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J. Biol. Chem., Vol. 281, Issue 18, 12929-12940, May 5, 2006
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2 Subunits to Specialized Plasma Membrane Microdomains*
1
¶2
From the
Departments of Physiology, Biochemistry and Molecular Biology, and ¶Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201
Received for publication, July 8, 2005 , and in revised form, February 24, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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dimers) with the 1 isoform of the catalytic () subunit are expressed in all cells. Additionally, most cells express Na+ pumps with a second isoform. For example, astrocytes and arterial myocytes also express Na+ pumps with the 2 isoform. The 2 pumps localize to plasma membrane (PM) microdomains overlying "junctional" sarco-/endoplasmic reticulum (S/ER), but the 1 pumps are more uniformly distributed. To study 2 targeting, we expressed 1/2 and 2/1 chimeras and 1-90 and 1-120 amino acid N-terminal peptides in primary cultured mouse astrocytes. Immunocytochemistry revealed that 2/1 (but not 1/2) chimeras markedly reduced native 2 (i.e. were "dominant negatives"). N-terminal (1-120 and 1-90 amino acids) 2 (and 3), but not 1 peptides also targeted to the PM-S/ER junctions and were dominant negative for native 2 in astrocytes and arterial myocytes. Thus 2 and 3 have the same targeting sequence. Ca2+ (fura-2) signals in astrocytes expressing the 1-90 2 peptide were comparable to signals in cells from 2 null mutants (i.e. functionally dominant negative): 1 µM ATP-evoked Ca2+ transients were augmented, and 100 nM ouabain-induced amplification was abolished. Amino acid substitutions in the 1-120 1 and 2 constructs, and in full-length 1, revealed that Leu-27 and Ala-35 are essential for targeting/tethering the constructs to PM-S/ER junctions. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Functional Na+ pumps are dimers (2-4). The catalytic () subunits contain the Na+, K+, ATP, and cardiotonic steroid binding sites (2-4). The subunits (molecular mass of 
110 kDa) have 10 membrane-spanning helices (M1-M10), cytoplasmic N- and C-terminal segments, and a large cytoplasmic loop between M4 and M5 (Fig. 1A) (2). The subunit tertiary structure has been deduced by homology modeling (5) based on the known structure of the sarco-/endoplasmic reticulum (S/ER) Ca2+ pump, SERCA (see Fig. 1B) (6).
There are four isoforms of the subunit, 1-4 (2). Most cells express two different isoforms, 1 and either 2 or 3, but sperm express 1 and 4 (7). For example, astrocytes express 1 and 2, and most neurons express 1 and 3 (8-10). In rodents, 1 has an unusually low affinity for ouabain, whereas the high ouabain affinity binding sites in 2 and 3 have been retained through evolution (2, 11).
Na+ pumps with 1 subunits distribute differently from those with 2 subunits in astrocytes or 3 in neurons (9, 13), and they apparently serve different functions (14). In particular, Na+ pumps with 2 or 3 subunits cluster, along with the Na+/Ca2+ exchanger, in PM microdomains at PM-S/ER junctions (9). Moreover, in astrocytes 2 is structurally (13) and functionally (14) linked to the Na+/Ca2+ exchanger and thereby helps to modulate Ca2+ transport and Ca2+ signaling. In neurons, 3 likely plays a similar role (13). In contrast, 1 is more uniformly distributed in the PM but may be excluded from PM-S/ER junctional regions ("PLasmERosomes") (9, 13). Astrocytes (14), like cardiac myocytes (15), express 1 and 2 in a 4:1 ratio, and 1 appears to be the "housekeeper" that maintains the low [Na+]CYT in bulk cytoplasm.
Because 1 and 2 are differently distributed in the PM of astrocytes and other cell types, the two isoforms must be targeted and tethered by different mechanisms. There is no information on the sorting of 2, but immunocytochemical and co-immunoprecipitation data indicate that it is tethered to a complex containing the cytoskeletal protein, ankyrin-B (13, 16). In epithelia, 1 also is tethered to an ankyrin (12, 17), probably ankyrin-G (18).
