Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor

  1. Moo Yeol Lee*,†,
  2. Hong Song*,
  3. Junichi Nakai‡,
  4. Masamichi Ohkura§,
  5. Michael I. Kotlikoff,
  6. Stephen P. Kinsey*,
  7. Vera A. Golovina*, and
  8. Mordecai P. Blaustein*,‖,**
  1. Departments of *Physiology and
  2. ‖Medicine, University of Maryland School of Medicine, Baltimore, MD 21201;
  3. ‡Laboratory for Memory and Learning, RIKEN Brain Science Institute, Hirosawa, Wako, Saitama 351-0198, Japan;
  4. §First Department of Pharmacology, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Yoshino, Nobeoka, Miyazaki 882-8508, Japan; and
  5. Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14835

  1. Communicated by Joseph F. Hoffman, Yale University School of Medicine, New Haven, CT, July 10, 2006 (received for review March 28, 2006)

 
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Abstract

Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent “junctional” sarcoplasmic/endoplasmic reticulum (jS/ER) constitute specialized Ca2+ signaling complexes in many cell types. We examined the possibility that some Ca2+ signals arising in the junctional space between the PM and jS/ER may represent cross-talk between the PM and jS/ER. The Ca2+ sensor protein, GCaMP2, was targeted to different PM domains by constructing genes for fusion proteins with either the α1 or α2 isoform of the Na+ pump catalytic (α) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that α2(f)GCaMP2, like native Na+ pumps with α2-subunits, sorted to PM domains that colocalized with subjacent S/ER; α1(f)GCaMP2, like Na+ pumps with α1-subunits, was more uniformly distributed. The GCaMP2 moieties in both constructs were tethered just beneath the PM. Both constructs detected global Ca2+ signals evoked by serotonin (in arterial smooth muscle cells) and ATP, and by store-operated Ca2+ channel-mediated Ca2+ entry after S/ER unloading with cyclopiazonic acid (in Ca2+-free medium). When cytosolic Ca2+ diffusion was markedly restricted with EGTA, however, only α2(f)GCaMP2 detected the local, store-operated Ca2+ channel-mediated Ca2+ entry signal. Thus, α1 Na+ pumps are apparently excluded from the PM microdomains occupied by α2 Na2+ pumps. The jS/ER and adjacent PM may communicate by Ca2+ signals that are confined to the tiny junctional space between the two membranes. Similar methods may be useful for studying localized Ca2+ signals in other subPM microdomains and signals associated with other organelles.

Ca2+ signals control numerous processes in all cells. This control requires both spatial and temporal regulation of the Ca2+ signals that rely on a repertoire of Ca2+ entry, storage, release, and extrusion mechanisms. The plasma membrane (PM) contributes, in part, by its organization into specialized microdomains, some of which are involved in Ca2+ homeostasis and signaling (1–4).

In skeletal and cardiac muscles there is a special relationship between certain PM microdomains containing dihydropyridine receptors/Ca2+ channels and the adjacent sarcoplasmic/endoplasmic reticulum (S/ER) Ca2+ stores with their complement of ryanodine receptors/Ca2+ channels. These PM–S/ER units play a unique role in excitation–contraction coupling (5). A comparable structural and functional relationship exists between certain PM microdomains and the underlying “junctional” S/ER (jS/ER) in other cell types, including neurons (6, 7) and vascular smooth muscle cells (8). Furthermore, coimmunoprecipitation data from the brain provide evidence of large structural complexes that include PM, S/ER, and cytoskeletal proteins in glia and neurons (4, 9).

Functionally, the PM-jS/ER units, called “PLasmERosomes” (1, 9) or, in vascular smooth muscle, the “buffer barrier” (3), appear to be specialized for (i) release of Ca2+ from the S/ER stores (2, 10); (ii) refilling the stores through PM store-operated Ca2+ channels (SOCs) (4, 11); and (iii) regulating, by Na+/Ca2+ exchange, Ca2+ concentrations in the stores ([Ca2+]S/ER) and in bulk cytosol ([Ca2+]CYT) (3, 11, 12).

From functional observations, including those from skeletal and cardiac muscle, we infer that there is a tiny cytosolic compartment, the “junctional space” (JS), between the PM and jS/ER. Ion concentrations in the JS or other subPM regions may differ, at least transiently, from those in bulk cytosol (1, 12, 13). Thus, PLasmERosomes may facilitate Ca2+ transfer between the extracellular fluid and S/ER lumen, via the JS, without traversing the bulk cytosol (3, 14, 15).

