(Received for publication, September 29, 1995; and in revised form, January 29, 1996)
From the
Molecular analyses have resulted in the isolation of two chicken
stomach AE2 anion exchanger cDNAs, AE2-1 and AE2-2. The 4.3-kilobase (kb) AE2-1 cDNA contains
an open reading frame that encodes a predicted polypeptide of
135
kDa that is homologous to AE2 anion exchangers from other species. The
partial
1.7-kb AE2-2 cDNA, which differs from the AE2-1 cDNA in two regions, would be predicted to encode an AE2
polypeptide with an alternative N-terminal cytoplasmic tail.
Examination of the distribution of these variant transcripts has
revealed that AE2 transcripts ranging in size from
4.4 to
7.3 kb accumulate in various adult tissues. However, in the
stomach, the unique sequence at the 5`-end of AE2-1 is
preferentially associated with transcripts that range in size from
4.5 to
4.9 kb, while the unique sequence at the 5`-end of AE2-2 is preferentially associated with the
7.3-kb AE2 RNA species. In situ hybridization analyses have
further revealed that AE2 transcripts accumulate to very high
levels within the acid-secreting epithelial cells of the profound gland
in the stomach and, to a lesser extent, within the mucus-secreting
cells of the superficial gland that line the stomach lumen. This result
suggests that AE2 anion exchangers are involved in the regulation of
intracellular pH in each of these gastric epithelial cell types.
Plasma membrane electroneutral anion exchangers are involved in the regulation of intracellular pH (1) and cell volume(2) . The best characterized anion exchanger is the erythroid AE1 anion exchanger (band 3 protein), which primarily mediates the one-for-one exchange of bicarbonate for chloride(3) . Molecular studies have indicated that variant AE1 anion exchangers are encoded by the AE1 gene in mouse(4) , rat(5) , human(6) , and chicken (7, 8, 9) . These variant AE1 transcripts differ only at their 5`-ends and encode polypeptides with alternative N-terminal cytoplasmic domains. These molecular analyses coupled with immunolocalization studies (10, 11) have suggested that specific AE1 variants mediate the electroneutral anion exchange activity that has been localized to the basolateral membrane of the acid-secreting A-intercalated cells of the kidney collecting duct.
Electroneutral anion exchange activities have also been characterized in a variety of other cell types, including gastric parietal cells (12) , cardiac Purkinje fibers(13) , and renal mesangial cells(14, 15) . The genes that encode most of these anion exchange activities are not known. However, immunolocalization studies using AE2 anion exchanger-specific peptide antibodies have suggested that the AE2 anion exchanger mediates the basolateral chloride/bicarbonate exchange activity of the acid-secreting parietal cells of the mammalian stomach (16) . The studies described here have revealed that variant chicken AE2 anion exchanger transcripts are generated by a complex pattern of alternative transcriptional initiation and differential RNA splicing. These transcripts are expressed in a wide variety of tissues including the proventriculus, the equivalent of the mammalian stomach, where they accumulate to very high levels. Examination of the cell type-specific pattern of expression of AE2 transcripts in the proventriculus has revealed that they primarily accumulate within the epithelial cells of the profound gland and, to a lesser extent, within the mucus-secreting cells that line the stomach lumen. Previous studies have suggested that the epithelial cells of the profound gland are functionally equivalent to the parietal cells of the mammalian stomach, secreting acid into the stomach lumen(17) . The high level of AE2 expression observed in this acid-secreting cell type suggests that the AE2 gene encodes the basolateral anion exchanger of these cells. The detection of AE2 transcripts within the mucus-secreting cells of the stomach further suggests that AE2 anion exchangers mediate the apical bicarbonate-secreting activity that has been characterized in this epithelial cell type in other species(18) .
Figure 1: Nucleotide and predicted amino acid sequences of the chicken AE2-1 anion exchanger. The nucleotide and amino acid sequences of the chicken AE2-1 anion exchanger are illustrated. This AE2 variant contains an open reading frame that initiates at nucleotide 233 and extends to nucleotide 3889.