The sorting of Na+ pump 1 and the homologous H+, K+-ATPase subunit have been studied extensively in polarized intestinal and renal epithelia. Here, this Na+ pump localizes to the basolateral membrane, and the H+, K+-ATPase sorts to the apical membrane (19). The sorting signal apparently involves a region of transmembrane helix 4 (M4; see Fig. 1A) and the flanking extracellular and cytoplasmic domains (19).
The precise role of the small, highly glycosylated subunits is uncertain. Experiments on Na+ pump 1 indicate, however, that association with the subunit is required to chaperone to the PM, as well as for the functional maturation of the pump (20). The 1 subunit (three isoforms have been identified (2)) interacts with a highly conserved amino acid (aa) sequence, SYGQ, in the extracellular loop between M7 and M8 (21) (aa 896-899 in mouse 1; see Fig. 1A). This sequence is present in most subunit isoforms from chickens (21) to mice and humans.
A sorting mechanism based on interaction with cannot explain the differences between 1 and 2 localization, because both 1 and 2 can co-assemble with either 1or 2 (10, 22, 23). This promiscuous co-assembly may be a consequence of the aforementioned conservation of the SYGC sequence in the isoforms.
In some epithelia, cycling of 1 subunits into the PM may be governed by protein kinase C-mediated phosphorylation, and several N-terminal potential phosphorylation sites have been recognized (24, 25). These sites are, however, identical in the mouse 1, 2, and 3 isoforms and, thus, cannot explain isoform-specific targeting.
Here we report that 1 and 2Na+ pumps are sorted by different mechanisms in astrocytes. The 2 sorting signal is located in the N-terminal cytoplasmic segment of the molecule. This selective sorting and membrane insertion of 2 apparently does not depend upon phosphorylation or upon association with .
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2 alleles (homozygous knockouts, 2-/- or "KO") were generated by mating heterozygotes (15). Standard PCR methods were used for genotyping genomic DNA from fetus tails. Cortical protoplasmic type-1 astrocytes were prepared from WT and KO mouse fetuses on days 18 or 19 (E18-19) as described (14, 26). The cells were plated onto poly-L-lysine-coated 25-mm glass coverslips (
50,000 cells/coverslip) for immunocytochemistry and Ca2+ imaging, or onto 10-cm Petri dishes for immunoblots. Experiments were performed on subconfluent cultures on days 7-9 in vitro. All animal protocols were approved by our Institutional Animal Care and Use Committee. Primary Cultured Rat Arterial Myocytes—Arterial myocytes were dissociated from the mesenteric arteries of male Sprague-Dawley rats (100-150 g). The cells were plated onto 25-mm coverslips and were grown in primary culture for 4-6 days as described (27).
Generation of Na+ Pump Subunit Chimeras and Related Constructs—Plasmids containing the DNA for rat Na+ pump 1, 2, and 3 subunits (pGEM 1, pBluescript II SK(+) 2 and pGEM 3 (from Dr. R. Mercer, Washington University, St. Louis)) were used. First, the Na+ pump 1 and 2 subunit cDNAs were subcloned into adenoviral (Adv) vectors to generate pAdv/1 and pAdv/2. A "FLAG tag" (f) was added to the C terminus of each subunit using PCR-based approaches. Second, domain-swaps of 1 and 2 were performed to create the pAdv/1-2f and pAdv/2-1f chimeras (e.g. N-terminal 1(1-130 or 1-333 aa) and C-terminal 2 with a FLAG tag; and, conversely, N-terminal 2(1-130 or 1-333 aa) and C-terminal 1 with a FLAG tag) (Fig. 1, A and C). Chimeras pAdv/1-2f and pAdv/2-1f were also subcloned into the pEGFP-C1 vector to generate constructs containing an N-terminal GFP tag (G) as well as the C-terminal FLAG tag (Fig. 1C). All sequences were confirmed by sequencing.
