The Cardiac Na+-Ca2+ Exchanger Binds to the Cytoskeletal Protein Ankyrin*

Na+-Ca2+ exchange is the major pathway of Ca2+ efflux during excitation-contraction coupling in cardiac muscle. The Na+-Ca2+ exchanger is present in cardiac transverse tubules with an apparent high density (Frank, J. S., Mottino, G., Reid, D., Molday, R. S., and Philipson, K. D. (1992) J. Cell Biol. 117, 337- 345). The mechanism for this localization is unknown but may involve interactions with the cytoskeleton. In the present study, we examined the interaction of the Na+-Ca2+ exchanger with the cytoskeletal protein ankyrin. On immunoblots of isolated canine cardiac sarcolemma, an antibody raised against purified rab- bit red blood cell-ankyrin (RBC-ankyrin) recognized a 220-kDa protein, which is the same size as RBC- ankyrin. Alkaline extraction of sarcolemma removed this protein. The Na+-Ca2+ exchange protein, purified from recombinant baculovirus-infected insect cells, bound L261-labeled-RBC-ankyrin with a KO of 42 2 3 nM. 12SI-RBC-ankyrin was co-precipitated by antibod- ies to the Na+-Ca2+ exchanger after preincubation

1 To whom correspondence should be addressed: Cardiovascular Reicine, Los Angeles, CA 90024-1760. Tel.: 310-825-7679;Fax: 310-206-5777. ' The abbreviations used are: SR, sarcoplasmic reticulum; Caps, 3-(cyclohexy1amino)-1-propanesulfonic acid; Mops, 3-(N-morpholino)propanesulfonic acid; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; RBC-ankyrin, red blood cell ankyrin; PAGE, poly-Relaxation results from sequestration of myoplasmic Ca2+ into the SR by a n ATP-dependent Ca2+ pump and from extrusion of Ca2+ across the sarcolemma by Na+-Ca2+ exchange. The Na+-Ca2+ exchanger uses the energy of the Na' gradient to remove Ca2+ from the cell. The stoichiometry is 3Na+ for 1Ca2+ (Philipson, 1990). Using immunolocalization techniques, Frank et al. (1992) reported recently that the Na+-Ca2+ exchanger is present in the sarcolemma of transverse tubules at a high density. This specific location of the exchanger may be of much functional importance for understanding the Ca2+ fluxes associated with excitation-contraction coupling. The mechanism for this localization is unknown, but the Na+-Ca2+ exchanger may interact with specific elements of the cytoskeleton to restrict its diffusion in the plane of the membrane.
Ankyrins are a multigene family of proteins that may serve as a link between several integral membrane proteins and a spectrin-based membrane cytoskeleton (Bennett, 1992). The anion exchanger of erythrocytes (Drenckhahn et al., 1988), Na+-Kt-ATPase (Morrow et al., 1989;Nelson and Veshnock, 1987) and amiloride-sensitive sodium channel of kidney (Smith et al., 19911, and the voltage-dependent sodium channel of brain (Srinivasan et al., 1988) are reported to interact with ankyrins in binding assays and are co-localized with ankyrins in tissues. In the present study, we examined the interaction of the Na+-Ca2+ exchanger with ankyrin.
1970) was used. Proteins from SDS-PAGE were transferred onto nitro-Immunoblots-A 6.5% polyacrylamide Laemmli system (Laemmli, cellulose for 30 min at 100 V in a Bio-Rad Mini Trans-Blot apparatus. Immunoreactions were detected using goat anti-rabbit IgG conjugated to horseradish peroxidase with 3,3'-diaminobenzidine as substrate. The affinity-purified polyclonal antibody to rabbit erythrocyte ankyrin was raised in rabbits and has been described previously (Bennett and Davis, 1982). Purification of the Na*-Ca*+ Exchanger-Na+-Ca2+ exchanger protein was expressed in Sf9 cells using a baculovirus vector as described previously (Li et al., 1992). Cells infected with recombinant virus 3E3 at a multiplicity of infection of 2 were harvested 3 days post-infection. The cell pellet was lysed by sonication and alkaline-extracted using 10 m M Caps, pH 12 (Philipson et al., 1988). The partially purified exchanger protein was then solubilized in SDS-PAGE sample buffer and purified by elution from a Bio-Rad Prep Cell following the directions of the manufacturer (Li et al., 1992). Binding Assay-Purified exchanger protein (approximately 0.1 pg) was dried on PVDF membrane discs (6 mm diameter). The discs were then incubated for 1 h at 37 "C in binding buffer (50 m M Hepes, pH 7.4, 50 m M NaCI, 1 m M EDTA, 1 m M NaN3, 0.5 m M dithiothreitol, and 40 mg/ml bovine serum albumin). The binding was performed in 50 1. 11 of binding buffer containing various concentrations of lZ5I-labeled ankyrin at 4 "C for 2 h. The discs were washed three times with 2 ml of binding buffer containing 2 mgiml bovine serum albumin at 4 "C, and each disc was counted in a y counter. Assay were performed in duplicate and controls for nonspecific binding to the filter discs were subtracted for each point.
Immunoprecipitation-Cardiac sarcolemmal vesicles (100 pg) were solubilized in 25 m M decyl maltoside, 140 m M NaCI, and 10 m M Mops, pH 7.4. Nonsoluble material was removed by centrifugation in a Beckman Airfuge. The supernatant was incubated with lZ5I-ankyrin on ice for 2 h. The solubilized sarcolemmd'251-ankyrin mixture was then added to anti-Na+-Ca2+ exchanger polyclonal or monoclonal antibodies (Philipson et Frank et al., 1992) immobilized on protein Aagarose. The protein A-agarose had been prepared by incubation of 100 acrylamide gel electrophoresis; BSA, bovine serum albumin.

