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Am J Physiol Heart Circ Physiol 281: H2575-H2584, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 6, H2575-H2584, December 2001

SAP97 interacts with Kv1.5 in heterologous expression systems

Mitsunobu Murata, Peter D. Buckett*, Jun Zhou*, Michael Brunner, Eduardo Folco, and Gideon Koren

Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PDZ domain-containing proteins such as SAP97 and ZO-1 have been implicated in the targeting and clustering of ion channels. We have explored the interactions of these polypeptides with a cardiac voltage-gated potassium channel. Immunocytochemistry in cardiac myocytes revealed colocalization of SAP97 and Kv1.5, both at the intercalated disks and the lateral membranes. Transient transfection experiments in COS-7 cells revealed that SAP97 and Kv1.5 polypeptides formed perinuclear clustered complexes that could be coimmunoprecipitated. Mutation of the three COOH-terminal amino acid residues of Kv1.5 (T-D-L to A-A-A) abolished these interactions. Whereas in most COS-7 cells the SAP97-Kv1.5 complexes were retained in the ER, functional analyses in Xenopus oocytes showed that Kv1.5-encoded outward potassium currents were augmented by coexpression with SAP97. By contrast, cotransfected ZO-1 and Kv1.5 polypeptides in COS-7 cells could not be coprecipitated nor did the coinjection of ZO-1 augment the Kv1.5-encoded currents in oocytes. Collectively, our results suggest that SAP97 may play an important role in the modulation of Kv1.5 channel function in cardiac myocytes.

cardiac myocytes; K+ channels; ZO-1


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ELECTRICAL PROPERTIES of cardiac myocytes are determined to a large extent by the expression of voltage-gated potassium channels. The organization of these channels on the cell surface and their subcellular localization allow cells to increase the efficiency of their response to extracellular signals. More than 70 PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins have been described and their interactions with membrane receptors, ion channels, and other signaling proteins have been elucidated (11). The name PDZ derives from the three proteins that contain this domain: the mammalian postsynaptic density protein-95 kDa (PSD-95), the Drosophila disk large tumor suppressor Dlg, and the mammalian tight junction protein ZO-1 (12, 21). Proteins possessing PDZ domains frequently contain other interaction modules, such as the SH3 domain. Hence, these proteins serve as a scaffold for the assembly of different polypeptides into macromolecular signaling complexes. Shaker-type potassium channels and N-methyl-D-aspartate (NMDA) receptors have been identified as proteins that interact with the PDZ domains of PSD-95 (17, 28, 34). These investigators characterized a short COOH-terminal end motif (tS/T-X-V), which is necessary for the binding of NMDA receptors and Shaker-like channels to PDZ domains. The binding to PDZ domains determines the subcellular localization and the clustering of these polypeptides in neurons (14, 15, 17). Additional members of the PSD-95 family have been implicated in the formation of synaptic complexes (4, 5, 16, 27).

Of the different channel types expressed in cardiac myocytes, potassium channels are the most diverse. These channels participate in determining the resting membrane potential, the action potential duration, the duration of the refractory period, and the automaticity (9). The regional and cell-specific distribution of these channels contributes to regional variations in shape and duration of the action potential in the heart (2, 9). Very little is known about the mechanisms that regulate the targeting of cardiac Kv channels to specific sites of the plasma membrane. Two recent studies addressed the membrane distribution of these polypeptides. Mays et al. (26) showed that Kv1.5 was localized to the intercalated disks of human atrial and ventricular myocytes. Barry et al. (3) showed a clear membrane distribution of Kv1.5, Kv1.2, Kv2.1, and Kv4.2 channel polypeptides with high staining intensity at the intercalated disks of rat cardiocytes. However, in contrast with neuronal cells, the mechanisms underlying the targeting and localization of Kv channels to the intercalated disks of cardiac myocytes have not been elucidated. Here we show that SAP97 colocalizes with Kv1.5 channel polypeptides in cardiac myocytes. It also coprecipitates and colocalizes with it in a COS-7 cell heterologous expression system. Furthermore, SAP97 enhances Kv1.5-encoded currents in coinjection experiments using Xenopus oocytes. The results suggest that SAP97 may play an important role in determining the plasma membrane expression and distribution of Kv1.5 in cardiac myocytes.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning, cell culture, and transfection. Rat SAP97 cDNA was subcloned into pCDNA3 as an EcoRI/EcoRI fragment and ZO-1 cDNA was subcloned into pCDNA3 as an EcoRI/ApaI fragment. Kv1.1 (20) and Kv1.5 (24) cDNAs were subcloned as HindIII/BamHI or BstEIV/ApaI fragments in pCDNA3 with a FLAG tag at the NH2 terminus. The FLAG epitope-tagged Kv1.5 A-A-A (FL-Kv1.5-A) mutant was generated by polymerase chain reaction (PCR) using the following primers: forward, 5'-GCCGATCCATTCTTCATCGTGGAGACC-3'; and reverse, 5'-CGCGGATCCTTACGCAGCAGCTTCACGGCTAGTGTCCAG-3'; and a 0.75-kb ClaI/BamHI fragment from the PCR product was used to replace an equivalent fragment in wild-type FL-Kv1.5 cDNA. COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37°C in a 5% CO2 incubator. Transient transfection experiments were performed as described previously (23).