Here we report that Ca2+ signals can be detected in the JS that are not observed elsewhere in the cell. To identify such signals, we used a new, high-affinity (K d = 146 nM), Ca2+-sensitive protein, GCaMP2 (16). GCaMP2 was fused to Na+ pump α1- and α2-subunits. Na+ pumps are expressed as αβ dimers. The catalytic subunit, α, contains the Na+, K+, ATP, and ouabain binding sites; the smaller β-subunit may chaperone α to the PM (17). There are four α-subunit isoforms (18). Na+ pumps with an α1 and those with an α2 are both expressed in astrocytes and in arterial myocytes (19–21). In both cell types, α2 Na+ pumps are confined to PM microdomains that overlie the jS/ER, whereas α1 Na+ pumps are more uniformly distributed in the PM (22–24). Therefore, we hypothesized that the GCaMP2 fused to α2-subunits [α2(f)GCaMP2], but not that fused to α1-subunits [α1(f)GCaMP2], might selectively detect PLasmERosome Ca2+ signals.

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Results

Expression of Na+ Pump α-Subunit–GCaMP2 Fusion Proteins.

GCaMP2 is a circularly permuted GFP fused to calmodulin and the calmodulin-binding peptide M13 (16). It emits a weak signal at the [Ca2+]CYT in resting cells (≈100 nM) and becomes brightly fluorescent when [Ca2+] is elevated. Thus, GCaMP2 may be suitable for studying Ca2+ signals in various cell types (16).

In preliminary studies, plasmids containing the GCaMP2 gene, were transfected into primary cultured rat mesenteric artery smooth muscle cells (ASMCs) to express GCaMP2 in the cytosol. The cells were then loaded with fura-2, and vasoconstrictor-evoked Ca2+ transient signals detected simultaneously by fura-2 and GCaMP2 were compared (Fig. 6, which is published as supporting information on the PNAS web site). Cells transfected with GCaMP2 were modestly fluorescent at rest. The ATP-evoked and serotonin (5HT)-evoked GCaMP2 signals were, however, robust and comparable to fura-2 signals in the same cells. GCaMP2 readily detected the low-frequency (≈5–10 per minute) Ca2+ oscillations that these vasoconstrictors often evoked. These findings suggested that a PM-targeted GCaMP2 might be useful as a near-membrane indicator.

To target GCaMP2 to specific subPM regions, we constructed genes to express fusion proteins consisting of an Na+ pump α1- or α2-subunit, a Flag epitope tag, and GCaMP2 (Fig. 1 A). The fusion proteins should be expressed in the PM with the GCaMP2 moieties located in the subPM cytosol, tethered to the C terminus of the respective α-subunit (Fig. 1 B). Because fusion protein fluorescence was weak at resting [Ca2+]CYT, visualization of transfected cells was enhanced by using pIRES2-DsRed2 as the expression vector (Fig. 1 A). DsRed2 was expressed in the cytosol and leaked out of cells permeabilized with saponin, whereas the membrane-bound GCaMP2 was retained and could be used for calibration with Ca2+–EGTA buffers (Fig. 7, which is published as supporting information on the PNAS web site).

Fig. 1.
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Fig. 1.

Construction, expression, and distribution of PM domain-targeted GCaMP2 fusion proteins. (A) Schematic representation of α1- and α2(f)GCaMP2 (Upper). Constructs were cloned into the pIRES2-DsRed2 vector (Lower). (B) Expressed Na+ pump C terminus is in the cytosol, so that GCaMP2 detects signals in the subPM cytosol. To test for PM expression (D), a second Flag-tag (f) was inserted at proline-120 in the loop between TM1 and TM2 [α1- or α2(f120)(f)GCaMP2]. (C) Expression of α1- and α2(f)GCaMP2 detected by immunoblot. Lane 1, nontransfected HEK293 cell extract; lanes 2 and 3, extracts of α1- and α2(f)GCaMP2-expressing HEK293 cells, respectively; lane 4, rat brain extract (positive control). The 153-kDa fusion proteins were detected with anti-GFP (cross-reacts with GCaMP2) and monoclonal anti-Flag antibodies (both 10 μg of protein per lane) as well as with Na+ pump α isoform-specific antibodies (1 μg of protein per lane for α1; 10 μg of protein per lane for α2) (27). (D and E) Distribution patterns of α1(f)GCaMP2 (D) and α2(f)GCaMP2 (E) detected by immunocytochemistry. Nonpermeabilized astrocytes expressing α1- or α2(f120)(f)GCaMP2 were stained with polyclonal anti-Flag (a and d) and monoclonal anti-SERCA2 (b) antibodies and DAPI nuclear stain (c). In some cases, as indicated, the cells were permeabilized with Brij 58 before exposure to anti-SERCA2 antibodies (e) (27). Insets in Dd, De, Ed, and Ee (enlargements of boxed regions) are also presented as color overlays (green, Flag; red, SERCA2; yellow, colocalization). (Scale bars: 10 μm.)


