and a residual state with a conductance of ~30 pS. Transitions between the ground state (fully closed) and each of these open conformations can be observed. As well, transitions can be observed from the main state to a residual state. The 60-pS substate appears to be favored when the channel is phosphorylated (19, 29), but the prevalence of ~60-pS transitions between the main state and residual state complicates interpretation of such data.
Recent studies comparing behavior of Cx43-wt and mutant (Cx43-S368A) isoforms have strengthened the conclusion that phosphorylation can induce changes in channel unitary conductance (21). Phosphorylation of Cx43 by PKC induces a dramatic increase in prevalence of the 60-pS substate and decrease in prevalence of the main state. These changes in channel behavior likely contribute to the decrease in GJC induced during PKC activation, but simultaneous changes in Po and N may also occur. Decreases in unitary conductance result from increasing the channel's pore length, increasing selectivity to current-carrying ions, increasing access resistance, decreasing pore diameter, or a combination of these effects. Phosphorylation of Cx43 by PKC therefore may induce a conformational change at the Cx43 COOH terminus that acts to partially occlude or lengthen the channel pore, increase its selectivity, or increase access resistance to the pore. In contrast to these results with PKC, the unitary conductance profile of Cx43 was not altered by v-Src or MAPK activation. Phosphorylation of Cx43 by PKC takes place at serine 368, a site close to the end of the COOH-terminal tail, whereas v-Src and MAPK induce phosphorylation of Cx43 at the proximal end of the COOH-terminal region (amino acid residues 247-282). Differing effects of phosphorylation in proximal vs. distal regions of the COOH terminus may suggest differing roles for these regions.
It is reasonable to hypothesize that phosphorylation of the
COOH-terminal tail of Cx43 could alter channel selectivity or access
resistance. The introduction of negatively charged moieties at the
entry to the pore region of the gap junction channel could act to
selectively draw cations through the channel while impeding the
progress of anions. Our data suggest that if such changes in
selectivity are occurring, their impact on electrical vs. dye coupling
are quite different. The j of channels observed in the presence of v-Src and activated MAPK were not different, suggesting that the decrease in electrical coupling was due to a decrease in
Po. If this were the only effect of these
kinases, one might expect dye coupling to decrease in parallel to the
decrease in junctional conductance. Such was not the case. Lucifer
yellow dye coupling was reduced to a far greater extent than NBD-TMA dye coupling, and the latter was reduced to a far greater extent than
would be predicted by changes in junctional conductance in wtS1, wtS3,
and EGF-treated wtC1 cells. These results suggest that selectivity of
the channel was affected by these treatments but in a fashion that was
undetectable relative to
j. This suggestion merits
further attention in experiments that measure gj
and dye diffusion simultaneously, an approach beyond the scope of the current study.
Considerable debate surrounds the mechanistic basis of v-Src-mediated reduction of GJC. Although v-Src activity has been associated with increased predominance of phosphoserines and phosphotyrosines on Cx43, decreases in GJC were most commonly associated with tyrosine phosphorylation (10-12, 23). Swenson et al. (30) demonstrated that v-Src decreased GJC of Cx43 by phosphorylation of Tyr265. Lin et al. (22) corroborated these results and determined that Tyr247 phosphorylation was also involved. In contrast, Zhou et al. (35) determined that Tyr247 and Tyr265 were not required for v-Src-mediated gating of Cx43. Instead, they provided evidence that MAPK phosphorylation of serines 255, 279, and 282 was necessary. In their model, they transfected Xenopus oocytes with Cx43 cRNA, permitted channels to form, and then injected v-Src cRNA to induce uncoupling. This differs from the other studies that chronically coexpressed v-Src and Cx43 (22, 30). It is possible that acute activation of v-Src has different effects on MAPK activity than chronic v-Src presence. In the stably transfected v-Src cells, the constant presence of Src activity may result in negative-feedback regulation of elements of the MAPK cascade, thus decreasing the sensitivity of this cascade to v-Src activation. If this were true, then the role that MAPK may play in v-Src-induced gating of Cx43 channels would be greatly diminished in the chronically active v-Src cell model. The possibility of cross talk between these signaling cascades prompted us to investigate the mechanisms of MAPK-induced decreases in GJC in conjunction with v-Src gating mechanisms. As stated previously, no differences were observed.
It is clear that regulation of gap junctions by growth factor-mediated signaling cascades is complicated. We have shown here that mechanisms of growth signaling-mediated decreases in GJC caused by v-Src and MAPK are different from those previously described for PKC activation (21) and that this is the result of specific phospho-amino acid residue localization in the COOH terminus. Although regulation of Cx43 by growth signaling cascades is most commonly studied, many other connexins are considered phosphoproteins as well (for review see Ref. 20), and these connexins can be differentially regulated by similar phosphorylating conditions (18). Furthermore, two different cell types expressing similar connexins may not have the same GJC response to a signal cascade agonist (1). These issues have been further complicated by the ability of connexins to form heteromeric/heterotypic channels (8) and the possibility that Cx hemichannels may also be regulated by growth signaling (7) and that gap junction proteins may influence growth independently from their formation of functional channels (27).
Such complexities make it essential to develop an understanding of
relationships between phosphorylation of Cx43 and the impact that this
can have on gap junction channel function. We provide evidence here
that Cx43 phosphorylation can acutely alter electrical coupling through
changes in Po, independent of changes in
j. Concomitant changes in GJC likely involve enhanced
permselectivity as well as reduced Po. The
overall effect that this has on cell physiology is not understood;
however, mechanistic understanding of channel phosphorylation provides
a starting point for the study of the physiological impacts that these
changes will have at the cellular level.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants HL-58732 to J. M. Burt and CA-52098 to A. F. Lau.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. M. Burt, Dept. of Physiology, Arizona Health Sciences Center, Rm. 4103, Univ. of Arizona, Tucson, AZ 85724 (E-mail: jburt{at}u.arizona.edu).
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. Section 1734 solely to indicate this fact.
First published October 16, 2002;10.1152/ajpcell.00214.2002
Received 10 May 2002; accepted in final form 10 October 2002.
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