Immunocytochemistry. Adult rat ventricular myocytes were prepared as described previously (35). Freshly isolated cardiocytes or COS-7 cells were fixed and permeabilized in 2% paraformaldehyde and 0.5% saponin in phosphate-buffered saline (PBS) (pH 7.4) for 30 min. The cells were postfixed in a 1:1 methanol-acetone mixture for 10 min and blocked with 5% BSA for 1 h. Cells were dual labeled with the primary antibodies in a sequential manner. The antibodies used were mouse monoclonal anti-PSD-95 family (Upstate Biotechnology), which may recognize several PDZ domain-containing proteins, including PSD-95, SAP-97, and chapsyn/PSD-93, affinity-purified rabbit polyclonal anti-SAP97 (a kind gift of Dr. M. Sheng), rabbit polyclonal anti-Kv1.5 (1), mouse monoclonal anti-BiP (Transduction Laboratories), and mouse monoclonal anti-N-cadherin (Transduction Laboratories). The secondary antibodies used were fluorescein isothiocyanate-conjugated goat anti-mouse or anti-rabbit and rhodamine-conjugated donkey anti-mouse or anti-rabbit IgGs.

Immunoprecipitation. Three to four days after the transfections, COS-7 cells were harvested with the use of a lysis buffer composed of (in mM) 150 NaCl, 10 Tris (pH 8.0), 0.5 EDTA, 1 iodoacetamide, and 1% Triton X-100 and a mixture of protease inhibitors. The immunoprecipitation experiments were carried out as described previously (23). The immunopellets were electrophoresed in 8% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), blotted onto PVDF membranes, and probed with the following antibodies: anti-PSD-95, anti-SAP97, rabbit polyclonal anti-ZO-1 antibody (Zymed), rabbit polyclonal anti-Kv1.5 antibody, or mouse monoclonal anti-FLAG (Sigma). Crude membrane and cytosol fractions were prepared from mouse heart ventricles by differential centrifugation. With the use of a Polytron, ventricles were homogenized in Tris-EDTA buffer (pH 7.4) with 1 mM iodoacetamide and a cocktail of protease inhibitors. Debris was pelleted by centrifugation at 1,000 g and the supernatant was spun at 40,000 g for 1 h. The crude membrane pellet was washed once and dissolved in Triton lysis buffer.