Expression of α1- and α2(f)GCaMP2 in HEK293 cells was verified by immunoblot: 111- and 153-kDa bands were both detected with anti-NASE (α1) antibodies, but only a 153-kDa band was observed with anti-HERED (α2) antibodies (Fig. 1 C). The 153-kDa bands correspond to the expressed fusion proteins, α1- and α2(f)GCaMP2 (Fig. 1 C). The 111-kDa bands correspond to native α1 or α2. Native α2 protein was detected in the brain but not in HEK293 cells, because kidney epithelia express only α1 (25). The 153-kDa α1- and α2(f)GCaMP2 bands were also detected with anti-Flag and anti-GFP antibodies (Fig. 1 C, bottom two panels).

Distribution and Properties of α1- and α2(f)GCaMP2.

To verify that the constructs were expressed in the PM, a second Flag tag was introduced into the extracellular loop between transmembrane (TM) helices 1 and 2 in α1- and α2(f120)(f)GCaMP2 (Fig. 1 B). Anti-Flag antibodies cross-reacted with these constructs in nonpermeabilized astrocytes (Fig. 1 D and E). In contrast, anti-NASE or anti-HERED (data not shown) (24), whose epitopes are located on the large cytoplasmic loop between TM4 and TM5 (Fig. 1 B) (26), and anti-SERCA2 (S/ER Ca2+ pump) exhibited cross-reactivity only in permeabilized cells (Fig. 1 D and E).

Immunocytochemistry was used to determine the distribution of α1- and α2(f120)(f)GCaMP2 in large, primary cultured mouse astrocytes. In these cells, which exhibit good spatial resolution, α2(f120)(f)GCaMP2 was expressed in the PM in a reticular pattern (Fig. 1 Ed) similar to that of native α2, which localizes to PM microdomains that overlie S/ER (22–24). Indeed, overlays (Fig. 1 E) revealed that α2(f120)(f)GCaMP2 and SERCA2 colocalized. In contrast, α1(f120)(f)GCaMP2, like native α1 (22–24), appeared to be more uniformly distributed (Fig. 1 Dd). Because of X–Y spatial resolution limits (≈250 × 250 nm), however, this result did not resolve the question of whether α1(f120)(f)GCaMP2 was excluded from the α2(f120)(f)GCaMP2-containing microdomains that overlie the S/ER (but see Ca 2+ Signals in EGTA-Loaded Cells).

In saponin-permeabilized cells at 33°C, α1- and α2(f)GCaMP2 exhibited K d values of 247 and 259 nM and Hill coefficients of 3.0 and 3.1, respectively (Fig. 7). Because GCaMP2 in solution has a K d of 146 nM and a Hill coefficient of 3.8 (16), Ca2+ binding was somewhat altered in the Na+ pump fusion proteins. Nevertheless, Fig. 2(see Comparison of Ca2+ Signals Obtained with fura-2 and GCaMP2) shows that global Ca2+ signals detected by α2(f)GCaMP2 are comparable to those measured with fura-2 (and with GCaMP2 itself) (Fig. 6).

Fig. 2.
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Fig. 2.

Fura-2 and GCaMP2 signals evoked by 5HT in ASMCs transfected with α2(f)GCaMP2. (A) DsRed2, GCaMP2, and fura-2 images. (Scale bar: 20 μm.) (B) Time course of Ca2+ signals detected by fura-2 (Upper) and GCaMP2 (Lower) within the blue and green boxes in the fura-2 image in A. (C) Pseudocolor fura-2 ratio (Upper) and GCaMP2 fluorescence (Lower) images from the same field as in A. a and b were captured at time points “a” and “b” indicated on the graphs in B. Relative color scale indicates low (L) and high (H) [Ca2+]. Similar results were obtained in >20 such experiments.


Comparison of Ca2+ Signals Obtained with fura-2 and GCaMP2.

ASMCs transfected with α2(f)GCaMP2 were loaded with fura-2. The field in Fig. 2 A contains a number of fura-2-loaded ASMCs, but only one transfected cell (identified by DsRed2 expression), which exhibited weak GCaMP2 fluorescence under these resting conditions. When the cells were stimulated with 10 μM 5HT (a nearly maximal dose), robust increases in [Ca2+]CYT were detected with fura-2 in all cells (Fig. 2 B Upper and C Upper). The Ca2+ signal detected by α2(f)GCaMP2 was very similar to the fura-2 signal in the transfected cell (Fig. 2 B). The small differences between these signals are likely due to differences between GCaMP2 and fura-2 Ca2+ binding kinetics and localization (see Figs. 6–8, which are published as supporting information on the PNAS web site). Virtually identical results were obtained with α1(f)GCaMP2 (data not shown). Moreover, α1(f)GCaMP2 and α2(f)GCaMP2 in ASMCs both also responded to a nearly maximal dose of ATP (10 μM) in a similar fashion (Fig. 8). Comparable results were obtained in astrocytes (data not shown).

