Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na<sup>+</sup>-K<sup>+</sup>-ATPase
MATERIALS AND METHODS
Construction of viral vectors.
For this study, we used WT canine PLM and the following PLM mutants: Ser63 to Ala (S63A), Ser68 to Ala (S68A), and Ser63/Ser68 to Ala double mutant (AA PLM). The Ser to Ala mutation rendered the respective site unphosphorylatable. The WT and mutant PLM was obtained by cleaving GFP from the PLM-GFP plasmid vector using standard molecular biology methods. They were then subcloned into the pShuttle CMV vector. Adenoviruses were generated with AdEasy Vector system according to the manufacturer's instructions (Stratagene). After amplification, the virus was purified on a CsCl gradient and stored at −80°C.
HeLa cell culture and expression of PLM mutants.
HeLa cells stably expressing the ouabain-resistant rat NKA-α1 (a generous gift from Dr. Joshua Berlin, University of Medicine and Dentistry of New Jersey–New Jersey Medical School; Ref. 18) were maintained in DMEM with 5% FBS, penicillin/streptomycin, and 1 μM ouabain. The medium was replaced every 3 days, and the cells were split via mild trypsinization when confluent. Twenty-four hours before experiments, cells were plated on a 25 × 25 coverslip with a total of 5 ml medium in a culture dish (2% FBS). PLM WT and phosphorylation-site mutants were expressed in these HeLa cells by exposure to the appropriate adenovirus at a multiplicity of infection of 10–50.
Confocal microscopy.
Perfusion chambers containing the cultured cells on the coverslips were mounted on the stage of an inverted microscope. Confocal images were measured with a ×40 oil immersion objective lens. For GFP measurements, excitation was at 488 nm (< 5%) and emission was recorded at 520 nm.
Western blot.
HeLa cells were lysed in ice-cold buffer containing 1% NP-40 and the following (in mmol/l): 150 NaCl, 10 Tris (pH 7.4), 2 EGTA, 50 NaF, 0.2 NaVO3, and protease inhibitors (Calbiochem). Lysates were size fractionated on 15% SDS-PAGE. Proteins were transferred to 0.20 μm nitrocellulose membranes and blocked in 5% nonfat dry milk in TBS-Tween for 2 h at room temperature. The blots were then incubated overnight at 4°C with custom-made primary antibodies (1:5,000 dilution) that detect total PLM (C2 antibody), PLM phosphorylated at Ser 68 (CP68), or PLM phosphorylated at Ser 63 (CP63; a kind gift from Dr. Randall Moorman, University of Virginia; Ref. 20).
PLM dephosphorylation was performed as previously described (3): 100 μg of cell lysate were dephosphorylated with 0.3 U PP2A and 0.3U PP1 (from Calbiochem) for 20 min at 37°C in reaction buffer containing 50 mM Tris·HCl, 1 mM dithiotreitol, and 1 mM MgCl2 (pH 7.5). The reaction was terminated by the addition of 1 μM microcystin-LR (a control reaction was also included that was preincubated with microcystin-LR for 10 min).
Intracellular [Na]i measurements in HeLa cells.
Cells were incubated with 10 μM sodium-binding benzofuran isophthalate (SBFI)-AM in the presence of Pluronic F-127 (0.05% wt/vol) for 90 min at room temperature. After the external dye was washed out, SBFI-AM was allowed to further deesterify for 20 min in normal Tyrode solution. Dual excitation measurements (at 340 and 380 nm; F340 and F380) were performed on selected area for a group of ∼20 cells (7). The F340-to-F380 ratio was calculated after background subtraction and converted to [Na]i by calibration at the end of each experiment (using divalent-free solutions with 0, 10, or 20 mM extracellular [Na]) in the presence of 10 μM gramicidin and 100 μM strophanthidin (7). All measurements were at room temperature.
Na efflux through the NKA.
NKA-mediated Na efflux was determined as the rate of pump-mediated [Na]i decline (7). Myocytes were Na loaded by inhibiting the NKA in a K-free solution containing the following (in mM): 145 NaCl, 2 EGTA, 10 HEPES, and 10 glucose (pH 7.4). [Na]i decline was measured upon pump reactivation in a solution containing the following (in mM): 130 TEA-Cl, 15 KCl, 1 MgCl2, 2 EGTA, 10 HEPES, and 10 glucose (pH 7.4). One micromolar of ouabain was used in most experiments to inhibit the endogenous human NKA. Since cell volume does not change with this protocol (8), [Na]i decline reflects the Na efflux. The rate of [Na]i decline (−d[Na]i/dt) was plotted vs. [Na]i and fitted with the following: Jpump = Vmax/{1 + (K0.5/[ Na]i){↑nH}}, where Vmax is the maximum Na extrusion rate of the pump, K0.5 is the [Na]i for the half-maximal activation of the pump, and nH is the Hill coefficient. Forskolin (20 μM) and phorbol 12,13-dibutyrate (PDBu; 300 nM) were used to activate PKA and PKC, respectively. They were applied in the K-free solution, 10–15 min before reactivating the pump. As a control, the −d[Na]i/dt vs. [Na]i curve is superimposable when two consecutive control runs are done in HeLa cells (similar to the case in myocytes, data not shown).
Statistical analysis.
Data are expressed as means ± SE. Statistical discriminations were performed with Student's t-test (paired when appropriate) with P < 0.05 considered significant.
Construction of viral vectors.
For this study, we used WT canine PLM and the following PLM mutants: Ser63 to Ala (S63A), Ser68 to Ala (S68A), and Ser63/Ser68 to Ala double mutant (AA PLM). The Ser to Ala mutation rendered the respective site unphosphorylatable. The WT and mutant PLM was obtained by cleaving GFP from the PLM-GFP plasmid vector using standard molecular biology methods. They were then subcloned into the pShuttle CMV vector. Adenoviruses were generated with AdEasy Vector system according to the manufacturer's instructions (Stratagene). After amplification, the virus was purified on a CsCl gradient and stored at −80°C.
HeLa cell culture and expression of PLM mutants.
HeLa cells stably expressing the ouabain-resistant rat NKA-α1 (a generous gift from Dr. Joshua Berlin, University of Medicine and Dentistry of New Jersey–New Jersey Medical School; Ref. 18) were maintained in DMEM with 5% FBS, penicillin/streptomycin, and 1 μM ouabain. The medium was replaced every 3 days, and the cells were split via mild trypsinization when confluent. Twenty-four hours before experiments, cells were plated on a 25 × 25 coverslip with a total of 5 ml medium in a culture dish (2% FBS). PLM WT and phosphorylation-site mutants were expressed in these HeLa cells by exposure to the appropriate adenovirus at a multiplicity of infection of 10–50.
Confocal microscopy.
Perfusion chambers containing the cultured cells on the coverslips were mounted on the stage of an inverted microscope. Confocal images were measured with a ×40 oil immersion objective lens. For GFP measurements, excitation was at 488 nm (< 5%) and emission was recorded at 520 nm.
Western blot.
HeLa cells were lysed in ice-cold buffer containing 1% NP-40 and the following (in mmol/l): 150 NaCl, 10 Tris (pH 7.4), 2 EGTA, 50 NaF, 0.2 NaVO3, and protease inhibitors (Calbiochem). Lysates were size fractionated on 15% SDS-PAGE. Proteins were transferred to 0.20 μm nitrocellulose membranes and blocked in 5% nonfat dry milk in TBS-Tween for 2 h at room temperature. The blots were then incubated overnight at 4°C with custom-made primary antibodies (1:5,000 dilution) that detect total PLM (C2 antibody), PLM phosphorylated at Ser 68 (CP68), or PLM phosphorylated at Ser 63 (CP63; a kind gift from Dr. Randall Moorman, University of Virginia; Ref. 20).
PLM dephosphorylation was performed as previously described (3): 100 μg of cell lysate were dephosphorylated with 0.3 U PP2A and 0.3U PP1 (from Calbiochem) for 20 min at 37°C in reaction buffer containing 50 mM Tris·HCl, 1 mM dithiotreitol, and 1 mM MgCl2 (pH 7.5). The reaction was terminated by the addition of 1 μM microcystin-LR (a control reaction was also included that was preincubated with microcystin-LR for 10 min).
Intracellular [Na]i measurements in HeLa cells.
Cells were incubated with 10 μM sodium-binding benzofuran isophthalate (SBFI)-AM in the presence of Pluronic F-127 (0.05% wt/vol) for 90 min at room temperature. After the external dye was washed out, SBFI-AM was allowed to further deesterify for 20 min in normal Tyrode solution. Dual excitation measurements (at 340 and 380 nm; F340 and F380) were performed on selected area for a group of ∼20 cells (7). The F340-to-F380 ratio was calculated after background subtraction and converted to [Na]i by calibration at the end of each experiment (using divalent-free solutions with 0, 10, or 20 mM extracellular [Na]) in the presence of 10 μM gramicidin and 100 μM strophanthidin (7). All measurements were at room temperature.
Na efflux through the NKA.