Electrophysiological studies. Xenopus oocytes were harvested from mature Xenopus laevis females and dissociated in Ca2+-free Barth's solution containing 1 mg/ml type II collagenase (Worthington). Isolated follicle-free stage 5 and 6 oocytes were selected and injected with different amounts of cRNAs. For coexpression experiments, Kv1.5 cRNA was mixed with either SAP97 or ZO-1 cRNAs before injections. The total volume of the cRNAs injected was constant (46 nl) in each group to minimize the variation resulting from on-site leakage. The injected oocytes were maintained at 18-19°C in Barth's solution. The Barth's solution, supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml), contained (in mM) 88 NaCl, 1.0 KCl, 2.4 NaHCO3, 0.3 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 15 HEPES, and 5 sodium pyruvate, pH 7.6. Kv1.5-encoded currents were measured with a two-microelectrode voltage clamp using a GeneClamp amplifier (model 500, Axon Instruments) controlled by pCLAMP software (version 6.0.4, Axon Instruments) through a Digidata interface (model 1200, Axon Instruments). The bath solution contained (in mM) 96 NaCl, 2 KCl, 0.5 CaCl2, 0.5 MgCl2, and 10 HEPES, pH 7.5. Currents were elicited by a series of depolarization pulses from the holding potential of -70 to +60 mV with an increment of 10 mV for 200 ms, followed by a repolarization to -40 mV to elicit the tail currents. A P/4 leakage substraction protocol was used to minimize the leakage and capacitative currents. Data were expressed as means ± SD. Student's t-test and analysis of variance were used to evaluate the statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Colocalization of SAP97 and Kv1.5 in cardiac myocytes. The involvement of PSD-95 and other PDZ domain-containing proteins in the assembly of macromolecular signaling complexes in neurons has been clearly established. By contrast, very little is known about the formation, molecular composition, and targeting of signaling complexes containing K+ channels in cardiac tissue. SAP97, a PDZ domain-containing protein abundantly expressed in the heart, was shown to interact with Shaker-like K+ channels in heterologous systems. We used immunocytochemistry to study the membrane distribution of SAP97 and Kv1.5 in cardiac myocytes using anti-SAP97, anti-PSD-95, and anti-Kv1.5 antibodies. Control experiments using preimmune serum, rabbit IgG, isotype-specific mouse IgG, or secondary antibodies did not show significant staining under the experimental conditions used. Experiments using affinity-purified anti-SAP97 antibody revealed that SAP97 is expressed at both the intercalated disks and the lateral membranes (Fig. 1A). A similar distribution was observed using the monoclonal anti-PSD-95 antibody (Fig. 1K), although the background staining with this antibody was higher. The distribution pattern of SAP97 differed from that of ZO-1, another PDZ domain-containing protein expressed in the heart: ZO-1 localized primarily to the intercalated disks, with no staining detectable in lateral membranes (not shown). More importantly, the staining with anti-Kv1.5 antibody revealed that the membrane distribution of this channel was similar to that of SAP97: the immunoreactive Kv1.5 polypeptide was detectable both at the intercalated disks and the lateral membranes (Fig. 1D). To further prove that the distribution of Kv1.5 and SAP97 includes both the intercalated disks and the lateral membrane, we costained cardiac myocytes with anti-N-cadherin and either anti-Kv1.5 or anti-SAP97 antibodies. The results showed that, whereas the N-cadherin antibody stained only the intercalated disks (Fig. 1, B, E, and H), SAP97 and Kv1.5 immunoreactive polypeptides were detected both in the intercalated disks and lateral membranes (Fig. 1, A, C, D, and F). Interestingly, Z-series confocal microscopy revealed a patchy distribution of Kv1.5 on the cell surface that clearly differs from that of N-cadherin (Fig. 1, G-I). To further prove the colocalization of SAP97 and Kv1.5, we performed costaining experiments using rabbit polyclonal anti-Kv1.5 and mouse monoclonal anti PSD-95 antibodies. The results (Fig. 1, J-L) confirmed the colocalization of Kv1.5 and SAP97 in cardiac myocytes. Although anti-PSD95 cross-reacts with additional PDZ domain-containing proteins, we had to use this antibody because the monoclonal anti-Kv1.5 antibody failed to detect Kv1.5 in cardiac myocytes. Taken together, these experiments revealed codistribution of Kv1.5 and SAP 97 in the lateral membranes and the intercalated disks.


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  		Fig. 1.   Confocal images of FLAG epitope-tagged Kv1.5 (FL-Kv1.5) and SAP97 in adult rat cardiac myocytes. Cells were reacted with rabbit anti-SAP97 (A), rabbit anti Kv1.5 (D, G, and J), mouse anti-postsynaptic density-95 (PSD-95) (K), and mouse anti-N-cadherin (B, E, and H) antibodies. C, F, I, and L are superimpositions of the images in A and B, D and E, G and H, and J and K, respectively. The mouse anti-PSD-95 antibody may recognize several PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins, including PSD-95, SAP97, and chapsyn/PSD-93.