More detailed examination revealed, however, that the α1- and α2(f)GCaMP2 signals were not identical. Fig. 3 shows data from two astrocytes with well resolved S/ER. In the cell transfected with α1(f)GCaMP2 (Fig. 3 A a–d), the resting GCaMP2 signal is relatively uniform (Fig. 3 Ac) and has no obvious relationship to the underlying S/ER [stained with 3,3′-dihexyloxacarbocyanine iodide (DiOC)] (Fig. 3 Ad). In the cell transfected with α2(f)GCaMP2 (Fig. 3 A e–h), however, the pattern of GCaMP2 fluorescence (Fig. 3 Ag) parallels the S/ER distribution (Fig. 3 Ah). These data fit with the colocalization of α2(f)GCaMP2 and native α2 Na+ pumps, but not α1(f)GCaMP2 or native α1 Na+ pumps, with SERCA2 (Fig. 1 D and E) (22–24). The C-terminal GCaMP2 apparently does not alter normal α isoform sorting. Here, too, spatial resolution limits preclude determination of whether Na+ pumps with an α1-subunit are excluded from the PM microdomains at PM–S/ER junctions.

Fig. 3.
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Fig. 3.

Spatially distinct ATP-evoked Ca2+ signals detected by α1- and α2(f)GCaMP2. (A) Small portions of astrocytes expressing α1(f)GCaMP2 (a–d) or α2(f)GCaMP2 (e–h). (a and e) DsRed2 images. (b and f) GCaMP2 images. (c and g) Enlargements of white-boxed areas in b and f. (d and h) S/ER stained with DiOC in the same area as c and g. (Scale bars: 25 μm in a, b, e, and f; 3 μm in c, d, g, and h.) (B) α1(f)GCaMP2 (Upper) or α2(f)GCaMP2 (Lower) fluorescence (F/F 0) response to 10 μM ATP. Black records were obtained from the entire DiOC image areas. Red, blue, and green records were obtained from the areas within the red, blue, and green circles, respectively, in the DiOC images. Representative images from one of five replicate experiments are shown.


Stimulation with 10 μM ATP induced large Ca2+ signals in both cells; the black records in Fig. 3 B show spatially averaged signals from the entire imaged areas in Fig. 3 A c and g. The green circles in Fig. 3 A d and h encompass small regions of S/ER, whereas the red and blue circles encompass nearby regions apparently devoid of S/ER. α1(f)GCaMP2 detected ATP-evoked Ca2+ signals in all three small regions (red, blue, and green records in Fig. 3 B Upper). In contrast, α2(f)GCaMP2 detected a signal only in the green-circled region (green record in Fig. 3 B Lower), consistent with the restricted expression of this construct (Figs. 1 D and 3A f–h).

Ca2+ Signals in EGTA-Loaded Cells.

In subsequent experiments, EGTA, a high-affinity Ca2+ buffer with slow reaction kinetics, was used to limit the spread of the Ca2+ signal (27–29) and to help identify the sites of signal origin. In transfected ASMCs loaded with 50 μM EGTA [i.e., preincubated with 50 μM EGTA-membrane-permeable acetoxymethyl ester (AM) for 60 min at 25°C], neither α1(f)GCaMP2 nor α2(f)GCaMP2 detected a significant 10 μM ATP- or 5HT-evoked Ca2+ signal, even though small cytosolic fura-2 signals were observed in these cells (Fig. 9, which is published as supporting information on the PNAS web site). ATP- and 5HT-evoked Ca2+ signals involve primarily inositol trisphosphate-mediated S/ER Ca2+ release. Thus, it appears that EGTA greatly reduced diffusion of Ca2+ to the PM, even at the PM–S/ER junctions, where the two membranes are ≤20 nm apart (6–8).