To transfect the 1/2 and 2/1 chimera constructs into astrocytes, the cultured cells were treated with purified adenoviral vector solutions. A multiplicity of infection of 10 plaque forming units/cell induced 80% transfection of cells on coverslips in 48 h. Transfection efficiency was even higher (>80-90%) for cells in 10-cm dishes used for immunoblots. All cells were studied after 48 h.
In a few experiments, we transfected cells with plasmids containing normal or mutated, full-length 1 with a C-terminal FLAG tag and the Ca2+-reporter protein, GCaMP2 (28, 29). A FLAG epitope was also, in some cases, inserted at Pro-120 in the first extracellular loop (Fig. 1A). The site-specific mutations (see "Results") were generated with "QuikChange" (Stratagene, La Jolla, CA) using the manufacturer's directions, and the constructs were cloned into the pIRES2-DsRed2 vector (Clontech, Mountain View, CA) to facilitate visualization of transfected cells before immunostaining. These constructs were transfected using Lipofectamine 2000 (see next section). The transfected cells expressed the 1f/GCaMP2 construct and DsRed2 independently and simultaneously.
N-terminal Truncations of Na+ Pump Subunits—DNA coding for the N-terminal 1-90 and 1-120 aa sequences for 1, 2, and 3 were also generated (Fig. 1C; Fig. 1B shows the sequences). The first (N-terminal) cytoplasmic domain of the Na+ pump 1, 2, or 3 sequence was cloned into pFLAG-CMV-5a vector (Sigma) by PCR (e.g. 1(1-90)f and 2(1-90)f, or 1(1-120)f, 2(1-120)f, and 3(1-120)f) (Fig. 1, C and E). To enable direct visualization of the truncated peptides in fluorescent imaging studies, GFP was fused to the N termini of some constructs (e.g. G2(1-120)f; Fig. 1C). In some experiments (see "Results"), QuikChange was used to introduce site-directed mutations in the truncated peptides.
Lipofectamine 2000 (Invitrogen) was used to transfect the fusion protein vectors and truncated subunits into astrocytes and arterial myocytes. We used 4 µg of DNA plus 8 µl of Lipofectamine in 2.5 ml of culture medium per 25-mm coverslip and 24 µg of DNA plus 60 µl of Lipofectamine in 13 ml of culture medium per 10-cm dish. The cells were incubated for 48 h (5% CO2, 37 °C) and then assayed for transgene expression by immunocytochemistry or GFP and Ca2+ imaging. Approximately 25-35% of the cells on coverslips were transfected by this method; transfection efficiency was 50% for cells cultured in 10-cm dishes and used for immunoblotting.
Immunoblot Analysis of Na+ Pump Subunit Isoforms and Constructs—Mouse WT and KO astrocytes were cultured in 10-cm dishes for 2 weeks. In some instances the cells were transfected with Na+ pump subunit constructs as described below. The cells were harvested, and the membrane proteins were prepared and analyzed by immunoblotting using published methods (13, 14). Isoform-specific monoclonal or polyclonal antibodies raised against the Na+ pump 2 subunit (Fig. 1D) and anti-FLAG antibodies (Fig. 1E) were employed for these studies. For quantitative assessment (14), subunit bands were normalized with the glia-specific protein, GFAP (glial fibrillary acidic protein).
Immunocytochemistry—The astrocytes and arterial myocytes on coverslips were fixed (45 min, 20 °C) in fixative consisting of 0.45% (w/v) formaldehyde, 75 mM cyclohexylamine (free base), 75 mM NaCl, 10 mM EGTA, 10 mM MgCl2, and 10 mM PIPES, pH 6.5. Unless otherwise stated, the cells were permeabilized (10 min at 20-25 °C) in fresh fixative containing 0.5% Brij 58 (Sigma); in some cases this step was omitted to avoid permeabilization (see "Results"). Na+ pump subunit isoform-selective monoclonal and polyclonal antibodies and anti-SERCA2b antibodies were used for these studies. Further details of our immunocytochemical methods are published (13, 14).