Biochemical and immunocytochemical evidence for interactions of FL-Kv1.5 with SAP97. We then investigated whether SAP97 and Kv1.5 interact in vivo. We were unable to consistently detect this interaction in coimmunoprecipitation experiments from heart extracts, presumably because of the low level of expression of Kv1.5. Thus we decided to examine the Kv1.5-SAP97 interaction in overexpression experiments in COS-7 cells, using SAP97 and NH2-terminal FL-Kv1.5 cDNAs subcloned into pCDNA3. Initially, the cells were transfected with SAP97 cDNA and the cell lysates were precipitated with anti-PSD-95 family antibody (anti-PSD-95). To determine the specificity of this antibody in COS-7 cells, the immunoprecipitates were size separated and immunoblotted with either anti-PSD-95 or an affinity-purified rabbit polyclonal anti-SAP97 antibody. Anti-PSD-95 antibody reacted with two ~120-kDa polypeptides in transfected COS-7 cells (Fig. 2A, lane 2), but it did not react with the ZO-1 polypeptides (Fig. 2A, lane 3). Importantly, affinity-purified anti-SAP97 antibody reacted with the same two 120-kDa polypeptides (Fig. 2A, lane 5), confirming the observation of Kim et al. (16) that SAP97 cDNA transfected into COS-7 cells codes for polypeptides with an apparent molecular mass of 120 kDa. The second band is likely due to posttranslational modifications of SAP97. Western blot analysis of cardiac cell lysates revealed that anti-PSD-95 antibody also reacted with two polypeptides with apparent molecular mass of ~120 kDa, identical to those of SAP97 expressed in COS-7 cells (Fig. 2B, lanes 1-3). Differential centrifugation of heart extracts revealed that most of the SAP97 polypeptides segregated to the crude membrane fraction (Fig. 2C, lane 2) rather than the cytosol (Fig. 1C, lane 1).


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  		Fig. 2.   The expression of SAP97 in transfected COS-7 cells and mouse heart detected by PSD-95 antibody. A: COS-7 cells were transfected with either vector alone (pCDNA3; lanes 1 and 4), SAP97 (lanes 2 and 5) or with ZO-1 (lanes 3 and 6) cDNAs. Cell lysates were reacted with anti-PSD-95 antibody for immunoprecipitation. The precipitates were immunoblotted with either anti-PSD-95 antibody (left) or anti-SAP97 antibody (right). B: crude cardiac myocyte lysates [20 µg (lane 1), 40 µg (lane 2), and 80 µg (lane 3)] were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-PSD-95 antibody. C: Western blot analysis of SAP97 in cytosol and membrane fractions from mouse heart. The fractions were prepared by differential centrifugation.