Figure 2: Comparison of the nucleotide sequences at the 5`-ends of the AE2-1 and AE2-2 anion exchanger cDNAs. The nucleotide sequences at the 5`-ends of the AE2-1 and AE2-2 cDNAs are illustrated in A. The putative translation initiation codons of the variant cDNAs are in boldface. The in-frame stop codon immediately preceding the translation initiation site of the AE2-1 cDNA is underlined. The sequence of the 57-nucleotide insert that is present in AE2-2 and absent in AE2-1 is illustrated in B.
Hybridization signals using different RNA probes were quantitated by counting silver grains over at least 30 randomly chosen areas of the profound gland, the superficial gland, and the mucosal tissue separating these glands, as well as background grains in the emulsion. Dark-field images resulting from the hybridization with each probe were collected with a Kodak DCS 420 digital camera. These images were imported into Adobe Photoshop, and silver grains were counted. The grain densities over specific regions of the stomach represent the number of grains over a fixed area of tissue minus the number of grains over the same area of emulsion alone. Essentially identical results were obtained in two separate experiments.
The second cDNA, AE2-2, was 1749 nucleotides in size, and sequence analysis revealed that the 3`-end of this partial cDNA corresponded to nucleotide 1763 of the AE2-1 cDNA. The sequence of AE2-2 was identical to AE2-1 from nucleotide 222 to its 3`-end with the exception of an insert of 57 nucleotides that initiated at nucleotide 621 of AE2-2 (Fig. 2B). This insert encodes 19 amino acids (amino acids 186-204 in AE2-2) that are absent in AE2-1 (Fig. 3). In addition, the 221 nucleotides at the 5`-end of AE2-2 were different than the 292 nucleotides at the 5`-end of AE2-1 (Fig. 2A). These results suggest that these cDNAs were derived from variant chicken AE2 transcripts that had been generated by alternative transcriptional initiation and differential RNA splicing.
Figure 3: Comparison of the sequences of the cytoplasmic domains of the chicken AE2-1 and AE2-2 anion exchangers with those of AE2 anion exchangers from other species. The amino acid sequences of the cytoplasmic domains of the chicken AE2-1 and AE2-2 anion exchangers are compared with those of AE2 anion exchangers from rat (RAE2), mouse (MAE2), rabbit (RABAE2), and human (HAE2). Dots indicate amino acids in the AE2 anion exchangers that are identical to the chicken AE2-1 polypeptide. Regions where amino acids are present in one sequence and absent in another are indicated by dashes. The predicted sequence encoded by the partial AE2-2 anion exchanger cDNA terminates at amino acid 561.
Both of the variant AE2 cDNAs would be predicted to initiate translation from an AUG codon that is present in their unique 5`-sequences (Fig. 2A). The AE2-1 variant contains 20 unique amino acids at its N terminus, while the AE2-2 variant contains a larger unique sequence of 52 amino acids at its N terminus (Fig. 3). The putative translation initiation site of the AE2-1 variant is preceded immediately upstream by an in-frame stop codon (Fig. 2A).
The variant chicken AE2 anion exchangers
share significant homology with AE2 polypeptides from other species.
This homology is most striking in the C-terminal transmembrane domain,
where the chicken AE2-1 polypeptide is 90% identical to AE2
anion exchangers from human(20, 21) ,
mouse(22) , rat(23) , and rabbit(24) . This
region of the predicted polypeptide possesses 10 hydrophobic stretches
that may span the membrane 12-14 times (data not shown). These
membrane-spanning regions are virtually identical to the homologous
regions of previously characterized AE2 anion exchangers. Those
substitutions that occur are primarily conservative in nature. The only
region of the transmembrane domain that has significantly diverged from
other AE2 anion exchangers is a putative extracellular loop that lies
between the fourth and fifth hydrophobic regions (data not shown). This
region also exhibits extensive variability among AE2 anion exchangers
from other
species(20, 21, 22, 23, 24) .