Cells on coverslips were imaged with a Nikon Diaphot inverted microscope (Nikon Corp., Melville, NY) equipped with a long working distance PlanApo 60X water immersion objective lens (numerical aperture 1.2) and a CoolSNAP charge-coupled device camera (Photometrics, Tucson, AZ). Illumination was provided by a diffraction grating-based Till Photonics Polychrome II illumination system (Applied Scientific Instrumentation, Inc., Eugene, OR). Images were acquired and analyzed with a Meta Imaging System (Universal Imaging, West Chester, PA).
Identification of Expressed Proteins—Na+ pump subunit isoform-selective antibodies as well as the GFP- and the FLAG tags were used to detect the expressed protein constructs. Monoclonal antibodies directed against cytoplasmic N-terminal peptide sequences in 1 (McK1 (30)) and 2 (McB2 (31)) and polyclonal antibodies directed against peptide sequences in the large cytoplasmic loop between M4 and M5 (11) in 1 (NASE) and 2 (HERED) were employed. In non-permeabilized cells, the extracellular C-terminal FLAG tag of the 120-aa peptide (Fig. 1, A and C), or a FLAG tag inserted between amino acids 120 and 121 in the full-length protein, was used to determine insertion into the PM.
N-terminal GFP-tagged constructs were also expressed in astrocytes. In live imaging experiments, these constructs could be visualized directly. Thus, following fura-2 loading, Ca2+ signaling in transfected and non-transfected cells could be compared on a single coverslip.
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Statistical Analysis—Summarized fura-2 imaging ([Ca2+]) data are presented as means ± S.E.; n is the number of cells studied. Data comparisons were made with two-way analysis of variance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2 isoform is localized to PM microdomains that overlie the ER, whereas the 1 isoform is more uniformly distributed in several different cell types (9, 32). This differential distribution is readily apparent when wild-type (WT) astrocytes are double-labeled with 1 and 2 isoform-specific antibodies (Fig. 2A). Based on this observation, our goal was to identify the one or more regions of the Na+ pump 2 subunit (Fig. 1, A and B) that are involved in targeting and tethering this subunit isoform to its appropriate PM location in astrocytes.
Detection of Native Subunit Isoforms and Transfected Constructs—To examine the difference between 1 and 2 targeting, we constructed 1/2 and 2/1 isoform chimeras and truncated segments (Fig. 1, C-E). To facilitate identification, these proteins were constructed with an N-terminal-fused green fluorescent protein, GFP (G), and/or a C-terminal FLAG tag (f). As we shall see, these tags apparently did not interfere with the targeting, tethering, or function of the constructs. Transfected and native (endogenous) subunits also could be identified with antibodies raised against isoform-specific epitopes located at the N terminus or in the large cytoplasmic loop between trans-membrane helices 4 and 5 (Figs. 1A, 1D, and 2A) (11, 30, 31). These epitopes were especially useful for studies of transfected astrocytes from 2-/- (KO) mice with no native 2, or for detecting repression of endogenous 2 expression.
Western blots probed with anti-2-selective antibodies (McB2 and HERED; see Fig. 1A) show the expressed proteins in WT and KO cells (Fig. 1D). Both antibodies detected 2 in non-transfected and full-length 1f-transfected WT cells, but not in KO cells. Both antibodies also detected 2 in KO cells transfected with full-length 2f. When KO cells were transfected with 1(1-130)/2f or 1(1-333)/2f, anti-HERED, but not McB2 antibodies, cross-reacted with the membrane proteins (Fig. 1D). Conversely, when KO cells were transfected with 2(1-130)/1f or 2(1-333)/1f, McB2, but not anti-HERED, antibodies cross-reacted with the membrane proteins (Fig. 1D). These results are expected because of the different locations of the McB2 and anti-HERED antibody epitopes (Fig. 1A).