We then tested whether SAP97 interacts with FL-Kv1.5 in cotransfection experiments of COS-7 cells. The cell lysates were first reacted with anti-Kv1.5 antibody. The immunoprecipitates were then size separated using SDS-PAGE and immunoblotted with anti-PSD-95, anti-Kv1.5, and anti-FLAG antibodies. The results (Fig. 3A) showed that anti-Kv1.5 antibody could immunoprecipitate a polypeptide with an apparent molecular mass of 70 kDa that reacted with both anti-Kv1.5 and anti-FLAG antibodies in immunoblot assays (Fig. 3A, middle and bottom, lanes 3 and 4). The mobility of the Kv1.5 polypeptide expressed in COS-7 cells is faster than that expressed in the heart (or in GH3 cells), which most likely indicates different posttranslational modifications. Indeed, immunocytochemical studies in COS-7 cells (see below) indicated that most of the Kv1.5 polypeptides remained in the endoplasmic reticulum (ER) and therefore could not complete the biosynthetic pathway that occurs in either GH3 cells or the heart. Importantly, anti-Kv1.5 antibody coprecipitated two polypeptides with an apparent molecular mass of 120 kDa that reacted with anti-PSD-95 antibody in immunoblot assays (Fig. 3A, top, lane 4). In the absence of cotransfected SAP97, the anti-Kv1.5 antibody did not precipitate these polypeptides (Fig. 3A, lanes 1-3). Anti-FLAG antibody could also coprecipitate SAP97 and Kv1.5 complexes (see Fig. 4A). To confirm these interactions between Kv1.5 and SAP97, lysates from cotransfected COS-7 cells were first reacted with anti-PSD-95 antibody, and the precipitates were then size separated using SDS-PAGE and immunoblotted with anti-Kv1.5 and anti-FLAG antibodies. The results revealed that anti-PSD-95 could coprecipitate a 70-kDa polypeptide that reacted in immunoblot assays with anti-Kv1.5 and anti-FLAG antibodies only in the presence of cotransfected SAP97 (Fig. 3B, lane 4) and not in its absence (Fig. 3B, lanes 1 and 3). In contrast to the observed interaction of SAP97 with Kv1.5, the cotransfection of ZO-1 with either FL-Kv1.5 or FL-Kv1.1 into COS-7 cells did not result in the coimmunoprecipitation of ZO-1 polypeptides with anti-FLAG antibody (Fig. 3C, in lanes 1 and 2), although both ZO-1 and FL-Kv channels were detectable in the COS-7 lysates (Fig. 2C, lanes 3 and 4). Thus ZO-1 does not interact directly with either FL-Kv1.5 or FL-Kv1.1 polypeptides in this system.


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  		Fig. 3.   Coimmunoprecipitation assays from lysates of COS-7 cells cotransfected with SAP97 and FL-Kv1.5. A: COS-7 cells were transfected with vector alone (pcDNA3; lane 1), SAP97 (lane 2), FL-Kv1.5 (lane 3), or SAP97 and FL-Kv1.5 (lane 4) cDNAs. Half of the cell lysates were reacted with anti-Kv1.5 antibody (AK1.5). The proteins were immunoblotted with 0.5 µg/ml anti-PSD-95 (top) and with either anti-Kv1.5 (1:2,500 dilution) (middle) or anti-FLAG (10 µg/ml) antibodies (bottom). B: remaining half of the cell lysates were immunoprecipitated with anti-PSD-95 antibody and blotted with anti-Kv1.5 (top), anti-FLAG (middle), and anti-PSD-95 (bottom) antibodies. Molecular mass standards are shown in kilodaltons. C: COS-7 cells were transfected with either ZO-1 and FL-Kv1.1 (lanes 1 and 3) or ZO-1 and FL-Kv1.5 (lanes 2 and 4). Cell lysates were then precipitated with anti-FLAG antibody (lanes 1 and 2; IP with anti-FLAG). Lanes 3 and 4 were directly loaded with the transfected cell lysates (Lysates). The membrane was then blotted with anti-ZO-1 (1:2,000 dilution) (top) and anti-FLAG (bottom) antibodies.



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  		Fig. 4.   Coimmunoprecipitation assays from lysates of COS-7 cells cotransfected with SAP97 and either FL-Kv1.5 or FLAG epitope-tagged Kv1.5 A-A-A (FL-Kv1.5-A). A: COS-7 cells were transfected with vector alone (pCDNA3; lane 1), FL-Kv1.5 (lane 2), FL-Kv1.5-A (lane 3), FL-Kv1.5 and SAP97 (lane 4), FL-Kv1.5-A and SAP97 (lane 5), or SAP97 alone (lane 6). Half of the cell lysates were reacted with anti-FLAG antibody and the proteins were immunoblotted with 0.5 µg/ml anti-PSD-95 (top) or 10 µg/ml anti-FLAG (bottom) antibodies. B: the remaining half of the cell lysates was immunoprecipitated with anti-PSD-95 antibody and blotted with anti-FLAG (top) or anti-PSD-95 (bottom) antibodies. Molecular mass standards are shown in kilodaltons.