A more appropriate test, however, is to measure localized Ca2+ entry across the PM (27–29). Because SOCs, like α2 Na+ pumps, localize to PM–S/ER junctions in ASMCs and astrocytes (1, 4, 11), we examined SOC-mediated Ca2+ entry (SOCE). Fig. 4 A shows images from a large astrocyte expressing α2(f)GCaMP2, in which elements of the S/ER could be readily visualized with DiOC (Fig. 4 Ac). This cell was preloaded with 50 μM EGTA. There was little change in fluorescent signal over the S/ER and a slight decline in the signal in nearby regions devoid of S/ER when the S/ER was unloaded with the S/ER Ca2+ pump inhibitor, cyclopiazonic acid (CPA), in Ca2+-free medium (Fig. 4 B, black and red records, respectively). When external Ca2+ was restored, inducing SOCE, the GCaMP2 fluorescent signal over the S/ER increased substantially but was little changed in S/ER-free regions (Fig. 4 B, black and red records, respectively; also compare Fig. 4 Cb with Fig. 4 Ca). In contrast, no SOCE-associated signal was observed in similarly treated cells expressing α1(f)GCaMP2 (data not shown). The absence of a signal in these cells could be due either to the lack of response of cells expressing α1(f)GCaMP2 or to the absence of α1(f)GCaMP2 in the PM microdomains within PLasmERosomes where SOCE is confined (4). This dilemma is addressed in Fig. 5.

Fig. 4.
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Fig. 4.

SOCE detected by α2(f)GCaMP2. Astrocytes expressing α2(f)GCaMP2 were loaded with EGTA. S/ER Ca2+ was released with 10 μM CPA in Ca2+-free (0-Ca2+) PSS; SOCE was activated by restoring normal PSS (without CPA). (Aa) GCaMP2 image. (Ab) Enlargement of white-boxed area in Aa. (Ac) S/ER stained with DiOC (same image area as in Ab). (Scale bars: 20 μm in a; 7 μm in b and c.) (B) Fluorescent Ca2+ signals (F/F 0) detected by GCaMP2. Bars at the bottom of each panel indicate periods when Ca2+ was removed or CPA was added. Black and red records were obtained from the areas within the white and red boxes, respectively, in Ac. (C) Pseudocolor GCaMP2 fluorescent images from the same field as in Ab. Ca and Cb were captured at time points “a” and “b” in B, respectively. Relative color scale indicates low (L) and high (H) [Ca2+]. Representative images from one of six replicate experiments are shown.


Fig. 5.
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Fig. 5.

Effects of EGTA loading on Ca2+ signals detected by fura-2, furaptra, α1(f)GCaMP2, and α2(f)GCaMP2 during S/ER Ca2+ store emptying and refilling; see Fig. 4 legend for protocol. (A) Fura-2 signals from nontransfected ASMCs loaded with fura-2 alone (Left) or fura-2 plus EGTA (Right). Data are representative of results from >10 (no EGTA) and 4 (+EGTA) replicate experiments. (B) GCaMP2 signals from ASMCs expressing α1(f)GCaMP2 (Left) or α2(f)GCaMP2 (Right). Lower records show responses generated in cells loaded with 50 μM EGTA. Data are representative of results from >10 (no EGTA) and 7 (+EGTA) replicate experiments each for α1- and α2(f)GCaMP2. (C) Simultaneous furaptra (Upper) and GCaMP2 (Lower) signals generated by unloading and refilling the S/ER in EGTA-loaded ASMCs expressing α1(f)GCaMP2 (Left) or α2(f)GCaMP2 (Right). Data are representative of results from three replicate experiments each for α1- and α2(f)GCaMP2.


Fig. 5 A shows the effect of EGTA on the Ca2+ signals detected with fura-2 in nontransfected ASMCs. In the absence of an EGTA preload, store unloading with CPA in Ca2+-free medium elicited a modest rise in [Ca2+]CYT (Fig. 5 A Left). Restoration of external Ca2+ then induced a much larger rise in [Ca2+]CYT associated with SOCE. These fura-2 signals were greatly attenuated, but not abolished, in ASMCs preloaded with 50 μM EGTA-AM (Fig. 5 A Right), as expected if EGTA buffers the cytosolic Ca2+.

In ASMCs (and astrocytes; data not shown) expressing either α1- or α2(f)GCaMP2 the fluorophore detected very small, CPA-induced S/ER Ca2+ unloading signals and much larger SOCE signals (Fig. 5 B Upper). In this case, preincubation with EGTA-AM prevented α1(f)GCaMP2 from detecting SOCE signals but had little effect on the SOCE signals detected by α2(f)GCaMP2 (Fig. 5 B Lower). It is possible that the S/ER did not unload and refill in the EGTA-loaded cells that expressed α1(f)GCaMP2. Therefore, the latter experiment was repeated in cells loaded with the low-affinity Ca2+ sensor furaptra (K d = 76 μM) (30). Under these circumstances, S/ER emptying and refilling could be monitored directly with the furaptra while SOCE was measured, simultaneously, with the appropriate α-subunit–GCaMP2 construct. The furaptra signals (Fig. 5 C Upper) reveal that the S/ER did unload when CPA was applied and did refill when CPA was washed out and that external Ca2+ was restored in cells expressing α1(f)GCaMP2 as well as those expressing α2(f)GCaMP2. Nevertheless, only α2(f)GCaMP2 detected the SOCE signal in EGTA-loaded cells (Fig. 5 C Lower). As noted in Discussion, this result has important implications not only for elucidation of local signaling at PM–S/ER junctions but also for PM microdomain organization and Na+ pump α-subunit isoform distribution.