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1/2 and 2/1 Chimeras in WT and KO Astrocytes—Chimeric constructs (see Fig. 1C for nomenclature) containing the 1 N-terminal segment, 1(1-130)/2f (Fig. 2, B-E) or 1(1-333)/2f (not shown) were expressed in WT and KO cells. The FLAG tag in the expressed constructs could be detected with FLAG epitope-specific antibodies in transfected cells (Fig. 2, B-E). The cross-reactivity with anti-NASE antibodies (Fig. 2, B-D) indicates that endogenous 1 is well expressed in all transfected and non-transfected cells even though this epitope is not present in the 1/2 chimera constructs (Fig. 1, A, C, and D). In KO astrocytes, 2-specific anti-HERED antibodies labeled only the transfected cells (e.g. Fig. 2E, arrowhead in the right-hand panel). The WT cells transfected with these constructs also normally express endogenous 2, which could be labeled specifically with McB2 antibodies (Fig. 2C) that do not cross-react with the 1/2 constructs (Fig. 1D).
Chimeric constructs with an 2 N-terminal segment, 2(1-130)/1f, 2(1-333)/1f, and G2(1-333)/1f also were expressed in WT and KO cells (Fig. 2, F-J). In 2 KO cells, 2/1 construct expression could be detected with anti-McB2 antibodies (Fig. 2J, see Fig. 1) as well as with FLAG (not shown) or GFP (Fig. 2H).
The HERED epitope (Fig. 1A) detects native 2 in all non-transfected WT astrocytes (Fig. 2K), but is not present in the 2/1 chimeric constructs (Fig. 1, A, C, and D). It was very surprising, however, that staining for this epitope could not be detected in WT cells transfected with either 2(1-130)/1f (Fig. 2G, arrowheads) or 2(1-333)/1f (not shown). In other words, either the epitope was "masked," or these 2/1 constructs indirectly repressed expression of native, full-length 2 (which we term a "dominant negative" effect). These constructs presumably bind competitively to the targeting complex and thereby lead to degradation of native 2.
The dominant negative effect of the 2/1 chimeric constructs was confirmed by immunoblot (Fig. 3). When Lipofectamine was used for transfection (50% transfection efficiency), the expression of native (full-length) 2 was reduced by 50% (band densities normalized with GFAP, an unrelated glial cell marker, Fig. 3A). In contrast, expression of the McB2 (2) epitope, which is contained in the 2/1 construct as well as in native 2 (see Fig. 1, A and C), was comparable in cells transfected with the 2/1 and the 1/2 constructs (Fig. 3A). When adenoviral vectors, with much higher transfection efficiency, were used for transfection, native 2 was almost completely suppressed (Fig. 3B).
The 2/1 chimeras appeared to be expressed primarily in a reticular (honeycomb) pattern (e.g. Fig. 2H), similar to the distribution of native 2 (Fig. 2, A (panels a and c) and K (32)). This distribution resembled the distribution of SERCA2b in the same cells (Fig. 2H), but overlays (not shown) indicated a broader distribution of the 2/1 chimeras. This suggests that these chimeras are expressed, in part, in PM microdomains that overlie "junctional" (sub-PM) ER (32) or are confined within the ER. In contrast, the 1/2 chimeras appeared to be more uniformly distributed in the PM (Fig. 2, B and D), reminiscent of the distribution of native 1 (Fig. 2A, panel b (32).
These results raise the possibility that the 2/1 chimeras may be sorted and tethered to the normal 2 as well as 1 distribution sites. Accordingly, the 2 sorting sequence may be contained within the N-terminal 1-130 aa segment. In subsequent studies, the dominant negative assay with anti-HERED antibodies (Fig. 2G) was used to identify the 2 sorting sequence.
Expression of 2 N-terminal Segments—The fact that the N-terminal 2 chimeras were dominant negative for native 2 raised the possibility that the N-terminal segment, alone, might have a similar effect. To explore this possibility, the effects of expressing 1 and 2 N-terminal 1-90 and 1-120 aa peptides (Fig. 1, C and E) in WT cells were examined.