NKA-mediated Na efflux was determined as the rate of pump-mediated [Na]i decline (7). Myocytes were Na loaded by inhibiting the NKA in a K-free solution containing the following (in mM): 145 NaCl, 2 EGTA, 10 HEPES, and 10 glucose (pH 7.4). [Na]i decline was measured upon pump reactivation in a solution containing the following (in mM): 130 TEA-Cl, 15 KCl, 1 MgCl2, 2 EGTA, 10 HEPES, and 10 glucose (pH 7.4). One micromolar of ouabain was used in most experiments to inhibit the endogenous human NKA. Since cell volume does not change with this protocol (8), [Na]i decline reflects the Na efflux. The rate of [Na]i decline (−d[Na]i/dt) was plotted vs. [Na]i and fitted with the following: Jpump = Vmax/{1 + (K0.5/[ Na]i){↑nH}}, where Vmax is the maximum Na extrusion rate of the pump, K0.5 is the [Na]i for the half-maximal activation of the pump, and nH is the Hill coefficient. Forskolin (20 μM) and phorbol 12,13-dibutyrate (PDBu; 300 nM) were used to activate PKA and PKC, respectively. They were applied in the K-free solution, 10–15 min before reactivating the pump. As a control, the −d[Na]i/dt vs. [Na]i curve is superimposable when two consecutive control runs are done in HeLa cells (similar to the case in myocytes, data not shown).
Statistical analysis.
Data are expressed as means ± SE. Statistical discriminations were performed with Student's t-test (paired when appropriate) with P < 0.05 considered significant.
Construction of viral vectors.
For this study, we used WT canine PLM and the following PLM mutants: Ser63 to Ala (S63A), Ser68 to Ala (S68A), and Ser63/Ser68 to Ala double mutant (AA PLM). The Ser to Ala mutation rendered the respective site unphosphorylatable. The WT and mutant PLM was obtained by cleaving GFP from the PLM-GFP plasmid vector using standard molecular biology methods. They were then subcloned into the pShuttle CMV vector. Adenoviruses were generated with AdEasy Vector system according to the manufacturer's instructions (Stratagene). After amplification, the virus was purified on a CsCl gradient and stored at −80°C.
HeLa cell culture and expression of PLM mutants.
HeLa cells stably expressing the ouabain-resistant rat NKA-α1 (a generous gift from Dr. Joshua Berlin, University of Medicine and Dentistry of New Jersey–New Jersey Medical School; Ref. 18) were maintained in DMEM with 5% FBS, penicillin/streptomycin, and 1 μM ouabain. The medium was replaced every 3 days, and the cells were split via mild trypsinization when confluent. Twenty-four hours before experiments, cells were plated on a 25 × 25 coverslip with a total of 5 ml medium in a culture dish (2% FBS). PLM WT and phosphorylation-site mutants were expressed in these HeLa cells by exposure to the appropriate adenovirus at a multiplicity of infection of 10–50.
Confocal microscopy.
Perfusion chambers containing the cultured cells on the coverslips were mounted on the stage of an inverted microscope. Confocal images were measured with a ×40 oil immersion objective lens. For GFP measurements, excitation was at 488 nm (< 5%) and emission was recorded at 520 nm.
Western blot.
HeLa cells were lysed in ice-cold buffer containing 1% NP-40 and the following (in mmol/l): 150 NaCl, 10 Tris (pH 7.4), 2 EGTA, 50 NaF, 0.2 NaVO3, and protease inhibitors (Calbiochem). Lysates were size fractionated on 15% SDS-PAGE. Proteins were transferred to 0.20 μm nitrocellulose membranes and blocked in 5% nonfat dry milk in TBS-Tween for 2 h at room temperature. The blots were then incubated overnight at 4°C with custom-made primary antibodies (1:5,000 dilution) that detect total PLM (C2 antibody), PLM phosphorylated at Ser 68 (CP68), or PLM phosphorylated at Ser 63 (CP63; a kind gift from Dr. Randall Moorman, University of Virginia; Ref. 20).
PLM dephosphorylation was performed as previously described (3): 100 μg of cell lysate were dephosphorylated with 0.3 U PP2A and 0.3U PP1 (from Calbiochem) for 20 min at 37°C in reaction buffer containing 50 mM Tris·HCl, 1 mM dithiotreitol, and 1 mM MgCl2 (pH 7.5). The reaction was terminated by the addition of 1 μM microcystin-LR (a control reaction was also included that was preincubated with microcystin-LR for 10 min).
Intracellular [Na]i measurements in HeLa cells.
Cells were incubated with 10 μM sodium-binding benzofuran isophthalate (SBFI)-AM in the presence of Pluronic F-127 (0.05% wt/vol) for 90 min at room temperature. After the external dye was washed out, SBFI-AM was allowed to further deesterify for 20 min in normal Tyrode solution. Dual excitation measurements (at 340 and 380 nm; F340 and F380) were performed on selected area for a group of ∼20 cells (7). The F340-to-F380 ratio was calculated after background subtraction and converted to [Na]i by calibration at the end of each experiment (using divalent-free solutions with 0, 10, or 20 mM extracellular [Na]) in the presence of 10 μM gramicidin and 100 μM strophanthidin (7). All measurements were at room temperature.
Na efflux through the NKA.
NKA-mediated Na efflux was determined as the rate of pump-mediated [Na]i decline (7). Myocytes were Na loaded by inhibiting the NKA in a K-free solution containing the following (in mM): 145 NaCl, 2 EGTA, 10 HEPES, and 10 glucose (pH 7.4). [Na]i decline was measured upon pump reactivation in a solution containing the following (in mM): 130 TEA-Cl, 15 KCl, 1 MgCl2, 2 EGTA, 10 HEPES, and 10 glucose (pH 7.4). One micromolar of ouabain was used in most experiments to inhibit the endogenous human NKA. Since cell volume does not change with this protocol (8), [Na]i decline reflects the Na efflux. The rate of [Na]i decline (−d[Na]i/dt) was plotted vs. [Na]i and fitted with the following: Jpump = Vmax/{1 + (K0.5/[ Na]i){↑nH}}, where Vmax is the maximum Na extrusion rate of the pump, K0.5 is the [Na]i for the half-maximal activation of the pump, and nH is the Hill coefficient. Forskolin (20 μM) and phorbol 12,13-dibutyrate (PDBu; 300 nM) were used to activate PKA and PKC, respectively. They were applied in the K-free solution, 10–15 min before reactivating the pump. As a control, the −d[Na]i/dt vs. [Na]i curve is superimposable when two consecutive control runs are done in HeLa cells (similar to the case in myocytes, data not shown).
Statistical analysis.
Data are expressed as means ± SE. Statistical discriminations were performed with Student's t-test (paired when appropriate) with P < 0.05 considered significant.
RESULTS
Measurement of NKA-mediated Na efflux in HeLa cells.
HeLa cells were loaded with SBFI and fluorescence was measured as described above. Fluorescence signals from a selected area of ∼20 cells were recorded, and [Na]i was calculated. Figure 1A shows a representative experiment where two [Na]i loading/decline cycles were monitored under identical conditions to verify that consecutive control runs result in identical results. In all experiments, 1 μM ouabain was used to block endogenous human NKA. A three-step calibration of the F340-to-F380 ratio was done at the end of the experiment. Similar to what we found in myocytes, consecutive control runs produce similar results (mean data not shown).
Measurement of Na-K-ATPase (NKA)-mediated Na efflux in a group of HeLa cells. A: NKA was first blocked (K-free, 145 mM Na external solution) causing intracellular Na concentration ([Na]i) loading of the cells and then NKA was reactivated in 15 mM K, Na-free solution. The protocol was then repeated under identical conditions to verify that consecutive control runs produce similar results; 1 μM ouabain (Oua) was used throughout the experiment to inhibit the endogenous human NKA. A: 3-step calibration of the F340-to-F380 ratio was done at the end. B: contribution of the different efflux pathways to Na extrusion in HeLa cells. Na efflux measured in the presence of 1 μM ouabain (NKA + leak) is mediated by the expressed rat NKA-α1 and NKA-independent pathways (leak component). The leak component (leak; measured in the presence of 10 mM ouabain) is small and not affected by application of either forskolin or phorbol 12,13-dibutyrate (PDBu) in HeLa cells. The difference between these 2 curves is the rate of Na efflux via the exogenous rat NKA-α1 (NKA). −d[Na]i/dt is the rate of [Na]i decline. The maximum Na extrusion rate of the pump (Vmax) and the [Na]i for the half-maximal activation of the pump (K0.5) shown at right represent the values derived by fitting the curves at left with a Hill equation.
In parallel experiments, we measured the rate of Na pump-independent Na efflux (leak) as the rate of [Na]i decline in the presence of 10 mM ouabain, which blocks both the exogenous and endogenous NKA. The leak Vmax averages 2.0 ± 0.3 mM/min and accounts for <25% of total Na efflux (Fig. 1B). Furthermore, the leak component was not affected by application of forskolin or PDBu in HeLa cells (not shown). The leak component was subtracted from total Na efflux to calculate the rate of NKA-mediated Na efflux (Fig. 1B).
Effect of PLM expression on [Na]i homeostasis in HeLa cells.