The association of PSD-95 with Shaker-like channels or NMDA receptors occurs through the interaction of the first two PDZ domains of PSD-95 with a short motif (tT/S-X-V) located at the COOH-terminal end of the channel or receptor proteins. Thus, to further characterize the specificity of the interaction of SAP97 with Kv1.5, we mutated the three COOH-terminal amino acid residues of Kv1.5 (T-D-L) to A-A-A (FL-KV1.5-A). Cotransfection experiments of FL-Kv1.5 or FL-Kv1.5-A with SAP97 in COS-7 cells showed that anti-FLAG antibody was able to coprecipitate SAP97 when cotransfected with FL-Kv1.5 (Fig. 4A, top, lane 4) but not with FL-Kv1.5-A (Fig. 4A, top, lane 5). Similarly, anti PSD-95 antibody was able to coprecipitate FL-Kv1.5 (Fig. 4B, top, lane 4) but not FL-Kv1.5-A (Fig. 4B, top, lane 5) with SAP97 in cotransfection experiments in COS-7 cells (Fig. 4B). Collectively, these results indicate that the T-D-L sequence located at the COOH terminus of Kv1.5 is critical for its interaction with SAP97.

We then used immunocytochemistry to further describe these complexes. The results show that the transient transfection of FL-Kv1.5 cDNA into COS-7 cells resulted in a diffuse intracellular reticular staining, a pattern characteristic of proteins located in the endoplasmic reticulum, with minimal linear accumulation on the cell membrane (Fig. 5A) (10). Only 20.4 ± 1.0% (n = 3) of the cells exhibited membrane staining. Indeed, costaining with anti-BiP antibody confirmed that most of the Kv1.5 polypeptides colocalized with this ER resident protein (Fig. 5, B and C), whose level of expression was higher in transfected than in nontransfected cells (not shown). The transfection of SAP97 cDNA alone revealed similar codistribution of SAP97 with BiP (Fig. 5, D-F). Untransfected cells showed low background fluorescence. The coexpression of Kv1.5 and SAP97 resulted in only a slight increase in the relative amount of cells showing membrane staining (29.3% ± 3.1, n = 3; P = not significant) but did not change the ER localization of the bulk of Kv1.5 and SAP97 proteins (Fig. 5, G-I). These results are similar to the observations of Tiffany et al. (32), which showed that the interactions of SAP97 and Kv1.2 occurred in the ER and attenuated the membrane expression of Kv1.2 and other Kv1 polypeptides. However, in contrast with Kim et al. (18), we observed a plaquelike distribution of SAP97 even in the absence of cotransfected Kv1.5 (Fig. 5E), indicating that Kv1.5 is not essential for the formation of these plaques. Our observations, and those of Tiffany et al. (32), suggest that SAP97 associates with Kv1.5 in the ER and does not promote significant additional trafficking of Kv1.5 to the membrane in COS-7 cells.


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  		Fig. 5.   Confocal images of FL-Kv1.5 and SAP97 in transfected COS-7 cells. COS-7 cells were transfected with FL-Kv1.5 (A-C), SAP97 (D-F), or both (G-I) cDNAs. Three days after transfection, the cells were reacted with rabbit anti-Kv1.5 (A and H), mouse anti-PSD-95 (E and G), and mouse anti-BiP (B and D) antibodies. C, F, and I are superimpositions of the images in A and B, D and E, and G and H, respectively.