The N-terminal 1-120 aa 1 and 2 peptides are both expressed in the WT astrocytes, as indicated by cross-reactivity to anti-FLAG antibodies (Fig. 4A, panels a-e). In both cases, all transfected as well as non-transfected cells cross-reacted with anti-NASE (Fig. 4A, panels a' and c'), demonstrating the presence of native 1 in all cells. However, only the non-transfected cells and those transfected with 1(1-120)f cross-reacted with anti-HERED antibodies (Fig. 4A, panels b' and d'). Thus, the 2(1-120)f construct was dominant negative for native 2 (Fig. 4A, panel d', arrowheads). Neither of these constructs detectably reduced expression of 1 (Fig. 4A, panels a' and c').
An even shorter construct, 2(1-90)f, with no transmembrane helices (Fig. 1, A, C, and E) also was dominant negative for native 2 (Fig. 4B, panels a and a', arrowheads). The implication is that a sequence within the N-terminal 90 amino acids preferentially binds to an appropriate "partner" and thereby displaces native 2, which is then retrieved and degraded.
As a control for the role of the N-terminal sequence in sorting, the entire initial cytosolic segment (i.e. the N terminus, through Gln-90, Fig. 1, A and B) was deleted from 2 (=
N(1-90)2f). This peptide, which contains all ten transmembrane helices, also was expressed in WT astrocytes (detected with anti-FLAG antibodies). N(1-90)2f, failed to act as a dominant negative for the expression of native 2 (Fig. 4B, panels b and b', arrowhead). In this case, native 2 was detected with McB2 antibodies, because N(1-90)2f contains the HERED epitope but lacks the McB2 epitope (Fig. 1A). This is further evidence that only a portion of the N-terminal segment plays an essential role in targeting and tethering of 2; apparently, no other parts of the molecule are required.
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Non-permeabilized WT cells transfected with 2(1-120)f, but not 2(1-90)f, which lacks a transmembrane helix (Fig. 1, A and C), cross-reacted with anti-FLAG antibodies (Fig. 4C, panels a and c). This demonstrates that 2(1-120)f is inserted into the PM. In contrast, anti-NASE and anti-SERCA antibodies, which cross-react with intracellular epitopes, stained all the permeabilized cells (Fig. 4A, panels c' and e'), but none of the non-permeabilized cells (Fig. 4C, panels a' and b').
As 2(1-90)f has no extracellular epitope, it is difficult to prove that this construct sorts to the appropriate 2 target sites. Like native 2 (Fig. 2A, panel c) and SERCA2b (Fig. 2H), however, both 2(1-90)f and 2(1-120)f distribute in a reticular pattern (Fig. 4, A (panels c-e) and B (panel a), insets). Moreover, 2(1-120)f co-localizes with SERCA2b (Fig. 4A, panels e*, e'*, and the overlay), even though SERCA2b is detectable only in permeabilized cells (Fig. 4, A (panel e') versus C (panel b)). Thus, "co-localization" does not mean that the two proteins are in the same membrane; rather, 2(1-120)f in the PM, and SERCA2b in the ER, overlie one another in membranes separated by only 10-15 nm (33) that together form "junctional" units, which we have called PLasmERosomes (14).
An important question is whether these dominant negative effects are limited to astrocytes. Mouse artery myocytes also express 1 and 2, but not 3Na+ pumps (34, 35); the 2Na+ pumps, but not 1, appear to play a critical role in the long term control of blood pressure and in hypertension (35-37). Therefore, we also expressed the 1-120 aa 1 and 2 peptides in primary cultured mouse mesenteric artery myocytes. Fig. 5 shows that 2(1-120)f (Fig. 5C, panels b and b', arrowheads), but not 1(1-120)f (Fig. 5B, panels b and b'), was also dominant negative for native 2 expression in arterial myocytes. Neither peptide affected 1 expression (Fig. 5, B (panels a and a') and C (panels a and a')). Thus, this mechanism for targeting and tethering 2 subunits applies to other tissues as well.