The WT PLM and mutated PLM used in this study do not contain any fluorescent marker to avoid potential contamination of the SBFI fluorescence signal. In parallel control experiments, the adenoviral transduction efficiency of PLM-GFP is >90% as indicated in Fig. 2A. Western blot experiments showed a comparable level of heterologous PLM expression, whether PLM was expressed alone or in a vector that also contained GFP (Fig. 2B). The noninfected (nonfluorescent) cells are displayed in a different layer and out of focus and are probably dead (Fig. 2A). Thus, during SBFI measurements, we selected areas that were devoid of out of focus and dead cells.
Effects of adenovirus-mediated phospholemman (PLM) expression in HeLa cells. A: image of HeLa cells taken under transmission light (left) and confocal GFP fluorescence from cells in the same area (right). Images were taken 24 h after adenovirus PLM-GFP transduction. Arrows indicate nonfluorescent cells. B: Western blot showing the PLM level (using the C2 antibody) in HeLA cells infected with GFP, PLM-GFP, and PLM. Representative of 3 experiments (Ctl, control). C: effect of acute PLM expression on resting [Na]i in HeLa cells. Mean data of resting [Na]i measured at the beginning of sodium-binding benzofuran isophthalate fluorescence experiments. The expression of both wild-type (WT) and AA PLM significantly increased resting [Na]i. D: [Na]i dependence of total Na efflux rate in HeLa cells with (red) or without (black) WT PLM. Dashed lines indicate the corresponding resting [Na]i (5.2 ± 0.5 mM in noninfected cells and 8.3 ± 1.0 mM in cells infected with WT PLM). **P < 0.01.
[ Na]i and NKA-mediated Na efflux were measured 24 h after adenoviral infection of WT or mutant PLM. [Na]i was 5.2 ± 0.5 mM (n = 12) in noninfected cells and significantly higher in HeLa cells expressing either WT or AA PLM (8.3 ± 1.0, n = 9, and 8.4 ± 0.7 mM, n = 10, respectively; Fig. 2C). The higher [Na]i in the presence of PLM is consistent with a decrease in Na efflux via NKA upon PLM association, as shown in myocyte studies (6). Indeed, the total Na efflux curve is right shifted upon WT PLM expression (K0.5: 10.7 ± 0.7 vs. 16.7 ± 0.8 mM), without any significant change in the Vmax (7.9 ± 0.7 vs. 8.2 ± 0.6 mM). Thus acute PLM expression decreases NKA activity and elevates [Na]i in HeLa cells. Moreover, the total efflux rate in HeLa cells without PLM at their [Na]i of 5.2 mM is similar to that in the presence of WT PLM at 8.3 mM [Na]i, the value measured under these conditions (1.4 vs. 1.6 mM/min; Fig. 2D). Because steady-state Na efflux is equal to Na influx, this indicates that PLM expression does not alter Na influx rate.
Effect of PKA activation on the NKA-mediated Na efflux in HeLa cells; role of PLM phosphorylation sites.
PKA was activated by 20 μM forskolin, and we determined the effect on NKA function by measuring the [Na]i dependence of NKA-mediated Na efflux (Fig. 3 and Table 1). In the absence of PLM, forskolin did not significantly affect either Vmax (5.8 ± 0.7 in control vs. 4.8 ± 0.7 mM/min with forskolin; n = 12), the apparent affinity of the pump for internal Na (K0.5 = 11.7 ± 0.9 in control and 11.2 ± 1.4 mM with forskolin) or nH (2.4 ± 0.1 vs. 2.6 ± 0.1). Figure 3A shows the Na-activation curve normalized to Vmax to emphasize the lack of effect on K0.5. Heterologous expression of WT PLM significantly inhibited NKA by decreasing the apparent Na affinity (K0.5 increased from 11.7 ± 0.9 in cells without PLM to 18.5 ± 0.7 mM with WT PLM; n = 11; Fig. 3B). Forskolin largely relieved NKA inhibition (K0.5 significantly decreased to 13.8 ± 1.2 mM; n = 11). The NKA Vmax was similar in HeLa cells with and without PLM (5.6 ± 0.6 vs. 5.8 ± 0.7 mM/min) and was not affected by PKA activation (5.7 ± 0.8 mM/min).
Effect of PKA activation on NKA function in HeLa cells: role of PLM phosphorylation sites. PKA was activated with 20 μM forskolin. A: effect of forskolin (FSK) on NKA-mediated Na extrusion in HeLa cells with no PLM. B: effect of forskolin in cells infected with WT PLM. The Na extrusion curve was normalized with respect to Vmax to emphasize the effect on K0.5. C: forskolin has no significant effect on NKA Vmax, independent of the presence of WT or nonphosphorylatable AA PLM. D: forskolin significantly increases the NKA apparent affinity for internal Na in HeLa cells expressing WT PLM but not in cells with no PLM or with AA PLM. **P < 0.01.
Table 1.
[Na]i dependence of NKA-mediated Na extrusion in Hela cells with WT PLM and mutants, and PKA/PKC-dependent effects
| No PLM | WT PLM | AA PLM | Ser63A | Ser68A | |
|---|---|---|---|---|---|
| Control condition | |||||
| Vmax, mM/min | 5.6 ± 0.4 | 6.5 ± 0.5 | 6.4 ± 0.5 | 6.3 ± 1.0 | 5.6 ± 0.8 |
| K0.5, mM [Na]i | 11.9 ± 0.6 | 17.5 ± 0.7* | 16.9 ± 0.6* | 17.1 ± 2.1* | 18.6 ± 1.6* |
| nH | 2.5 ± 0.1 | 2.7 ± 0.1 | 2.7 ± 0.1 | 2.6 ± 0.1 | 2.5 ± 0.1 |
| 10 μM forskolin | |||||
| Vmax, mM/min | 4.8 ± 0.7 | 5.7 ± 0.8 | 6.1 ± 0.5 | ||
| K0.5, mM [Na]i | 11.2 ± 1.4 | 13.8 ± 1.2† | 16.0 ± 0.8 | ||
| nH | 2.6 ± 0.1 | 2.8 ± 0.2 | 2.6 ± 0.1 | ||
| 300 nM PDBu | |||||
| Vmax, mM/min | 5.5 ± 1.0 | 6.2 ± 0.9 | 5.2 ± 1.0 | 5.3 ± 1.0 | 5.0 ± 0.5 |
| K0.5, mM [Na]i | 12.2 ± 0.8 | 13.4 ± 1.1† | 17.7 ± 1.6 | 14.0 ± 1.7† | 13.9 ± 1.3† |
| nH | 2.5 ± 0.1 | 2.6 ± 0.1 | 2.7 ± 0.1 | 2.5 ± 0.1 | 2.5 ± 0.1 |
Values are means ± SE. Data for untreated cells (control condition) were pulled from all experiments. Thus the values reported for control condition in the table might differ slightly from those reported in the text for paired comparisons with Na-K-ATPase (NKA) function parameters in cells treated with forskolin or phorbol 12,13-dibutyrate (PBDu). [Na]i, intracellular Na concentration; WT, wild type. The maximum Na extrusion rate of the pump (Vmax), the [Na]i for half-maximal activation of the pump (K0.5), and the Hill coefficient (nH) in first row are means of control conditions in forskolin and PDBu experiment.
Expression of nonphosphorylatable AA PLM mutant (Ser68 and Ser63 to alanine double mutation) also decreased the apparent Na affinity of NKA (vs. NKA only) similar to WT PLM (K0.5 = 16.3 ± 0.6 mM; n = 10; Fig. 3C). However, forskolin did not relieve NKA inhibition in HeLa cells expressing AA PLM (K0.5 = 16.0 ± 0.8 mM). In addition, PKA activation did not change the Vmax or nH of NKA after AA PLM overexpression in HeLa cells (Vmax: 6.9 ± 0.4 in control conditions vs. 6.1 ± 0.5 mM/min with forskolin; nH: 2.8 ± 0.2 vs. 2.6 ± 0.1).
Overall, the data in HeLa cells are consistent with those in cardiac myocytes (2, 6), indicating that PLM inhibits NKA activity by decreasing its [Na]i affinity and that this inhibitory effect is relieved by PKA phosphorylation of PLM phosphorylation sites. The nonphosphorylatable PLM works like a tonic inhibitor of NKA that cannot be relieved.
Effect of PKC activation on the NKA-mediated Na efflux in HeLa cells; role of PLM phosphorylation sites.
We used the same method to determine the effect of PKC activation on NKA function in HeLa cells and the role of the different PKC phosphorylation sites on PLM in mediating this effect (Fig. 4). PKC was activated with 300 nM PDBu. In the absence of PLM, PDBu failed to induce any changes in K0.5, Vmax, or nH (Fig. 4A and Table 1). This implies that PKC does not have a direct effect on NKA transport function under our experimental conditions here. PDBu increased the NKA Na affinity in cells expressing WT PLM (K0.5 decreased from 16.3 ± 1.4 mM in control condition to 13.4 ± 1.1 mM with PDBu; n = 9; Fig. 4B) but not in cells expressing AA PLM (K0.5 = 17.6 ± 1.3 vs. 17.7 ± 1.6 mM; n = 7; Fig. 4C). PDBu had no effect on Vmax and nH. Thus PLM phosphorylation is required for the PKC effects on NKA activity.