In contrast to the transmembrane domain, the N-terminal cytoplasmic
domain is only
70% identical to AE2 anion exchangers from other
species. This sequence divergence is most striking at the N termini of
the chicken AE2 variants, which exhibit little homology to previously
characterized AE2 polypeptides (Fig. 3).
Figure 4:
Tissue
distribution of chicken AE2 anion exchanger transcripts. 2
µg of poly(A) RNA isolated from adult chicken
heart, kidney, liver, skeletal muscle, brain, proventriculus, gizzard,
and intestine were electrophoresed on a formaldehyde-agarose gel and
transferred to nitrocellulose. The filter was incubated with a
P-labeled antisense RNA probe complementary to nucleotides
2783-2979 of the chicken AE2-1 cDNA. Following washing
of the filter, hybridizing species were detected by autoradiography. B is a shorter exposure of the autoradiogram shown in A. The migration of RNA markers in kilobases is
indicated.
To determine if the variant AE2-1 and AE2-2 cDNAs
corresponded to specific RNA species detected in the blotting analysis, P-labeled antisense RNA probes were generated that were
complementary to the unique sequences at the 5`-ends of the variant
cDNAs. Blotting analyses with these variant-specific probes revealed
that the probe specific for AE2-1 recognized a similar array
of transcripts (Fig. 5A) as those recognized by the
transmembrane domain probe. However, the AE2-1-specific probe
did not recognize the major
4.4-kb proventricular transcript
detected by the transmembrane domain probe (compare Fig. 4B and Fig. 5B). In contrast, the AE2-2-specific probe primarily recognized the
7.3-kb
transcript present in the proventriculus (Fig. 5C).
This probe also weakly hybridized to the
4.5-4.9-kb AE2 transcripts from each tissue. Longer exposure of these
autoradiograms indicated that both probes recognize a
7.3-kb
transcript in each tissue (data not shown).
Figure 5:
Tissue distribution of the AE2 transcripts that contain the unique sequences at the 5`-ends of
the AE2-1 and AE2-2 cDNAs. Identical RNA blots
containing 2 µg of poly(A) RNA isolated from adult
chicken heart, kidney, liver, skeletal muscle, brain, proventriculus,
gizzard, and intestine were incubated with a
P-labeled
antisense RNA probe complementary to nucleotides 14-278 of the AE2-1 cDNA (A and B) or with a
P-labeled antisense RNA probe complementary to nucleotides
1-194 of the AE2-2 cDNA (C). These probes are
complementary to sequences in the unique 5`-ends of the variant cDNAs.
Following washing of the filters, hybridizing species were detected by
autoradiography using an intensifying screen. The autoradiograms in A and C were exposed for the same length of time. B is a shorter exposure of the autoradiogram shown in A. The migration of RNA markers in kilobases is
indicated.
These data indicate that
the unique sequences at the 5`-ends of the AE2-1 and AE2-2 cDNAs are associated with multiple variant AE2 transcripts. However, the transcripts containing the unique
sequence at the 5`-end of AE2-1 are much more abundant in each
tissue we have examined than transcripts containing the unique sequence
at the 5`-end of AE2-2 (Fig. 5, compare A and C). Furthermore, the alternative 5`-ends of the variant AE2 cDNAs are differentially spliced onto the different size AE2 transcripts. This is best illustrated in the
proventriculus, where the unique sequence at the 5`-end of AE2-1 is preferentially spliced onto the 4.5-4.9-kb class of AE2 transcripts (Fig. 5A), while the unique
sequence at the 5`-end of AE2-2 is preferentially spliced onto
transcripts of
7.3 kb (Fig. 5C). In addition, AE2 transcripts in the proventriculus must possess 5`-ends in
addition to those of the AE2-1 and AE2-2 cDNAs since
neither the AE2-1- nor the AE2-2-specific probe
recognizes the major
4.4-kb proventricular transcript.