Functional Down-regulation of 2 by Expression of 2 N-terminal Segments—Genetically induced knock-out of 2(2-/-) (14) or selective inhibition with 100 nM ouabain (38) sensitizes astrocytes to agonists such as ATP and amplifies the responses to sub-maximal doses of agonists. Accordingly, reduced 2 activity should steepen or left-shift the agonist dose-response curves. This is, indeed, the case, as illustrated by the comparison of Ca2+ transient responses to ATP in WT and 2KO cells (Fig. 6A).
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2 constructs should have effects similar to 2 KO or low dose ouabain. This is exemplified in WT astrocytes transfected with the G2(1-90) construct (Fig. 1C). Fig. 6B (panel a) shows a field with two representative astrocytes: The cell on the right was transfected (indicated by the presence of GFP), whereas the cell on the left was not (Fig. 6B, panel b). ATP (0.1 µM) induced a small Ca2+ transient (measured with fura-2) in the non-transfected cell (Fig. 6B, panel c, top panel). When this cell was treated with 100 nM ouabain, the response to 0.1 µM ATP was greatly augmented. In contrast, the transfected cell exhibited a large Ca2+ transient in response to 0.1 µM ATP (as if 2 had already been inhibited), and the response was not augmented by 100 nM ouabain (Fig. 6B, panel c, bottom panel). Summarized data (Fig. 6C) indicate that the response to ATP was significantly augmented in cells transfected with G2(1-90), in which native (functional) 2 expression was, presumably, markedly reduced. Also, the usual augmentation of the ATP-evoked response by 100 nM ouabain in WT cells was attenuated in transfected cells (Fig. 6B, panel c, bottom panel versus top panel). This is expected in cells in which expression of the high ouabain affinity 2 receptor is reduced. (Recall that in rodents, 1 has a very low affinity and should not respond to 100 nM ouabain (2, 14, 35).) Thus, the effects observed in G2(1-90)-transfected cells were comparable to the results observed in ouabain-treated (i.e. 2-inhibited) cells or in astrocytes from 2-/- mice. Clearly, the reduced 2 function in the cells expressing G2(1-90) reflects the dominant negative effect of the nonfunctional, N-terminal 2 peptide on 2 expression.
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2 function. This was tested by transfecting G2f (with the complete 2 sequence; Fig. 1C) into KO astrocytes. Fig. 6D, panel a, shows two representative KO astrocytes. The one on the left was transfected and expressed G2f, as indicted by the GFP fluorescence in panel b. The response of the non-transfected cell to 0.1 µM ATP was not augmented by 100 nM ouabain (Fig. 6D, panel c, top panel). On the other hand, the transfected cell exhibited a greatly augmented response to 0.1 µM ATP when treated with 100 nM ouabain (Fig. 6D, panel c, bottom panel). This indicates that G2f was able to "rescue" the low dose ouabain effect in 2 KO cells. Because ouabain is hydrophilic, the response to ouabain implies that G2f was expressed in the PM. Thus, the N-terminal GFP and the C-terminal FLAG tag apparently do not interfere with normal Na+ pump sorting and function in the G2f construct.
Expression of 3 N-terminal Segments—There is evidence that the expression of Na+ pumps with 2 and 3 subunits is mutually exclusive. For example, neonatal rat cardiac muscle expresses 1 and 3, whereas 1 and 2 are present in adult cardiac muscle (39). In neurons, the Na+ pump 3 isoform is expressed with a distribution comparable to that of 2 in astrocytes: i.e. 3 is confined to PM microdomains that overlie the sub-PM ER, and it co-localizes with SERCA (9, 32). Neuronal 3 and astrocyte 2 both co-immunoprecipitate comparable cytoskeletal and ER proteins (13). Moreover, the N-terminal sequences of 2 and 3 are very similar (Fig. 1B). For these several reasons, we speculated that the 3 isoform might have the same sorting signal as 2, but a different tissue-specific promoter. Therefore, the effect of transfection with 3(1-120)f was tested on the expression of 2 in astrocytes. As shown in Fig. 7A, panels a-c, 3(1-120)f is expressed in astrocytes, and is distributed in a reticular pattern similar to that of SERCA2b (panels c and c' insets, and see Fig. 7B, panel a inset). Indeed, the 3 N-terminal segment, like its 2 counterpart, markedly reduced native 2, but not 1 expression (Fig. 7A, panel b' versus a'). Also, 3(1-120)f experiments on non-permeabilized cells demonstrate that 3(1-120)f is expressed in the PM (Fig. 7B, panel a). The similar distribution patterns of 3(1-120)f and SERCA2b (Fig. 7A, panel c and c'), therefore, indicates that they are expressed in different but adjacent membranes. The implication is that the targeting and tethering mechanisms for 2 and 3 are identical or very similar.