Na+-Ca2+ Exchanger Binds
to Ankyrin pl of antibodies with 100 pl of a 50% suspension of protein A-agarose for 1 h a t room temperature followed by five washes with 140 mM NaCl and 10 mhl Mops, pH 7.4. The immunoprecipitation reaction was allowed to proceed for 1 h a t room temperature, and the agarose beads were then washed five times as above. The washed beads were then assayed for bound ""I-ankyrin.
prepared as described previously (Frank et al., 1992). The cells were Immunofluorescence-Isolated rat and guinea pig myocytes were fixed for 10 min in 1.5% buffered formaldehyde, quenched in 0.2% sodium borohydrate, and then permeabilized by a 10-min exposure to 0.1% Triton X-100. Subsequently, the cells were incubated with 5% goat serum and 3% BSA in PBS solution for 45 min and then incubated in primary antibodies for 90 min. After four changes in PBSBSA the cells were incubated for 1 h in fluorescence-labeled goat anti-rabbit antibody. The cells were washed several times in PBS and mounted on slides with mounting medium (90% glycerol, 2% 1,4-diazabicyclo-(2,2,2)-octane, and anti-bleaching agents).

Ankyrin in Cardiac
Sarcolemma-Polyclonal antibodies raised against purified rabbit RBC-ankyrin reacted with ca- FR;. 2. Immunofluorescent localization of ankyrin in rat myocytes. Isolated myocytes were incubated with a polyclonal antibody against RBC-ankyrin and subsequently incubated with fluorescencelabeled goat anti rabbit IgG (magnification, X 1000).