Coexpression of SAP97 and FL-Kv1.5 enhances Kv1.5-encoded currents in Xenopus oocytes. We next investigated the functional effects of the interaction of SAP97 and Kv1.5. Transient transfection experiments in COS-7 cells are not adequate for this purpose because of the impossibility of accurately determining the SAP97/Kv1.5 expression ratio in each cell. Also, most of the SAP97 and Kv1.5 polypeptides are retained in the ER and thus are not suitable for electrophysiological studies. Therefore, to assess the functional effects of the interaction of SAP97 and Kv1.5, we carried out coinjection experiments of FL-Kv1.5 with either SAP97 or ZO-1 cRNAs into Xenopus oocytes. To ensure the validity of the results, we first measured the amplitude of the Kv1.5-encoded outward currents in oocytes injected with 4 (not shown), 8, and 16 fmol of FL-Kv1.5 cRNAs. As shown in Fig. 6A, the linearity of the current was maintained fairly well at different voltages with the amount of cRNA tested. Based on these results, 8 fmol of FL-Kv1.5 cRNA was used in coinjection experiments. Oocytes injected with FL-Kv1.5 cRNA expressed rapidly activating and slowly inactivating outward potassium current (Fig. 6B). Coinjection of SAP97 with FL-Kv1.5 resulted in a statistically significant twofold increase in the steady-state levels of the Kv1.5-encoded currents (Fig. 6, B and C). A similar increase was observed in two additional batches of oocytes coinjected, respectively, with 2:4 and 4:4 fmol of Kv1.5 to SAP97 cRNAs. However, in one batch of oocytes, which expressed exceptionally high levels of currents, the coinjection of SAP97 at 4:8 and 8:8 fmol failed to enhance the Kv1.5-encoded currents, although the 4:4 ratio did. Thus it appears that the enhancing effect of SAP97 is sensitive to the initial level of expression of Kv1.5: the lower the Kv1.5-encoded currents, the more pronounced the enhancing effect. In all coinjection groups, the maximum relative conductance (G/Gmax, Fig. 5D), voltage dependence, and kinetics of activation (data not shown) of the currents remained similar to those of the control group. In contrast with the observed effect of SAP97, the coinjection of ZO-1 cRNA caused either no change or even a slight decrease in the amount of Kv1.5-encoded currents (Fig. 6, B and C). These results, which are in agreement with the lack of interaction of ZO-1 with Kv1.5 in transfection experiments (see above), suggest that SAP97, but not ZO-1, is able to modulate the expression of Kv1.5.


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  		Fig. 6.   Augmentation of Kv1.5-encoded currents by coinjection of SAP97 cRNA into Xenopus oocytes. A: doubling the amount of the injected FL-Kv1.5 cRNA resulted in a twofold increase in the amplitude of the Kv1.5-encoded currents. Data are shown as means ± SD (n = 5 for the 8 fmol group and 3 for the 16 fmol group). B: macroscopic currents of oocytes injected with either FL-Kv1.5 (8 fmol), FL-Kv1.5 + SAP97 (8:8 fmol) or FL-Kv1.5 + ZO-1 (8:8 fmol) cRNAs. The currents were recorded by a two-electrode voltage-clamp technique, with voltage steps from -60 mV to +60 mV in 10-mV increments. The holding potential was -70 mV. C: a summary of the current levels at 20 mV in each group. Data are shown as means ± SD. *P < 0.05. n = 5-6. D: relative maximal conductance (G/Gmax) of the currents as a function of the membrane potential. The slope conductance was calculated assuming a reversal potential of -80 mV. No significant differences were found among the three groups (P > 0.05). Data were fitted by a Boltzmann function, with a slope factor and a half-conductance voltage of 11.6 and 0.8 mV in the Kv1.5 group, 11.4 and -2.0 mV in the SAP97 group, and 11.7 and -2.1 mV in the ZO-1 group (inset). Data are shown as means ± SD; n = 5-6.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The targeting of ion channels to discrete plasma membrane domains of cardiac myocytes is critical to cardiac cell excitation. PDZ domain-containing proteins have been shown to play an important role in the targeting of sodium channels, NMDA receptors, and voltage-gated potassium channels to discrete plasma membrane domains in neuronal cells (8, 29, 30), but very little is known about the function of this family of proteins in the organization of macromolecular signaling complexes in the heart. Recent studies (33) in cardiac myocytes revealed that the gap junction protein connexin43 associates with ZO-1 at the intercalated disks and that the overexpression of dominant negative mutants of ZO-1 abolishes the formation of recombinant gap junctions.