Effect of PKC activation on NKA function in HeLa cells; role of PLM phosphorylation sites. PKC was activated with 300 nM PDBu. A: PDBu did not alter NKA-mediated Na extrusion in HeLa cells with no PLM. B: PDBu stimulates NKA in cells infected with WT PLM. Na extrusion curve was normalized with respect to Vmax to emphasize the effect on K0.5. C: PDBu does not affect NKA Vmax, independent of the presence of WT or AA PLM. D: PDBu significantly increases the NKA apparent affinity for internal Na in HeLa cells expressing WT PLM but not in cells with no PLM or with AA PLM. **P < 0.05.
WT PLM expressed in HeLa cells is readily phosphorylated by PKC at both Ser63 and Ser68 (similar to the situation in cardiac myocytes), as demonstrated using phosphorylation-dependent antibodies (Fig. 5A). There was also a variable degree of baseline phosphorylation at both S63 and S68 (see untreated column in Fig. 5B). We investigated the separate effect of phosphorylation at each site on NKA function by mutating the other site to alanine. PDBu still induced phosphorylation of the S63A PLM mutant at Ser68 and of the S68A mutant at Ser63, as indicated by a 2- to 2.5-fold increase in the signal of the respective phospho-specific antibody (Fig. 5C; note that both alanine mutants display relatively high baseline signals, but these are independent of phosphorylation at that site). We expressed the Ser63A (Fig. 5D) or Ser68A (Fig. 5E) PLM mutants in HeLa cells and measured the effect of PDBu on NKA-mediated Na efflux. Expression of either PLM decreased the apparent Na affinity of NKA, to a similar extent as seen with WT PLM. PKC activation by PDBu significantly increased the apparent Na affinity of NKA in both cases (K0.5 decreases to 14.0 ± 1.7 mM for Ser63A, n = 9, and 13.9 ± 1.3 mM for Ser68A, n = 7). Thus PLM phosphorylation at either Ser68 or Ser63 can relieve the NKA inhibition by PLM to a similar extent and the effect does not seem additive.
Role of Ser63 and Ser68 phosphorylation sites in mediating the effect of PKC on NKA function. A: Western blot showing the time-dependent phosphorylation of Ser63 (CP63 antibody) and Ser68 (CP68 antibody) by PDBu. Experiments were done on HeLa cells infected with WT PLM. Representative of 3 experiments. B: Western blot showing the level of basal phosphorylation at Ser63 and Ser68 sites. Cells were treated with PP1a and PP2a (complete dephosphorylation), PDBu, and PKC. C2 antibody shows the total PLM level. Representative of 3 experiments. C: phosphorylation of S63A and S68A PLM mutants. Phosphorylation-dependent antibodies still recognize PLM mutated at the respective site, but the signal does not increase upon PKC activation with PDBu. Treatment with PDBu results in higher CP63 intensity in lysates from cells expressing the S68A PLM mutant and, reciprocally, higher CP68 signal for the S63A mutant (2- to 2.5-fold increase). Representative of 3 experiments. D: effect of 300 nM PDBu on NKA function in HeLa cells expressing the S63A PLM mutant (CON, control). *P < 0.05. E: effect of 300 nM PDBu on NKA function in HeLa cells expressing the S68A PLM mutant. *P < 0.05.
Measurement of NKA-mediated Na efflux in HeLa cells.
HeLa cells were loaded with SBFI and fluorescence was measured as described above. Fluorescence signals from a selected area of ∼20 cells were recorded, and [Na]i was calculated. Figure 1A shows a representative experiment where two [Na]i loading/decline cycles were monitored under identical conditions to verify that consecutive control runs result in identical results. In all experiments, 1 μM ouabain was used to block endogenous human NKA. A three-step calibration of the F340-to-F380 ratio was done at the end of the experiment. Similar to what we found in myocytes, consecutive control runs produce similar results (mean data not shown).
Measurement of Na-K-ATPase (NKA)-mediated Na efflux in a group of HeLa cells. A: NKA was first blocked (K-free, 145 mM Na external solution) causing intracellular Na concentration ([Na]i) loading of the cells and then NKA was reactivated in 15 mM K, Na-free solution. The protocol was then repeated under identical conditions to verify that consecutive control runs produce similar results; 1 μM ouabain (Oua) was used throughout the experiment to inhibit the endogenous human NKA. A: 3-step calibration of the F340-to-F380 ratio was done at the end. B: contribution of the different efflux pathways to Na extrusion in HeLa cells. Na efflux measured in the presence of 1 μM ouabain (NKA + leak) is mediated by the expressed rat NKA-α1 and NKA-independent pathways (leak component). The leak component (leak; measured in the presence of 10 mM ouabain) is small and not affected by application of either forskolin or phorbol 12,13-dibutyrate (PDBu) in HeLa cells. The difference between these 2 curves is the rate of Na efflux via the exogenous rat NKA-α1 (NKA). −d[Na]i/dt is the rate of [Na]i decline. The maximum Na extrusion rate of the pump (Vmax) and the [Na]i for the half-maximal activation of the pump (K0.5) shown at right represent the values derived by fitting the curves at left with a Hill equation.
In parallel experiments, we measured the rate of Na pump-independent Na efflux (leak) as the rate of [Na]i decline in the presence of 10 mM ouabain, which blocks both the exogenous and endogenous NKA. The leak Vmax averages 2.0 ± 0.3 mM/min and accounts for <25% of total Na efflux (Fig. 1B). Furthermore, the leak component was not affected by application of forskolin or PDBu in HeLa cells (not shown). The leak component was subtracted from total Na efflux to calculate the rate of NKA-mediated Na efflux (Fig. 1B).
Effect of PLM expression on [Na]i homeostasis in HeLa cells.
The WT PLM and mutated PLM used in this study do not contain any fluorescent marker to avoid potential contamination of the SBFI fluorescence signal. In parallel control experiments, the adenoviral transduction efficiency of PLM-GFP is >90% as indicated in Fig. 2A. Western blot experiments showed a comparable level of heterologous PLM expression, whether PLM was expressed alone or in a vector that also contained GFP (Fig. 2B). The noninfected (nonfluorescent) cells are displayed in a different layer and out of focus and are probably dead (Fig. 2A). Thus, during SBFI measurements, we selected areas that were devoid of out of focus and dead cells.
Effects of adenovirus-mediated phospholemman (PLM) expression in HeLa cells. A: image of HeLa cells taken under transmission light (left) and confocal GFP fluorescence from cells in the same area (right). Images were taken 24 h after adenovirus PLM-GFP transduction. Arrows indicate nonfluorescent cells. B: Western blot showing the PLM level (using the C2 antibody) in HeLA cells infected with GFP, PLM-GFP, and PLM. Representative of 3 experiments (Ctl, control). C: effect of acute PLM expression on resting [Na]i in HeLa cells. Mean data of resting [Na]i measured at the beginning of sodium-binding benzofuran isophthalate fluorescence experiments. The expression of both wild-type (WT) and AA PLM significantly increased resting [Na]i. D: [Na]i dependence of total Na efflux rate in HeLa cells with (red) or without (black) WT PLM. Dashed lines indicate the corresponding resting [Na]i (5.2 ± 0.5 mM in noninfected cells and 8.3 ± 1.0 mM in cells infected with WT PLM). **P < 0.01.
[ Na]i and NKA-mediated Na efflux were measured 24 h after adenoviral infection of WT or mutant PLM. [Na]i was 5.2 ± 0.5 mM (n = 12) in noninfected cells and significantly higher in HeLa cells expressing either WT or AA PLM (8.3 ± 1.0, n = 9, and 8.4 ± 0.7 mM, n = 10, respectively; Fig. 2C). The higher [Na]i in the presence of PLM is consistent with a decrease in Na efflux via NKA upon PLM association, as shown in myocyte studies (6). Indeed, the total Na efflux curve is right shifted upon WT PLM expression (K0.5: 10.7 ± 0.7 vs. 16.7 ± 0.8 mM), without any significant change in the Vmax (7.9 ± 0.7 vs. 8.2 ± 0.6 mM). Thus acute PLM expression decreases NKA activity and elevates [Na]i in HeLa cells. Moreover, the total efflux rate in HeLa cells without PLM at their [Na]i of 5.2 mM is similar to that in the presence of WT PLM at 8.3 mM [Na]i, the value measured under these conditions (1.4 vs. 1.6 mM/min; Fig. 2D). Because steady-state Na efflux is equal to Na influx, this indicates that PLM expression does not alter Na influx rate.
Effect of PKA activation on the NKA-mediated Na efflux in HeLa cells; role of PLM phosphorylation sites.