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Discussion

SubPM [Ca2+] Measurement.

The studies reported here are based on two critical factors. The first is the evidence that most cells express α1 and a second Na+ pump α-subunit isoform, usually α2 or α3 (18, 31), with different PM distributions (22–24). Moreover, the different α-subunit isoforms confer different kinetic properties, including Na+ affinities, on the Na+ pumps (32). Thus, the ionic composition of the microdomains of cytoplasm near the different Na+ pumps must be different. Colocalization of the Na/Ca exchanger (23) with Na+ pumps with an α2-subunit (or an α3-subunit, depending on cell type) raises the possibility that α2 (and α3) effects are “amplified” by their indirect influence on the Na/Ca exchanger and on Ca2+ homeostasis and signaling (33). This coupled mechanism is convenient because it is much easier to detect small changes in [Ca2+]CYT than [Na+]CYT in living cells and because Ca2+, which binds to proteins and is sequestered, diffuses more slowly than Na+ in the cytosol (34, 35).

The second factor is the ability to measure local subPM Ca2+ levels. Such studies require detection within a few nanometers of the PM. One solution is to use Ca2+ sensors with a lipophilic tail [e.g., fura-piperazine-C12H25 (FFP-18)] (36), but such dyes insert into all membranes. Another solution is to employ evanescent wave microscopy to detect signals within ≈100 nm of the PM (29, 37). We adopted a third approach: fusion of a new, high-affinity, brightly fluorescent Ca2+ sensor, GCaMP2 (16), to the Na+ pump α1- and α2-subunits. In addition, use of EGTA limited Ca2+ diffusion to within just a few nanometers of the “source” (27–29). These α isoforms sort to different PM microdomains (22–24), and EGTA enabled us to compare local changes in [Ca2+]CYT in the neighborhood of the α1 and α2 Na+ pumps.

Targeting GCaMP2.

We constructed genes for the α1- and α2(f)GCaMP2 fusion proteins (Fig. 1). Expression and distribution of the two constructs in the PM were verified by immunocytochemistry (Fig. 1 D) and by the GCaMP2 fluorescence (Figs. 3 A and 4A). Like native α2 (22), α2(f)GCaMP2 colocalized with underlying S/ER, whereas α1(f)GCaMP2, like native α1, appeared to be more uniformly distributed.

The global [Ca2+]CYT signals detected by both GCaMP2 constructs were comparable to those of fura-2. The fusion protein GCaMP2 moieties should detect submicromolar changes in subPM [Ca2+]CYT. Moreover, if the α1- and α2-subunit isoforms distribute differently in the PM, the GCaMP2 moieties fused to α1 and α2 might be expected to detect different local subPM Ca2+ signals. As Fig. 3 reveals, however, even if evoked, large Ca2+ signals arise locally, they often spread rapidly and become global signals. This spread masks the sites of signal initiation and obscures local differences in the Ca2+ signals.

Detection of Local (SubPM) Ca2+ Signals with EGTA and α2(f)GCaMP2.

To determine whether both GCaMP2 constructs could detect local Ca2+ signals in PLasmERosomes, we studied a signal, SOCE, that apparently arises at these PM–S/ER junctions (1, 4). But even SOCE signals may arise locally and spread broadly (4). Thus, there are two possible explanations for our observation that, under control conditions, SOCE signals were detected by both α1- and α2(f)GCaMP2 (Fig. 5 B Upper). Either α1(f)GCaMP2 distributed throughout the PM (including PLasmERosomes) or the SOCE signal spread from domains in which α1(f)GCaMP2 was not expressed to domains in which it was expressed.

This dilemma was resolved by introducing the “slow” Ca2+ buffer, EGTA (28). EGTA binds Ca2+ with a slow on-rate (K on ≈ 3–10 μM−1·s−1) (38, 39) but high affinity (K d = 150 nM at pH 7.2) (40) and thus strongly buffers the cytosolic Ca2+. Therefore, EGTA should have little effect on Ca2+ signals at their sites of origin but should minimize the spread of these signals and strongly attenuate the resultant global [Ca2+]CYT elevation. Indeed, when cells were preincubated with 50 μM EGTA/AM, the large, global fura-2 signals normally evoked by ATP, 5HT, and SOCE were all markedly attenuated.

The ATP- and 5HT-evoked signals detected by both α1- and α2(f)GCaMP2 were also markedly attenuated or abolished in EGTA-loaded cells. In contrast, EGTA had a very different effect on the SOCE signals detected by these two constructs: Although EGTA prevented α1(f)GCaMP2 from detecting the SOCE signal, it had negligible effect on the SOCE signal detected by α2(f)GCaMP2 (Fig. 5 B and C).