In this study, we have demonstrated that SAP97, a PDZ domain-containing protein abundantly expressed in the heart, localizes to the intercalated disks and the lateral membranes of adult cardiac myocytes. This plasma membrane distribution is identical to that of Kv1.5, which suggests that Kv1.5 and SAP97 may interact in cardiac tissue. However, we were unable to demonstrate this interaction in coimmunoprecipitation experiments from cardiac cell lysates because of the low level of expression of Kv1.5 in the heart. Thus we used transient transfection experiments in COS-7 cells to demonstrate the association of Kv1.5 with SAP97. Furthermore, we show that this interaction is specific, because a T-D-L to A-A-A mutation at the COOH-terminus of Kv1.5 abolished its association with SAP97. Although the tE-T-D-L COOH terminal sequence of Kv1.5 differs from the tS/T-X-V consensus motif defined by Sheng and others (17, 31), studies on the interaction of PSD-95 with the COOH-terminus of Kir2.3 (tE-S-A-I) demonstrated that its PDZ domains do accommodate a leucine residue at the COOH-terminus (7). Moreover, SAP97/hDlg has been shown to associate with adenomotous Polyposis coli, human papillomavirus E6, and adenovirus type 9 E4, allowing the SAP97 COOH-terminal consensus binding motif to be extended to tX-S/T-X-V/I/L (19, 22, 25).

Although our cotransfection experiments in COS-7 cells clearly demonstrated the specific association of SAP97 and Kv1.5, the immunocytochemical analysis of cotransfected COS-7 cells showed that the majority of SAP97 and Kv1.5 polypeptides were localized in the ER, with minimal accumulation on the cell membrane. This distribution, which resembles that reported (32) for other Kv1 channels in similar overexpression systems, differs from the plasma membrane localization of endogenous SAP97 and Kv1.5 in cardiac myocytes and probably reflects the inability of COS-7 cells to target high levels of exogenously expressed proteins to their proper intracellular destinations. Thus we used a different expression system to investigate the functional consequences of the interaction of Kv1.5 and SAP97. As shown in Fig. 6, we demonstrated that the coinjection of Kv1.5 with SAP97, but not with ZO-1, into Xenopus oocytes resulted in a significant increase of the steady-state levels of Kv1.5-encoded currents. Recently, Burke et al. (6) showed that the interaction between PSD-95 and Kv1.4 in oocytes leads to the immobilization of Kv1.4 channels in the plasma membrane. Thus the channel clusters formed in the presence of PSD-95 were stable in size, shape, and position (6). We hypothesize that the enhancement of the whole cell Kv1.5-encoded currents in oocytes by SAP97 may occur by a similar mechanism, involving the recruitment or retention of more functional channels (and/or channel-modulating proteins) to the membranes, rather than increasing the channel open probability or unitary conductance. Indeed, this model is supported by recent observations by Horio et al. (13), who showed that the coexpression of SAP97 and the Kir channel in oocytes enhanced the Kir-encoded current without changing the single channel conductance or open probability.

The level of expression and proper distribution of voltage-gated potassium channels are crucial for determining the shape and duration of the action potential and the refractoriness of cardiac myocytes (2, 9). PDZ domain-containing proteins have emerged as an important group of scaffolding proteins that interact with ion channels, receptors, and their modulators (8, 29). In this study, we have demonstrated that SAP97 interacts with Kv1.5 in heterologous expression systems and colocalizes with it in cardiac myocytes. Further studies are needed to elucidate the exact nature of these interactions and their functional significance as related to outward potassium currents expressed in the heart.


    ACKNOWLEDGEMENTS

We thank Momoyo Murata and Ming-Ying Pu for excellent technical support. We also thank M. Wyszynski and M. Sheng for the generous gift of anti-SAP97 antibody and the rat SAP97 cDNA, S. Ryeom and D. A. Goodenough for the ZO-1 cDNA and the rat anti-ZO-1 antibody, M. Seki for pcDNA3 with a FLAG epitope, and Y. Kim for the Marathon cDNA library.


    FOOTNOTES

* P. D. Buckett and J. Zhou contributed equally to this work.

G. Koren is a recipient of National Heart, Lung, and Blood Institute Grant HL-46005 and an Established Investigator Award from the American Heart Association. M. Murata is a recipient of a postdoctoral Fellowships Award from the Japan Heart Foundation.

Address for reprint requests and other correspondence: G. Koren, Cardiovascular Div., Brigham and Women's Hosp., 75 Francis St., Boston, MA 02115 (E-mail: koren{at}calvin.bwh.harvard.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 25 May 2001; accepted in final form 8 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(6):H2575-H2584
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


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