PKA was activated by 20 μM forskolin, and we determined the effect on NKA function by measuring the [Na]i dependence of NKA-mediated Na efflux (Fig. 3 and Table 1). In the absence of PLM, forskolin did not significantly affect either Vmax (5.8 ± 0.7 in control vs. 4.8 ± 0.7 mM/min with forskolin; n = 12), the apparent affinity of the pump for internal Na (K0.5 = 11.7 ± 0.9 in control and 11.2 ± 1.4 mM with forskolin) or nH (2.4 ± 0.1 vs. 2.6 ± 0.1). Figure 3A shows the Na-activation curve normalized to Vmax to emphasize the lack of effect on K0.5. Heterologous expression of WT PLM significantly inhibited NKA by decreasing the apparent Na affinity (K0.5 increased from 11.7 ± 0.9 in cells without PLM to 18.5 ± 0.7 mM with WT PLM; n = 11; Fig. 3B). Forskolin largely relieved NKA inhibition (K0.5 significantly decreased to 13.8 ± 1.2 mM; n = 11). The NKA Vmax was similar in HeLa cells with and without PLM (5.6 ± 0.6 vs. 5.8 ± 0.7 mM/min) and was not affected by PKA activation (5.7 ± 0.8 mM/min).
Effect of PKA activation on NKA function in HeLa cells: role of PLM phosphorylation sites. PKA was activated with 20 μM forskolin. A: effect of forskolin (FSK) on NKA-mediated Na extrusion in HeLa cells with no PLM. B: effect of forskolin in cells infected with WT PLM. The Na extrusion curve was normalized with respect to Vmax to emphasize the effect on K0.5. C: forskolin has no significant effect on NKA Vmax, independent of the presence of WT or nonphosphorylatable AA PLM. D: forskolin significantly increases the NKA apparent affinity for internal Na in HeLa cells expressing WT PLM but not in cells with no PLM or with AA PLM. **P < 0.01.
Table 1.
[Na]i dependence of NKA-mediated Na extrusion in Hela cells with WT PLM and mutants, and PKA/PKC-dependent effects
| No PLM | WT PLM | AA PLM | Ser63A | Ser68A | |
|---|---|---|---|---|---|
| Control condition | |||||
| Vmax, mM/min | 5.6 ± 0.4 | 6.5 ± 0.5 | 6.4 ± 0.5 | 6.3 ± 1.0 | 5.6 ± 0.8 |
| K0.5, mM [Na]i | 11.9 ± 0.6 | 17.5 ± 0.7* | 16.9 ± 0.6* | 17.1 ± 2.1* | 18.6 ± 1.6* |
| nH | 2.5 ± 0.1 | 2.7 ± 0.1 | 2.7 ± 0.1 | 2.6 ± 0.1 | 2.5 ± 0.1 |
| 10 μM forskolin | |||||
| Vmax, mM/min | 4.8 ± 0.7 | 5.7 ± 0.8 | 6.1 ± 0.5 | ||
| K0.5, mM [Na]i | 11.2 ± 1.4 | 13.8 ± 1.2† | 16.0 ± 0.8 | ||
| nH | 2.6 ± 0.1 | 2.8 ± 0.2 | 2.6 ± 0.1 | ||
| 300 nM PDBu | |||||
| Vmax, mM/min | 5.5 ± 1.0 | 6.2 ± 0.9 | 5.2 ± 1.0 | 5.3 ± 1.0 | 5.0 ± 0.5 |
| K0.5, mM [Na]i | 12.2 ± 0.8 | 13.4 ± 1.1† | 17.7 ± 1.6 | 14.0 ± 1.7† | 13.9 ± 1.3† |
| nH | 2.5 ± 0.1 | 2.6 ± 0.1 | 2.7 ± 0.1 | 2.5 ± 0.1 | 2.5 ± 0.1 |
Values are means ± SE. Data for untreated cells (control condition) were pulled from all experiments. Thus the values reported for control condition in the table might differ slightly from those reported in the text for paired comparisons with Na-K-ATPase (NKA) function parameters in cells treated with forskolin or phorbol 12,13-dibutyrate (PBDu). [Na]i, intracellular Na concentration; WT, wild type. The maximum Na extrusion rate of the pump (Vmax), the [Na]i for half-maximal activation of the pump (K0.5), and the Hill coefficient (nH) in first row are means of control conditions in forskolin and PDBu experiment.
Expression of nonphosphorylatable AA PLM mutant (Ser68 and Ser63 to alanine double mutation) also decreased the apparent Na affinity of NKA (vs. NKA only) similar to WT PLM (K0.5 = 16.3 ± 0.6 mM; n = 10; Fig. 3C). However, forskolin did not relieve NKA inhibition in HeLa cells expressing AA PLM (K0.5 = 16.0 ± 0.8 mM). In addition, PKA activation did not change the Vmax or nH of NKA after AA PLM overexpression in HeLa cells (Vmax: 6.9 ± 0.4 in control conditions vs. 6.1 ± 0.5 mM/min with forskolin; nH: 2.8 ± 0.2 vs. 2.6 ± 0.1).
Overall, the data in HeLa cells are consistent with those in cardiac myocytes (2, 6), indicating that PLM inhibits NKA activity by decreasing its [Na]i affinity and that this inhibitory effect is relieved by PKA phosphorylation of PLM phosphorylation sites. The nonphosphorylatable PLM works like a tonic inhibitor of NKA that cannot be relieved.
Effect of PKC activation on the NKA-mediated Na efflux in HeLa cells; role of PLM phosphorylation sites.
We used the same method to determine the effect of PKC activation on NKA function in HeLa cells and the role of the different PKC phosphorylation sites on PLM in mediating this effect (Fig. 4). PKC was activated with 300 nM PDBu. In the absence of PLM, PDBu failed to induce any changes in K0.5, Vmax, or nH (Fig. 4A and Table 1). This implies that PKC does not have a direct effect on NKA transport function under our experimental conditions here. PDBu increased the NKA Na affinity in cells expressing WT PLM (K0.5 decreased from 16.3 ± 1.4 mM in control condition to 13.4 ± 1.1 mM with PDBu; n = 9; Fig. 4B) but not in cells expressing AA PLM (K0.5 = 17.6 ± 1.3 vs. 17.7 ± 1.6 mM; n = 7; Fig. 4C). PDBu had no effect on Vmax and nH. Thus PLM phosphorylation is required for the PKC effects on NKA activity.
Effect of PKC activation on NKA function in HeLa cells; role of PLM phosphorylation sites. PKC was activated with 300 nM PDBu. A: PDBu did not alter NKA-mediated Na extrusion in HeLa cells with no PLM. B: PDBu stimulates NKA in cells infected with WT PLM. Na extrusion curve was normalized with respect to Vmax to emphasize the effect on K0.5. C: PDBu does not affect NKA Vmax, independent of the presence of WT or AA PLM. D: PDBu significantly increases the NKA apparent affinity for internal Na in HeLa cells expressing WT PLM but not in cells with no PLM or with AA PLM. **P < 0.05.
WT PLM expressed in HeLa cells is readily phosphorylated by PKC at both Ser63 and Ser68 (similar to the situation in cardiac myocytes), as demonstrated using phosphorylation-dependent antibodies (Fig. 5A). There was also a variable degree of baseline phosphorylation at both S63 and S68 (see untreated column in Fig. 5B). We investigated the separate effect of phosphorylation at each site on NKA function by mutating the other site to alanine. PDBu still induced phosphorylation of the S63A PLM mutant at Ser68 and of the S68A mutant at Ser63, as indicated by a 2- to 2.5-fold increase in the signal of the respective phospho-specific antibody (Fig. 5C; note that both alanine mutants display relatively high baseline signals, but these are independent of phosphorylation at that site). We expressed the Ser63A (Fig. 5D) or Ser68A (Fig. 5E) PLM mutants in HeLa cells and measured the effect of PDBu on NKA-mediated Na efflux. Expression of either PLM decreased the apparent Na affinity of NKA, to a similar extent as seen with WT PLM. PKC activation by PDBu significantly increased the apparent Na affinity of NKA in both cases (K0.5 decreases to 14.0 ± 1.7 mM for Ser63A, n = 9, and 13.9 ± 1.3 mM for Ser68A, n = 7). Thus PLM phosphorylation at either Ser68 or Ser63 can relieve the NKA inhibition by PLM to a similar extent and the effect does not seem additive.
Role of Ser63 and Ser68 phosphorylation sites in mediating the effect of PKC on NKA function. A: Western blot showing the time-dependent phosphorylation of Ser63 (CP63 antibody) and Ser68 (CP68 antibody) by PDBu. Experiments were done on HeLa cells infected with WT PLM. Representative of 3 experiments. B: Western blot showing the level of basal phosphorylation at Ser63 and Ser68 sites. Cells were treated with PP1a and PP2a (complete dephosphorylation), PDBu, and PKC. C2 antibody shows the total PLM level. Representative of 3 experiments. C: phosphorylation of S63A and S68A PLM mutants. Phosphorylation-dependent antibodies still recognize PLM mutated at the respective site, but the signal does not increase upon PKC activation with PDBu. Treatment with PDBu results in higher CP63 intensity in lysates from cells expressing the S68A PLM mutant and, reciprocally, higher CP68 signal for the S63A mutant (2- to 2.5-fold increase). Representative of 3 experiments. D: effect of 300 nM PDBu on NKA function in HeLa cells expressing the S63A PLM mutant (CON, control). *P < 0.05. E: effect of 300 nM PDBu on NKA function in HeLa cells expressing the S68A PLM mutant. *P < 0.05.