The
relative abundance of the variant AE2 transcripts varied
dramatically in the different tissues. This highly regulated pattern of
expression can be seen when comparing the AE2 variant
composition in the different compartments of the gastrointestinal
tract. The 4.4-kb AE2 transcript is very abundant in the
proventriculus (Fig. 4A), while it is undetectable in
the gizzard (Fig. 4A), the portion of the chicken
stomach that is primarily involved in mechanical aspects of digestion,
and in the intestine (Fig. 4A). In addition, the
7.3-kb transcript is the most prevalent transcript recognized by
the AE2-2-specific probe in the proventriculus, while the
4.5-4.9-kb species are the most prevalent transcripts
recognized by this probe in the other tissues (Fig. 5C).
Figure 6:
Structure of AE2 transcripts
containing the unique sequences at the 5`-ends of the AE2-1 and AE2-2 cDNAs. Poly(A) RNA isolated
from the proventriculus of a 1-day-old chicken was reverse-transcribed
using a primer complementary to nucleotides 986-1006 of the AE2-1 cDNA. This first strand cDNA was PCR-amplified (lanes 1 and 3) using sense primers corresponding to nucleotides
3-23 of the AE2-1 cDNA (lanes 1 and 2)
or nucleotides 113-133 of the AE2-2 cDNA (lanes 3 and 4). These sense primers were used in combination with
an antisense primer complementary to nucleotides 707-727 of the AE2-1 cDNA (nucleotides 693-713 of AE2-2). Lanes 2 and 4 correspond to control amplifications
carried out in the absence of first strand cDNA template. The locations
of the amplification primers and the probes used in this analysis are
illustrated by the arrows in D. The black boxes in D represent the unique sequences at the 5`-ends of the AE2 variants. The PCR products were electrophoresed on a 1.5%
agarose gel, stained with ethidium bromide (A), and
transferred to nitrocellulose. The nitrocellulose filters were
incubated either with a
P-end-labeled oligonucleotide
corresponding to probe 1 (B) or with a
P-end-labeled oligonucleotide corresponding to probe 2 (C). Following washing, hybridizing species were detected by
autoradiography. The markers correspond to pGEM-3 DNA digested with HinfI restriction endonuclease.
These results indicate
that transcripts initiating with the unique sequence at the 5`-end of
the AE2-2 cDNA (Fig. 6, lane 3) either contain
(slower migrating species) or lack (faster migrating species) the
57-nucleotide insert present in the AE2-2 cDNA. In contrast,
this alternatively spliced 57-nucleotide insert does not associate with
transcripts initiating with the unique sequence at the 5`-end of AE2-1. Sequence analysis of the PCR amplification products
supported this conclusion. The fact that the amplification products
contained only those sequences that were present in the AE2-1 and AE2-2 cDNAs indicates that extensive variability must
reside in other regions of the AE2 transcripts to account for
the wide range in sizes (4.4-7.3 kb) observed.
In situ hybridization studies have determined the cell
types in the proventriculus where the variant AE2 transcripts
accumulate. Initial analyses employed an antisense RNA probe that was
complementary to a portion of the C-terminal transmembrane domain and
the 3`-untranslated region of the variant AE2-1 cDNA
(nucleotides 3671-4295). Blotting analyses have indicated that
this antisense probe does not recognize the chicken AE1 or AE3 anion exchanger transcripts at the criterion used for the in situ studies (data not shown). 4-µm frozen tissue
sections from the proventriculus of a 21-day chicken embryo were
hybridized with this P-labeled antisense RNA, and
following processing of the tissue sections, the results were
visualized by dark-field microscopy (Fig. 7, A and D-F). This analysis revealed that AE2 transcripts accumulate to very high levels within the epithelial
cells of the profound gland (Fig. 7, A and F)
as well as within the epithelial cells that line the duct connecting
the profound gland with the stomach lumen (Fig. 7, D-F). AE2 transcripts also accumulate at much
lower levels in the mucus-secreting epithelial cells of the superficial
gland (Fig. 7A). There was no detectable accumulation
of AE2 transcripts in the mucosal tissue that separates these
two glands, which includes the lamina propria mucosa and the inner
muscularis mucosa, or in the outer muscle layer that separates the
profound glands from the serosa, the outer wall of the proventriculus (Fig. 7A).