Several important conclusions can be drawn from these observations. First, they demonstrate the feasibility of selectively targeting GCaMP2 to detect local cytosolic Ca2+ signals at their sites of origin. Also, as anticipated (1, 9), α1 Na+ pumps are apparently excluded from the PM microdomains occupied by α2 pumps at PM–S/ER junctions. Furthermore, SOCs, as well as α2 Na+ pumps, are confined to these PM microdomains in ASMCs (11) and astrocytes (4). These microdomains are components of PLasmERosomes (1), in which the PM and jS/ER are separated by only 12–20 nm (6–8). The cytosolic volume in the intervening JS is very small, on the order of 5 × 10−19 liters for a disk with a radius of 100 nm (1). Thus, ions entering or leaving this space may have a large effect on local ion concentrations, especially if diffusion between the JS and bulk cytosol is restricted (1). Indeed, calculations indicate that diffusion of Na+ between the JS and bulk cytosol must be restricted to permit α2 Na+ pumps (with K d for Na+ = 24 mM) (32) to extrude Na+ when bulk [Na+]CYT is only ≈6.5 mM (12). Clearly, the PLasmERosome components function as a unit that likely serves to shuttle Ca2+ directly between the extracellular fluid and the S/ER while limiting access to bulk cytosol (3, 15). For example, Ca2+ may be extruded here via the Na/Ca exchangers that are also confined to the PM microdomains in the PLasmERosomes (24). Conversely, Na/Ca exchangers may mediate Ca2+ entry and modulate [Ca2+]S/ER as a result of ouabain’s action on the α2 Na+ pumps located here (12, 21). Also, Ca2+ may enter here via SOCs, as we have shown, to refill the S/ER after depletion (4, 30).

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Materials and Methods

DNAs for Na+ Pump Catalytic α-Subunit–GCaMP2 Fusion Proteins.

Plasmids containing the coding sequences for rat Na+ pump α1 and α2 subunits [pGEM α1 and pBluescript II SK(+) α2] were gifts from R. Mercer (Washington University, St. Louis, MO). A Flag epitope tag (f) was added to the C terminus of α1 and α2 by PCR-based methods to generate α1(f) and α2(f).

GCaMP2 DNA was generated as described (16); it was fused to the 3′ ends of α1(f) and α2(f) to express, respectively, α1- and α2(f)GCaMP2 (Fig. 1 A and B). To help visualize transfected cells, the α1- and α2(f)GCaMP2 constructs were cloned into the pIRES2-DsRed2 vector (CLONTECH, Mountain View, CA). The α-subunit–GCaMP2 construct and DsRed2 were then expressed separately but simultaneously. All constructs were confirmed by sequencing.

Primary Cultured Cells.

ASMCs were prepared from mesenteric arteries of male Sprague–Dawley rats (100–150 g) (41). Mouse cortical protoplasmic type-1 astrocytes were prepared from fetuses on embryonic day 18 or 19 (42). ASMCs and astrocytes were plated onto 25-mm coverslips and were grown in primary culture for 4–6 and 7–9 days, respectively. All animal protocols were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee.

Transfection, Immunoblotting, and Immunocytochemistry.

HEK293 cells or the primary cultured cells were transfected with α1- or α2(f)GCaMP2 by using 4 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and 2 μg of DNA per milliliter of culture medium. The cells were used 48 h after transfection for both functional studies and immunocytochemistry. HEK293 cell membrane proteins were prepared and analyzed by immunoblotting (9, 12). Isoform-specific polyclonal antibodies raised against the Na+ pump α1 and α2 isoforms (26) and anti-Flag or anti-GFP (cross-reacts with GCaMP2) antibodies were used. Distribution of the expressed Na+ pump constructs in astrocytes was studied by immunocytochemistry (9, 24). Both nonpermeabilized and Brij 58-permeabilized cells were stained with anti-Flag and anti-SERCA2 antibodies (24).

Digital Imaging of Live Cells.

Coverslips with cultured ASMCs or astrocytes were mounted in a superfusion chamber on the microscope stage. Cells expressing α1- or α2(f)GCaMP2 were studied for 1–2 h during continuous superfusion (2 ml/min) with physiological salt solution (PSS) (140 mM NaCl/5 mM KCl/5 mM NaHCO3/1.8 mM CaCl2/1.4 mM MgCl2/1.2 mM NaH2PO4/11.5 mM glucose/10 mM Hepes, pH 7.4). Pharmacological reagents were added to the PSS (see Results). In some experiments, cells were loaded (60 min) with 3 μM fura-2/AM (22°C), 50 μM EGTA/AM (22°C), or 6 μM furaptra/AM (mag-fura-2; 37°C) in 1 ml of PSS with 0.5% BSA. When the cells were loaded with both EGTA and furaptra, the dye was loaded first to maximize loading of furaptra into the S/ER (43) and to minimize intraorganellar sequestration of EGTA. Extracellular reagents were removed by superfusing the cells with PSS (33°C for 30 min) before starting experiments. All experiments were performed at 33°C.