Measurement of NKA-mediated Na efflux in HeLa cells.
HeLa cells were loaded with SBFI and fluorescence was measured as described above. Fluorescence signals from a selected area of ∼20 cells were recorded, and [Na]i was calculated. Figure 1A shows a representative experiment where two [Na]i loading/decline cycles were monitored under identical conditions to verify that consecutive control runs result in identical results. In all experiments, 1 μM ouabain was used to block endogenous human NKA. A three-step calibration of the F340-to-F380 ratio was done at the end of the experiment. Similar to what we found in myocytes, consecutive control runs produce similar results (mean data not shown).
Measurement of Na-K-ATPase (NKA)-mediated Na efflux in a group of HeLa cells. A: NKA was first blocked (K-free, 145 mM Na external solution) causing intracellular Na concentration ([Na]i) loading of the cells and then NKA was reactivated in 15 mM K, Na-free solution. The protocol was then repeated under identical conditions to verify that consecutive control runs produce similar results; 1 μM ouabain (Oua) was used throughout the experiment to inhibit the endogenous human NKA. A: 3-step calibration of the F340-to-F380 ratio was done at the end. B: contribution of the different efflux pathways to Na extrusion in HeLa cells. Na efflux measured in the presence of 1 μM ouabain (NKA + leak) is mediated by the expressed rat NKA-α1 and NKA-independent pathways (leak component). The leak component (leak; measured in the presence of 10 mM ouabain) is small and not affected by application of either forskolin or phorbol 12,13-dibutyrate (PDBu) in HeLa cells. The difference between these 2 curves is the rate of Na efflux via the exogenous rat NKA-α1 (NKA). −d[Na]i/dt is the rate of [Na]i decline. The maximum Na extrusion rate of the pump (Vmax) and the [Na]i for the half-maximal activation of the pump (K0.5) shown at right represent the values derived by fitting the curves at left with a Hill equation.
In parallel experiments, we measured the rate of Na pump-independent Na efflux (leak) as the rate of [Na]i decline in the presence of 10 mM ouabain, which blocks both the exogenous and endogenous NKA. The leak Vmax averages 2.0 ± 0.3 mM/min and accounts for <25% of total Na efflux (Fig. 1B). Furthermore, the leak component was not affected by application of forskolin or PDBu in HeLa cells (not shown). The leak component was subtracted from total Na efflux to calculate the rate of NKA-mediated Na efflux (Fig. 1B).
Effect of PLM expression on [Na]i homeostasis in HeLa cells.
The WT PLM and mutated PLM used in this study do not contain any fluorescent marker to avoid potential contamination of the SBFI fluorescence signal. In parallel control experiments, the adenoviral transduction efficiency of PLM-GFP is >90% as indicated in Fig. 2A. Western blot experiments showed a comparable level of heterologous PLM expression, whether PLM was expressed alone or in a vector that also contained GFP (Fig. 2B). The noninfected (nonfluorescent) cells are displayed in a different layer and out of focus and are probably dead (Fig. 2A). Thus, during SBFI measurements, we selected areas that were devoid of out of focus and dead cells.
Effects of adenovirus-mediated phospholemman (PLM) expression in HeLa cells. A: image of HeLa cells taken under transmission light (left) and confocal GFP fluorescence from cells in the same area (right). Images were taken 24 h after adenovirus PLM-GFP transduction. Arrows indicate nonfluorescent cells. B: Western blot showing the PLM level (using the C2 antibody) in HeLA cells infected with GFP, PLM-GFP, and PLM. Representative of 3 experiments (Ctl, control). C: effect of acute PLM expression on resting [Na]i in HeLa cells. Mean data of resting [Na]i measured at the beginning of sodium-binding benzofuran isophthalate fluorescence experiments. The expression of both wild-type (WT) and AA PLM significantly increased resting [Na]i. D: [Na]i dependence of total Na efflux rate in HeLa cells with (red) or without (black) WT PLM. Dashed lines indicate the corresponding resting [Na]i (5.2 ± 0.5 mM in noninfected cells and 8.3 ± 1.0 mM in cells infected with WT PLM). **P < 0.01.
[ Na]i and NKA-mediated Na efflux were measured 24 h after adenoviral infection of WT or mutant PLM. [Na]i was 5.2 ± 0.5 mM (n = 12) in noninfected cells and significantly higher in HeLa cells expressing either WT or AA PLM (8.3 ± 1.0, n = 9, and 8.4 ± 0.7 mM, n = 10, respectively; Fig. 2C). The higher [Na]i in the presence of PLM is consistent with a decrease in Na efflux via NKA upon PLM association, as shown in myocyte studies (6). Indeed, the total Na efflux curve is right shifted upon WT PLM expression (K0.5: 10.7 ± 0.7 vs. 16.7 ± 0.8 mM), without any significant change in the Vmax (7.9 ± 0.7 vs. 8.2 ± 0.6 mM). Thus acute PLM expression decreases NKA activity and elevates [Na]i in HeLa cells. Moreover, the total efflux rate in HeLa cells without PLM at their [Na]i of 5.2 mM is similar to that in the presence of WT PLM at 8.3 mM [Na]i, the value measured under these conditions (1.4 vs. 1.6 mM/min; Fig. 2D). Because steady-state Na efflux is equal to Na influx, this indicates that PLM expression does not alter Na influx rate.
Effect of PKA activation on the NKA-mediated Na efflux in HeLa cells; role of PLM phosphorylation sites.
PKA was activated by 20 μM forskolin, and we determined the effect on NKA function by measuring the [Na]i dependence of NKA-mediated Na efflux (Fig. 3 and Table 1). In the absence of PLM, forskolin did not significantly affect either Vmax (5.8 ± 0.7 in control vs. 4.8 ± 0.7 mM/min with forskolin; n = 12), the apparent affinity of the pump for internal Na (K0.5 = 11.7 ± 0.9 in control and 11.2 ± 1.4 mM with forskolin) or nH (2.4 ± 0.1 vs. 2.6 ± 0.1). Figure 3A shows the Na-activation curve normalized to Vmax to emphasize the lack of effect on K0.5. Heterologous expression of WT PLM significantly inhibited NKA by decreasing the apparent Na affinity (K0.5 increased from 11.7 ± 0.9 in cells without PLM to 18.5 ± 0.7 mM with WT PLM; n = 11; Fig. 3B). Forskolin largely relieved NKA inhibition (K0.5 significantly decreased to 13.8 ± 1.2 mM; n = 11). The NKA Vmax was similar in HeLa cells with and without PLM (5.6 ± 0.6 vs. 5.8 ± 0.7 mM/min) and was not affected by PKA activation (5.7 ± 0.8 mM/min).
Effect of PKA activation on NKA function in HeLa cells: role of PLM phosphorylation sites. PKA was activated with 20 μM forskolin. A: effect of forskolin (FSK) on NKA-mediated Na extrusion in HeLa cells with no PLM. B: effect of forskolin in cells infected with WT PLM. The Na extrusion curve was normalized with respect to Vmax to emphasize the effect on K0.5. C: forskolin has no significant effect on NKA Vmax, independent of the presence of WT or nonphosphorylatable AA PLM. D: forskolin significantly increases the NKA apparent affinity for internal Na in HeLa cells expressing WT PLM but not in cells with no PLM or with AA PLM. **P < 0.01.
Table 1.
[Na]i dependence of NKA-mediated Na extrusion in Hela cells with WT PLM and mutants, and PKA/PKC-dependent effects
| No PLM | WT PLM | AA PLM | Ser63A | Ser68A | |
|---|---|---|---|---|---|
| Control condition | |||||
| Vmax, mM/min | 5.6 ± 0.4 | 6.5 ± 0.5 | 6.4 ± 0.5 | 6.3 ± 1.0 | 5.6 ± 0.8 |
| K0.5, mM [Na]i | 11.9 ± 0.6 | 17.5 ± 0.7* | 16.9 ± 0.6* | 17.1 ± 2.1* | 18.6 ± 1.6* |
| nH | 2.5 ± 0.1 | 2.7 ± 0.1 | 2.7 ± 0.1 | 2.6 ± 0.1 | 2.5 ± 0.1 |
| 10 μM forskolin | |||||
| Vmax, mM/min | 4.8 ± 0.7 | 5.7 ± 0.8 | 6.1 ± 0.5 | ||
| K0.5, mM [Na]i | 11.2 ± 1.4 | 13.8 ± 1.2† | 16.0 ± 0.8 | ||
| nH | 2.6 ± 0.1 | 2.8 ± 0.2 | 2.6 ± 0.1 | ||
| 300 nM PDBu | |||||
| Vmax, mM/min | 5.5 ± 1.0 | 6.2 ± 0.9 | 5.2 ± 1.0 | 5.3 ± 1.0 | 5.0 ± 0.5 |
| K0.5, mM [Na]i | 12.2 ± 0.8 | 13.4 ± 1.1† | 17.7 ± 1.6 | 14.0 ± 1.7† | 13.9 ± 1.3† |
| nH | 2.5 ± 0.1 | 2.6 ± 0.1 | 2.7 ± 0.1 | 2.5 ± 0.1 | 2.5 ± 0.1 |
Values are means ± SE. Data for untreated cells (control condition) were pulled from all experiments. Thus the values reported for control condition in the table might differ slightly from those reported in the text for paired comparisons with Na-K-ATPase (NKA) function parameters in cells treated with forskolin or phorbol 12,13-dibutyrate (PBDu). [Na]i, intracellular Na concentration; WT, wild type. The maximum Na extrusion rate of the pump (Vmax), the [Na]i for half-maximal activation of the pump (K0.5), and the Hill coefficient (nH) in first row are means of control conditions in forskolin and PDBu experiment.