Figure 7:
Identification of AE2 anion
exchanger-expressing cells in the chicken proventriculus by in situ hybridization. The proventriculus was isolated from a 21-day
chicken embryo, fixed in 4% paraformaldehyde, and frozen in embedding
medium. 4-µm cross-sections (A-C) or tangential
sections (D-F) of the proventriculus were cut. These
sections were hybridized with P-labeled antisense RNA
probes complementary to nucleotides 3671-4295 of the AE2-1 cDNA (A and D-F), nucleotides 14-278
of the AE2-1 cDNA (B), and nucleotides 1-194 of
the AE2-2 cDNA (C). The dark-field images in A-C are from tissue sections exposed for 90 days. The
images in D-F are from sections exposed for 30 days. The
regions of the superficial glands (S), the profound glands (P), and the ducts (D) that connect the profound
glands with the lumen are labeled. The mucosal tissue (M) that
separates the superficial and profound glands as well as the outer
muscle layer (ML) are also labeled. The arrow in F points to a duct from a profound gland that is opening into
the stomach lumen. The image in each panel corresponds to multiple
overlapping images that were collected using a Kodak DCS 420 digital
camera. The images were imported into Adobe Photoshop and merged. Bar = 100 µm.
The hybridization signal obtained with this antisense RNA probe was compared with the signal obtained with a sense probe corresponding to the same region of the AE2-1 cDNA. This analysis revealed that the antisense probe exhibited grain densities >10-fold higher in the epithelial cells of the profound gland and 2.5-fold higher in the epithelial cells of the superficial gland than that observed with the control sense probe. The high level of AE2 expression in the epithelial cells of the profound gland suggests that like the mammalian AE2 anion exchanger(16) , chicken AE2 anion exchangers mediate a basolateral anion exchange activity in the acid-secreting cells of the chicken stomach. In addition, the accumulation of AE2 transcripts in the mucus-secreting cells of the superficial gland suggests that AE2 anion exchangers may also mediate the apical bicarbonate-secreting activity that has been characterized in this epithelial cell type in other species(18) .
Similar in
situ analyses using antisense RNA probes complementary to the
unique sequences at the 5`-ends of the AE2-1 and AE2-2 cDNAs have investigated the cell type-specific pattern of
expression of transcripts containing these sequences. These studies
have revealed that the AE2-1-specific antisense probe (Fig. 7B) exhibited a hybridization signal 3-fold
higher than that observed with a control sense probe in the epithelial
cells of the profound gland. However, the signal with this AE2-1-specific antisense probe in the epithelial cells of the
superficial gland was identical to that observed with the control sense
probe. In addition, the AE2-2-specific antisense probe (Fig. 7C) exhibited levels of hybridization similar to
those of the control sense probe in the cells of the profound and
superficial glands. The inability to detect transcripts containing the
unique sequences of the AE2-1 and AE2-2 cDNAs in the
cells of the superficial gland suggests that AE2 variants
containing these sequences do not accumulate within the mucus-secreting
cells of this gland. However, it is possible that the in situ hybridization technique is not of sufficient sensitivity to detect
the AE2-1 and AE2-2 variants that may be present in
this cell type at low abundance. The hybridization signal observed with
the variant-specific probes (Fig. 7, B and C)
is much lower than that observed with the probe corresponding to a
portion of the transmembrane domain and the 3`-untranslated region of
the AE2-1 cDNA (Fig. 7A). This is consistent
with the blotting analysis that indicated that the major proventricular AE2 transcript of 4.4 kb does not hybridize with either
of the variant-specific probes ( Fig. 4and Fig. 5).