Alteration of Targeting by Site-specific Mutations in the N Termini of 1 and 2 Subunits—This similarity between the effects of the 2 and 3 N-terminal constructs, led us to contrast their amino acid sequences with that of the 1 N terminus. At only three positions are the 2 and 3 N termini identical, and different from 1: residues 27, 35, and 64 (using the numbering for mature 1, Fig. 1B). To test the possibility that these specific amino acid residues (aa) play a critical role in 2 targeting and tethering, each of the three residues in the 2(1-120)f construct was mutated, one at a time, to the corresponding aa in 1, i.e. L27M, A35S, and Q64A. Astrocytes were then transfected with these mutated constructs (e.g. 2(1-120, L27M)f). The results are clear: mutations L27M and A35S (Fig. 8A, panels b and c, arrowheads in lower panels), but not Q64A (Fig. 8A, panel d, arrowhead), abolished ("knocked out") the dominant negative activity of the N-terminal 2 construct. Thus, both Leu-27 and Ala-35 are essential for 2 targeting.
The complementary experiment was then performed: the aforementioned three 2/3 amino acids were mutated, one at a time and together, into 1(1-120)f. When these constructs were then transfected into astrocytes (Figs. 8B and 9), only the two 1(1-120)f peptides that contained both the M27L and S35A mutations were dominant negative for 2 (Fig. 8B, panels d and f, arrowheads in lower panels); the A64Q mutation was not needed (Fig. 8B, panel e). Furthermore, the two 1(1-120)f constructs with both M27L and S35A were sorted to the PM as indicated by the detection of the FLAG epitope (Fig. 9B, panels a and b), but not the SERCA2b epitope (Fig. 9B, panel a'), in non-permeabilized cells (Fig. 9B shows the three-mutation construct). Also, like native, full-length 2 and 2(1-120)f (Fig. 4A, panel e), 1(1-120, M27L, S35A, and A64Q)f distributed in the PM in a reticular pattern and co-localized with SERCA2b in the underlying ER (detected following permeabilization: Fig. 9A (panel c) and B (panel d)). The introduction of dominant negative activity into 1(1-120)f with the mutations, M27L and S35A, and the sorting to the PM microdomains that overlie adjacent (junctional) ER, confirm the key role of Leu-27 and Ala-35 in the targeting and tethering of 2.
Expression of Wild-type and Mutated Full-length 1 Constructs—We also tested full-length 1 with both the M27L and S35A mutations on 2 expression. This construct should contain the normal 1 targeting sequence (see "Discussion") as well as the 2 targeting sequence. In this case, we used an 1f construct with a C-terminal Ca2+-sensitive fusion protein (GCaMP2) that is expressed in the PM with the same broad distribution as native 1 (29). Cells transfected with the construct containing the WT 1 sequence (detected with anti-FLAG antibodies), like non-transfected cells, expressed the HERED (2) epitope (Fig. 10A); i.e. the normal 1 sequence was not dominant negative for 2. In contrast, cells transfected with mutated full-length 1 constructs containing the 2 amino acids, Leu-27 and Ala-35, did not express the HERED epitope (arrowheads in Fig. 10, B and C, panel a). Thus, even the full-length 1 with the M27L and S35A mutations was dominant negative for native 2.
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