Expression of nonphosphorylatable AA PLM mutant (Ser68 and Ser63 to alanine double mutation) also decreased the apparent Na affinity of NKA (vs. NKA only) similar to WT PLM (K0.5 = 16.3 ± 0.6 mM; n = 10; Fig. 3C). However, forskolin did not relieve NKA inhibition in HeLa cells expressing AA PLM (K0.5 = 16.0 ± 0.8 mM). In addition, PKA activation did not change the Vmax or nH of NKA after AA PLM overexpression in HeLa cells (Vmax: 6.9 ± 0.4 in control conditions vs. 6.1 ± 0.5 mM/min with forskolin; nH: 2.8 ± 0.2 vs. 2.6 ± 0.1).
Overall, the data in HeLa cells are consistent with those in cardiac myocytes (2, 6), indicating that PLM inhibits NKA activity by decreasing its [Na]i affinity and that this inhibitory effect is relieved by PKA phosphorylation of PLM phosphorylation sites. The nonphosphorylatable PLM works like a tonic inhibitor of NKA that cannot be relieved.
Effect of PKC activation on the NKA-mediated Na efflux in HeLa cells; role of PLM phosphorylation sites.
We used the same method to determine the effect of PKC activation on NKA function in HeLa cells and the role of the different PKC phosphorylation sites on PLM in mediating this effect (Fig. 4). PKC was activated with 300 nM PDBu. In the absence of PLM, PDBu failed to induce any changes in K0.5, Vmax, or nH (Fig. 4A and Table 1). This implies that PKC does not have a direct effect on NKA transport function under our experimental conditions here. PDBu increased the NKA Na affinity in cells expressing WT PLM (K0.5 decreased from 16.3 ± 1.4 mM in control condition to 13.4 ± 1.1 mM with PDBu; n = 9; Fig. 4B) but not in cells expressing AA PLM (K0.5 = 17.6 ± 1.3 vs. 17.7 ± 1.6 mM; n = 7; Fig. 4C). PDBu had no effect on Vmax and nH. Thus PLM phosphorylation is required for the PKC effects on NKA activity.
Effect of PKC activation on NKA function in HeLa cells; role of PLM phosphorylation sites. PKC was activated with 300 nM PDBu. A: PDBu did not alter NKA-mediated Na extrusion in HeLa cells with no PLM. B: PDBu stimulates NKA in cells infected with WT PLM. Na extrusion curve was normalized with respect to Vmax to emphasize the effect on K0.5. C: PDBu does not affect NKA Vmax, independent of the presence of WT or AA PLM. D: PDBu significantly increases the NKA apparent affinity for internal Na in HeLa cells expressing WT PLM but not in cells with no PLM or with AA PLM. **P < 0.05.
WT PLM expressed in HeLa cells is readily phosphorylated by PKC at both Ser63 and Ser68 (similar to the situation in cardiac myocytes), as demonstrated using phosphorylation-dependent antibodies (Fig. 5A). There was also a variable degree of baseline phosphorylation at both S63 and S68 (see untreated column in Fig. 5B). We investigated the separate effect of phosphorylation at each site on NKA function by mutating the other site to alanine. PDBu still induced phosphorylation of the S63A PLM mutant at Ser68 and of the S68A mutant at Ser63, as indicated by a 2- to 2.5-fold increase in the signal of the respective phospho-specific antibody (Fig. 5C; note that both alanine mutants display relatively high baseline signals, but these are independent of phosphorylation at that site). We expressed the Ser63A (Fig. 5D) or Ser68A (Fig. 5E) PLM mutants in HeLa cells and measured the effect of PDBu on NKA-mediated Na efflux. Expression of either PLM decreased the apparent Na affinity of NKA, to a similar extent as seen with WT PLM. PKC activation by PDBu significantly increased the apparent Na affinity of NKA in both cases (K0.5 decreases to 14.0 ± 1.7 mM for Ser63A, n = 9, and 13.9 ± 1.3 mM for Ser68A, n = 7). Thus PLM phosphorylation at either Ser68 or Ser63 can relieve the NKA inhibition by PLM to a similar extent and the effect does not seem additive.
Role of Ser63 and Ser68 phosphorylation sites in mediating the effect of PKC on NKA function. A: Western blot showing the time-dependent phosphorylation of Ser63 (CP63 antibody) and Ser68 (CP68 antibody) by PDBu. Experiments were done on HeLa cells infected with WT PLM. Representative of 3 experiments. B: Western blot showing the level of basal phosphorylation at Ser63 and Ser68 sites. Cells were treated with PP1a and PP2a (complete dephosphorylation), PDBu, and PKC. C2 antibody shows the total PLM level. Representative of 3 experiments. C: phosphorylation of S63A and S68A PLM mutants. Phosphorylation-dependent antibodies still recognize PLM mutated at the respective site, but the signal does not increase upon PKC activation with PDBu. Treatment with PDBu results in higher CP63 intensity in lysates from cells expressing the S68A PLM mutant and, reciprocally, higher CP68 signal for the S63A mutant (2- to 2.5-fold increase). Representative of 3 experiments. D: effect of 300 nM PDBu on NKA function in HeLa cells expressing the S63A PLM mutant (CON, control). *P < 0.05. E: effect of 300 nM PDBu on NKA function in HeLa cells expressing the S68A PLM mutant. *P < 0.05.
DISCUSSION
The present study shows that heterologous PLM decreases internal Na affinity of rat NKA-α1 stably expressed in HeLa cells. NKA inhibition results in a higher [Na]i in the presence of PLM. PLM phosphorylation by PKA and PKC restores the NKA Na affinity and thus relieves the pump inhibition. PKA and PKC activation does not affect NKA function in HeLa cells without PLM or in cells expressing mutant PLM that has both Ser63 and Ser68 sites unphosphorylatable (by Ser to Ala mutations), indicating that these are the only major sites that mediate the PKA- and PKC-dependent effects on NKA. Phosphorylation of either Ser63 or Ser68 by PDBu is sufficient to relieve the PLM-dependent NKA inhibition.
PLM is unique within the FXYD family in that it has multiple phosphorylation sites in the cytoplasmic tail. We have previously shown that PLM phosphorylation mediates the PKA and PKC-dependent regulation of NKA in cardiac myocytes. However, the role of Ser63 and Ser68 phosphorylation sites in mediating these kinase-dependent effects (K0.5 vs. Vmax) is not fully understood. Phosphorylation of PLM at Ser68 mediates the PKA-dependent increase in the Na affinity of both NKA α1- and α2-isoforms in mouse ventricular myocytes (2) and oocytes (1). PKC activation also increased PLM-dependent Na affinity of both NKA α1- and α2-isoforms, while increasing the Vmax of NKA-α2 (2). However, a role for PLM phosphorylation at the Ser63 site in relieving the NKA inhibition has not been demonstrated so far.
Here we determined the role of PLM phosphorylation sites (Ser63 and Ser 68) by measuring the effect of PKA and PKC activation on NKA function in HeLa cells stably expressing rat NKA-α1 with acute expression of PLM. HeLa cells have the advantage of being easy to culture and less problematic (compared to cardiac myocytes) for gene manipulation. For [Na]i measurements, fluorescence signals were collected from a region containing ∼20 cells. Although we did not use a fluorescent tag attached to PLM to prevent contamination of the SBFI signal, parallel adenoviral infections with GFP included in the vector (but not fused to PLM) showed a high expression efficiency (>90%) and Western blot data indicated that the level of PLM was comparable whether infected alone or in the vector with GFP. Because only ∼90% of the cells used for [Na]i measurements expressed PLM, the effect of PLM on NKA function measured here might be slightly underestimated. However, this does not affect the validity of our conclusions.
WT and mutant (S63A, S68A, and S63A-S68A double mutant) PLM were used here to determine how NKA was regulated by PLM upon Ser68 and Ser63 phosphorylation. WT and all PLM mutants inhibited NKA by decreasing the apparent pump affinity for internal Na. This result is consistent with previous data in cardiac myocyte (2, 6, 11), Hela cells (12), and oocytes (1, 4).
Activation of PKA and PKC shifted the apparent affinity for internal Na of rat NKA-α1 in HeLa cells expressing WT PLM by ∼4 mM. This is comparable to what we found in mouse cardiac myocytes (2, 6). However, in a previous study (11), we found that PLM phosphorylation by PKC increased NKA activity, but via increasing Vmax, while the affinity for internal Na was not changed. The reasons for the lack of K0.5 effect in that study are unclear. PKA and PKC activation did not affect NKA K0.5 in HeLa cells without PLM or in cells expressing PLM with the Ser63 and Ser68 sites rendered unphosphorylatable (AA PLM). Thus, if PKA and/or PKC phosphorylate PLM at other sites, this has no effect on NKA transport function in HeLa cells.