Similar in situ studies using probes specific for the chicken AE1 and AE3 anion exchangers have revealed that only
transcripts derived from the chicken AE2 anion exchanger gene
accumulate to detectable levels in the cells of the superficial and
profound glands (data not shown).
Molecular analyses have indicated that electroneutral anion
transporters are encoded by a multigene family, which includes the AE1, AE2, and AE3 anion exchangers. The
widespread distribution of transcripts derived from these genes (23) suggests that these anion exchangers are important in
regulating intracellular pH in a variety of cell types. Recent analyses
have suggested that the electroneutral anion exchange activity that has
been characterized in the basolateral membrane of mammalian gastric
parietal cells is mediated by the AE2 anion exchanger(16) .
Physiological studies have demonstrated that upon stimulation, parietal
cells secrete H across their apical membrane, thereby
acidifying the lumen of the stomach. To prevent intracellular
alkalinization during acid secretion, the anion exchange activity
localized in the basolateral membrane of parietal cells mediates the
exchange of intracellular bicarbonate for extracellular
chloride(12) . The demonstration that polypeptides recognized
by AE2-specific peptide antibodies accumulate in the basolateral
membrane of gastric parietal cells in both rabbit and rat (16) strongly suggests that the AE2 anion exchanger mediates
the basolateral anion exchange activity of this acid-secreting cell
type. The studies presented here indicate that variant AE2 transcripts accumulate to very high levels within the epithelial
cells of the chicken profound gland and, to a lesser extent, within the
mucus-secreting cells of the superficial gland. These results suggest
that these chicken AE2 gene products may not only mediate the
basolateral anion exchange activity of the acid-secreting cells in the
profound gland, they also may mediate the apical bicarbonate-secreting
activity that has been characterized in the gastric mucus-secreting
cells of other species(18) .
In situ hybridization studies have shown that variant AE2 anion exchanger transcripts accumulate to very high levels within the acid-secreting epithelial cells of the profound gland in the chicken stomach. Higher power magnification of regions within the profound gland indicates that all of the epithelial cells lining the profound gland accumulate AE2 transcripts (data not shown). This observation suggests that each of the epithelial cells of the gland mediates acid secretion, as had been previously proposed (17) . The high level of AE2 expression in the cells lining the ducts that connect the profound glands with the lumen was somewhat surprising since these epithelial cells are morphologically distinct from the epithelial cells of the profound gland(17) . Furthermore, histochemical staining has shown that the epithelial cells lining these ducts secrete mucopolysaccharides similar to those secreted by the epithelial cells of the superficial glands(17) . Although it is not known whether the cells of these ducts mediate acid secretion, the in situ results taken together with previous analyses indicate that they exhibit properties of both the acid-secreting and mucus-secreting cells of the stomach.
Previous immunolocalization studies failed to detect AE2 anion exchangers in the mucus-secreting cells of rabbit and rat gastric epithelia, suggesting that the apical bicarbonate-secreting activity of this cell type is not encoded by the AE2 gene in these species(16) . However, the in situ data presented here have shown that AE2 transcripts accumulate in the mucus-secreting cells of the chicken superficial gland, albeit at much lower levels than detected in the profound gland. This result suggests that one or more of the variant AE2 anion exchangers mediate the apical bicarbonate-secreting activity of these cells, which has been proposed to serve a protective function by buffering the epithelial lining of the stomach against the acidic environment of the lumen. Physiological studies have shown that luminal bicarbonate transport in the stomach ranges from 2 to 20% of luminal proton transport(18) . It is interesting to note that this large difference in the extent of luminal bicarbonate and proton transport is reflected by the differing levels of AE2 expression in the mucus-secreting and acid-secreting cells.
All of the chicken tissues
examined accumulate multiple AE2 transcripts that are
generated by a complex pattern of alternative transcriptional
initiation and differential RNA splicing. Both the abundance and the
repertoire of these transcripts vary dramatically between tissues.