Cells were activated with 5HT-containing (ASMCs, only) or ATP-containing PSS. SOCE was induced by unloading the S/ER with 10 μM CPA in Ca2+-free PSS and then restoring the Ca2+ (4, 30) and removing CPA. The Ca2+-free PSS contained 0.1 mM EGTA and no CaCl2.

Cells were imaged with a Nikon Eclipse 2000 inverted microscope equipped with a UV-Fluor ×40 (n.a. 1.4, oil) objective lens (Nikon, Melville, NY) and a Hamamatsu ORCA-ER CCD camera (Hamamatsu Photonics, Bridgewater, NJ). A Lambda DG-4 wavelength switcher with a xenon arc lamp (Sutter Instruments, Novato, CA) provided illumination. Excitation and emission wavelengths, respectively, used for the various fluorophores were as follows: fura-2 and furaptra, 340/380 nm (excitation ratio) and 535 nm; GCaMP2 and DiOC, 488 nm and 525 nm (or 535 nm when used simultaneously with fura-2 or furaptra); and DsRed2, 545 nm and 610 nm. Images were acquired and analyzed with a Meta Imaging System (Molecular Devices, West Chester, PA).

The S/ER distribution was examined at the end of some live imaging experiments. The cells were stained with DiOC (0.2 μg/ml for 3 min at 33°C), a stain for S/ER and mitochondria (44). The same wavelengths were used for GCaMP2 and DiOC imaging, but the DiOC emission was at least 1,000-fold brighter than that of α1- or α2(f)GCaMP2; thus, GCaMP2 fluorescence did not interfere with visualization of DiOC-stained organelles.

Reagents.

Fura-2/AM, EGTA/AM, DAPI, and DiOC were obtained from Molecular Probes (Eugene, OR); furaptra/AM was from TefLabs (Austin, TX). CPA, saponin, Brij 58, anti-Flag, and anti-GFP antibodies were purchased from Sigma-Aldrich (St. Louis, MO); anti-SERCA2 antibodies were obtained from Affinity Bioreagents (Golden, CO). All other reagents were “reagent-grade” or the highest grade available.

Statistics.

Figures show representative data from individual imaging experiments; each “experiment” refers to a single transfected coverslip. The numbers of replicate experiments are given in the figure legends.

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Acknowledgments

We thank Drs. A. McDonough (University of Southern California, Los Angeles, CA) and T. Pressley (Texas Tech University, Lubbock, TX) for gifts of antibodies. This work was supported by National Institutes of Health Grants NS-16106, HL-45215, and HL-78870 Project 1 (to M.P.B.); DK-65992 and HL-45239 (to M.I.K.); and NS-48263 and HL-78870 Project 2 (to V.A.G.); by Alzheimer’s Disease Association Grant IIRG-03-5665 (to V.A.G.); by the Japanese Ministry of Education, Culture, Sports, Science, and Technology and the Human Frontier Science Program (J.N.); and by the Japan Society for the Promotion of Science (M.O.).

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Footnotes

  • **To whom correspondence should be addressed at:
    Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201.
    E-mail: mblaustein{at}som.umaryland.edu

  • ↵ †Present address: Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322.

  • Author contributions: M.Y.L. and M.P.B. designed research; M.Y.L., H.S., and S.P.K. performed research; J.N., M.O., and M.I.K. contributed new reagents/analytic tools; V.A.G. assisted with methodology; M.Y.L. and V.A.G. analyzed data; and M.Y.L. and M.P.B. wrote the paper.

  • Conflict of interest statement: No conflicts declared.

  • Abbreviations::
    ASMC,
    arterial smooth muscle cell;
    CPA,
    cyclopiazonic acid;
    5HT,
    serotonin;
    JS,
    junctional space;
    PM,
    plasma membrane;
    PSS,
    physiological salt solution;
    S/ER,
    sarcoplasmic/endoplasmic reticulum;
    jS/ER,
    junctional S/ER;
    SOC,
    store-operated Ca2+ channel;
    SOCE,
    SOC-mediated Ca2+ entry;
    DiOC,
    3,3′-dihexyloxacarbocyanine iodide;
    TM,
    transmembrane;
    AM,
    membrane-permeable acetoxymethyl ester.
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