FIG. 3. Binding of Iz5I-ankyrin to
A purified Na '-Ca2 exchanger. Na . -Ca2-exchange protein was purified and bound to PVDF discs as described in "Ex-

'OI
nine cardiac sarcolemma on immunoblots. The antibodies recognize a sarcolemmal protein a t -220 kDa, the same size as purified rabbit RBC-ankyrin (Fig. 1). The altibodies also react weakly with a band a t -90 kDa. Cytoskeletal proteins can be removed from membranes by alkaline extraction (Steck and Yu, 1973). Treatment of sarcolemma with 10 mM Caps a t pH 12 completely removed the 220-kDa protein band (Fig. 1). Fig. 2 shows immunofluorescent labeling of an isolated rat myocyte stained with the anti-RBC-ankyrin antibody. The immunolabeling occurs in a striation pattern that is consistent with localization at the transverse tubular membrane. Labeling appears to be less intense at the peripheral sarcolemma except at the intercalated discs, where labeling is intense. No labeling was seen using nonimmune antibodies. Similar results were seen with isolated guinea pig myocytes. These results demonstrate the presence of ankyrin in cardiac myocytes. Association of the Na+-Ca2' Exchanger with Ankyrin-Na'-Ca2+ exchanger protein was purified from recombinant baculovirus-infected Sf9 cells (Li et al., 1992). A 115-kDa protein band was eluted using a Bio-Rad Prep Cell and dialyzed against 10 mM NaCI, 10 mM Mops. The Na+-Ca2-! exchanger protein was bound to the PVDF membrane discs and was recognized strongly by anti-Na+-Ca2+ exchanger antibodies (data not shown). Fig. 3A shows that lZ5I-RBC-ankyrin bound to the Na+-Ca2+ exchanger protein with a KD of41.6 ? 2.5 nM (n = 3).

DISCUSSION
The Na".-Ca2' exchanger plays an important role in excitation-contraction coupling in cardiac muscle. The Na+-Ca2-exchanger is clearly the dominant Ca2' efflux mechanism of myocardial cells (Bridge et al., 1990). A possible additional role in Ca2+ influx is controversial (Leblanc and Hume, 1990;Lederer et al., 1990;Sham et al., 1992). Applying rapid perfusion techniques to cultured cardiac myocytes, Langer and Rich (1992) have defined an intracellular Ca2' compartment dependent upon NaA-Ca2+ exchange. They suggested that this Ca2+-containing compartment is located in the diadic region of the myocytes. This model implies that exchange occurs across the sarcolemma of the transverse tubule. Frank et al. (1992)  used antibodies to the cardiac Na+-Ca2+ exchanger to immunolocalize the exchanger in isolated cardiac myocytes. The results showed strong labeling of the transverse tubular membrane and patchy labeling of the peripheral sarcolemma. It may be of much functional importance that the Na+-Ca2+ exchanger is abundantly located in sarcolemma closest to the release sites of Ca2+. A second immunolocalization study, however, has described a different fluorescence pattern with a more uniform distribution of exchangers in the sarcolemma (Kieval et al., 1992). The mechanism for possibly localizing the exchanger to specific regions of the sarcolemma is unknown but would be likely to involve interactions with the cytoskeleton. The cytoskeleton plays a major role in the regional localization of membrane proteins. Ankyrin is a peripheral membrane protein that links integral membrane proteins to other elements of the cytoskeleton. The present study demonstrates that the cardiac Na+-Ca2+ exchanger binds to ankyrin with high affinity. This was shown by direct binding and by immunoprecipitation experiments. The interaction of the Na+-Ca2+ exchanger with ankyrin may be responsible for any special localization of the exchanger. Flucher et al. (1990) reported that in skeletal muscle, the a1 and a2 subunits of the dihydropyridine receptor were co-localized in the triad junction with ankyrin. They suggested that the ankyrin might be involved in this organization of the triad. Using anti-RBC-ankyrin antibodies, we demonstrate the presence of ankyrin in cardiac sarcolemma by immunoblot and immunofluorescent techniques. The ankyrin is widely distributed in the sarcolemma but may be at higher levels in the transverse tubules. Such a distribution would be similar to what we observed previously for the distribution of the Na+-Ca2+ exchanger (Frank et al., 1992). Its specific role in sarcolemmal organization needs to be defined in greater detail. Ankyrin is coded by multiple genes and several isoforms exist (Lambert et al., 1990;Otto et al., 1991). The molecular details of the cardiac form of ankyrin has not been resolved.