Blotting analyses have revealed similar diversity among the transcripts
derived from the AE2 anion exchanger gene in rat (23) and human(20) , which range in size from 3.9
to
4.4 kb. The studies in rat also demonstrated that individual AE2 transcripts exhibit differences in tissue-specific
expression(23) . However, the data presented here are the first
to define the molecular basis for some of this observed heterogeneity.
The 7.3-kb AE2 transcripts we have detected in
chickens have not been observed in other species. Although a transcript
of
8 kb hybridized with a probe derived from the cytoplasmic
domain of the human AE2 anion exchanger(20) , this RNA
species did not hybridize with a transmembrane domain probe, suggesting
that it was not derived from the AE2 gene. Immunoblotting
analyses have shown that polypeptides of
145 and
165 kDa are
the primary AE2 species in the rat stomach. These species correspond to
differentially glycosylated forms of the polypeptide derived from
transcripts homologous to the smaller size class of chicken AE2 transcripts (
4.4-4.9 kb). Less abundant AE2
polypeptides much greater than 200 kDa in size were also detected in
the rat stomach (16) . Whether these higher molecular mass AE2
species result, at least in part, from translation of transcripts
homologous to the
7.3-kb chicken AE2 transcripts awaits
further analysis.
The variant AE2 anion exchanger transcripts encode polypeptides with alternative N-terminal cytoplasmic tails. Similar N-terminal cytoplasmic diversity has been observed among AE1(4, 5, 6, 7, 8, 9) and AE3 (23, 26, 27, 28) anion exchangers. Recent studies have indicated that the alternative sequences at the N termini of the variant chicken erythroid AE1 anion exchangers are involved in targeting these variant transporters to different membrane compartments within transfected human erythroleukemia cells(9) . Additional analyses will be required to ascertain whether specific AE2 variants mediate the basolateral and apical anion exchange activities, respectively, of the acid-secreting cells of the profound gland and the bicarbonate-secreting cells of the superficial gland. However, it is tempting to speculate that the alternative N-terminal cytoplasmic sequences of the variant AE2 anion exchangers may be involved in targeting these electroneutral transporters to opposite membrane domains in these epithelial cell types.
The mechanisms involved in directing plasma membrane transporters to specific membrane domains within cells are not well understood. One mechanism that has been proposed for restricting the distribution of membrane proteins is their association with elements of the membrane cytoskeleton(29) . Consistent with this hypothesis, immunolocalization studies have shown that the rat AE1 anion exchanger colocalizes with ankyrin in the basolateral membrane of A-intercalated cells of the kidney collecting duct(10) . Given the potential role of ankyrin in restricting the distribution of AE1 anion exchangers in the epithelial cells of the kidney, it is of interest that sequences that have been implicated in mediating the association of the human AE1 anion exchanger with ankyrin (30, 31) are highly homologous to regions that are conserved among all characterized AE2 anion exchangers (amino acids 398-449 and 477-485 of AE2-1). Whether these sequences are involved in mediating the association of AE2 anion exchangers with any of the ankyrin isoforms that have been characterized (32, 33, 34) is not known.
Recent data have suggested that AE2 anion exchangers do not associate with the membrane cytoskeleton via interaction with ankyrin. Immunocytochemical studies have shown that the membrane cytoskeletal elements ankyrin and fodrin do not colocalize with the AE2 anion exchanger in the epithelial cells of the choroid plexus(35) . Furthermore, the murine AE2 anion exchanger, unlike the murine AE1 and AE3 anion exchangers, could not be coimmunoprecipitated with the repeat domain of human erythroid ankyrin (ANK1) from human embryonic kidney cells cotransfected with these polypeptides(36) . These results, however, do not exclude the possibility that one or more of the variant chicken AE2 anion exchangers associate with the peripheral membrane cytoskeleton through interaction with one of the multiple ankyrin isoforms that are encoded by the three ankyrin genes(32, 33, 34) . Future studies will further investigate the potential role of the membrane cytoskeleton in restricting the distribution of the variant AE2 anion exchangers in the epithelial cells of the stomach.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U48889 [GenBank]and U48890[GenBank].