PKC readily phosphorylates WT PLM at both Ser63 and Ser68 sites and mutant S63A and S68A PLM at the nonmutated site in HeLa cells (Fig. 5, A-C). Phosphorylation at only one site is sufficient for increasing the NKA affinity for internal Na, as demonstrated in experiments with the S63A and S68A mutants, and the effect does not seem additive. Thus PLM phosphorylation at either Ser 63 or Ser68 is both necessary and sufficient to relieve the PLM-induced NKA inhibition.
PKA and PKC activation did not significantly affect the NKA maximum pumping rate under any condition. A previous study using Rb uptake (16) found that PKA and PKC modestly reduce the NKA Vmax in HeLa cells expressing rat NKA-α1 (by 22 and 27%, respectively). This effect might be due to NKA internalization and might have been enhanced in that study by the stronger kinase activation (1 μM PMA for PKC; forskolin plus IBMX for PKA) or their use of monensin as an ionophore. The lack of PKA and PKC effects on Vmax found here is in agreement with our own previous data in cardiac myocytes (2) and other data in oocytes (1) that show no Vmax effects for the α1-isoform of NKA. Both studies found, however, that PKC activation increases the Vmax of the NKA-α2, which may explain our previous finding that PDBu increases the total NKA Vmax in mouse cardiac myocytes (11).
In summary, we showed that 1) PLM phosphorylation at either Ser68 or Ser63 is necessary to fully mediate the enhancement of rat NKA-α1 activity induced by PKA and PKC activation; 2) PLM phosphorylation at other sites, if it occurs in HeLa cells, has no consequence on NKA function; and 3) PLM phosphorylation by PKC at either Ser63 or Ser68 is sufficient and redundant in relieving the NKA inhibition by PLM.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute Grants HL-30077, HL-64724, and HL-81562 (to D. M. Bers) and the American Heart Association (to F. Han and J. Bossuyt and 0735084N to S. Despa).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
Abstract
Phospholemman (PLM) is a major target for phosphorylation mediated by both PKA (at Ser68) and PKC (at both Ser63 and Ser68) in the heart. In intact cardiac myocytes, PLM associates with and inhibits Na-K-ATPase (NKA), mainly by reducing its affinity for internal Na. The inhibition is relieved upon PLM phosphorylation by PKA or PKC. The aim here was to distinguish the role of the Ser63 and Ser68 PLM phosphorylation sites in mediating kinase-induced modulation of NKA function. We expressed wild-type (WT) PLM and S63A, S68A, and AA (Ser63 and Ser68 to alanine double mutant) PLM mutants in HeLa cells that stably express rat NKA-α1 and we measured the effect of PKA and PKC activation on NKA-mediated intracellular Na concentration decline. PLM expression (WT or mutant) significantly decreased the apparent NKA affinity for internal Na and had no significant effect on the maximum pump rate (Vmax). PKA activation with forskolin (20 μM) restored NKA Na affinity in cells expressing WT but not AA PLM and did not affect Vmax in either case. Similarly, PKC activation with 300 nM phorbol 12,13-dibutyrate increased NKA Na affinity in cells expressing WT, S63A, and S68A PLM and had no effect in cells expressing AA PLM. Neither forskolin nor phorbol 12,13-dibutyrate affected NKA function in the absence of PLM. We conclude that PLM phosphorylation at either Ser63 or Ser68 is both necessary and sufficient for completely relieving the PLM-induced NKA inhibition.
phospholemman (plm) was first reported as an important phosphorylation substrate for both PKA and PKC (13, 17, 18) in the heart; however, its physiological role was poorly understood. More recent studies (4, 5, 21) indicate that PLM belongs to a family of proteins (FXYD gene family) that bind specifically to and regulate the Na-K-ATPase (NKA) in various tissues. PLM is the only FXYD protein highly expressed in the heart and is unique among the FXYD proteins in that it has multiple phosphorylation sites in the cytoplasmic domain.
PLM associates specifically and stably with rat NKA α1-, α2-, and α3-isoforms and reduces their apparent affinity for intracellular Na and extracellular K when coexpressed in oocytes (4). We (6, 11) have previously shown that PLM reduces the NKA activity, mainly by reducing the apparent pump affinity for internal Na, in intact cardiac myocytes. PLM phosphorylation relieves this inhibition and thus mediates the PKA/PKC-dependent stimulation of NKA (2, 6, 11). Biochemical studies (10) indicate that PKA phosphorylates PLM mostly at the Ser68 site (although some phosphorylation also occurs at the Ser63 site) and PKC phosphorylates both Ser63 and Ser68 sites. Removal of these phosphorylation sites in the shark PLM-like protein restores the apparent Na affinity of the NKA (14, 15). NKA is strongly stimulated in sarcolemmal membranes isolated from ischemic rat hearts compared with the controls, and this is accompanied by activation of PKA and PKC and subsequent phosphorylation of PLM (but not of the NKA α-subunit; Ref. 9). PKA activation by forskolin resulted in PLM phosphorylation at Ser68 and significantly increased the Na pump current in isolated guinea-pig cardiac myocytes (20).
PKC activation also increases the maximum Na extrusion rate of the NKA α2-isoform in mouse ventricular myocytes (6, 11). Furthermore, the PKA- and PKC-dependent effects on NKA and the phosphorylation state of Ser63 and Ser68 are additive (11), suggesting that the PKA/PKC-dependent pathways are independent in terms of NKA regulation. Thus the aim of this study was to investigate the role of PLM phosphorylation sites (Ser63 and Ser 68) in modulating NKA function. We measured the effect of PKA/PKC activation on the NKA-mediated intracellular Na concentration ([Na]i) decline in HeLa cells overexpressing rat NKA-α1 and determined how expression of PLM [wild type (WT) and its mutants] affect NKA regulation by PKA and PKC.
Values are means ± SE. Data for untreated cells (control condition) were pulled from all experiments. Thus the values reported for control condition in the table might differ slightly from those reported in the text for paired comparisons with Na-K-ATPase (NKA) function parameters in cells treated with forskolin or phorbol 12,13-dibutyrate (PBDu). [Na]i, intracellular Na concentration; WT, wild type. The maximum Na extrusion rate of the pump (Vmax), the [Na]i for half-maximal activation of the pump (K0.5), and the Hill coefficient (nH) in first row are means of control conditions in forskolin and PDBu experiment.
REFERENCES
References
- 1. Bibert S, Roy S, Schaer D, Horisberger JD, Geering K. Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes. J Biol Chem283: 476–486, 2008 [[PubMed]
- 2. Bossuyt J, Despa S, Han F, Hou Z, Robia SL, Lingrel JB, Bers DM. Isoform specificity of the Na/K-ATPase association and regulation by phospholemman. J Biol Chem284: 26749–26757, 2009
- 3. Bossuyt J, Ai X, Moorman RJ, Pogwizd SM, Bers DM. Expression and phosphorylation the Na-pump regulatory subunit phospholemman in heart failure. Circ Res97: 558–565, 2005 [[PubMed]
- 4. Crambert G, Fuzesi M, Garty H, Karlish S, Geering K. Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties. Proc Natl Acad Sci USA99: 11476–11481, 2002
- 5. Crambert G, Geering K. FXYD proteins: new tissue-specific regulators of the ubiquitous Na,K-ATPase. SciSTKE2003: RE1, 2003 [[PubMed]
- 6. Despa S, Bossuyt J, Han F, Ginsburg KS, Jia LG, Kutchai H, Tucker AL, Bers DM. Phospholemman-phosphorylation mediates the beta-adrenergic effects on Na/K pump function in cardiac myocytes. Circ Res97: 252, 2005 [[PubMed]
- 7. Despa S, Islam MA, Pogwizd SM, Bers DM. Intracellular [Na] and Na pump rate in rat and rabbit ventricular myocytes. J Physiol539: 133–143, 2002
- 8. Despa S, Islam MA, Weber CR, Pogwizd SM, Bers DM. Intracellular Na concentration is elevated in heart failure but Na/K pump function is unchanged. Circulation105: 2543–2548, 2002 [[PubMed]
- 9. Fuller W, Eaton P, Bell JR, Shattock MJ. Ischemia-induced phosphorylation of phospholemman directly activates rat cardiac Na/K-ATPase. FASEB J18: 197, 2004 [[PubMed]
- 10. Fuller W, Howie J, McLatchie LM, Weber RJ, Hastie CJ, Burness K, Pavlovic D, Shattock MJ. FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C. Am J Physiol Cell Physiol296: C1346–C1355, 2009
- 11. Han F, Bossuyt J, Despa S, Tucker AL, Bers DM. Phospholemman phosphorylation mediates the protein kinase C-dependent effects on Na/K pump function in cardiac myocytes. Circ Res99: 1376–1383, 2006 [[PubMed]
- 12. Lifshitz Y, Lindzen M, Garty H, Karlish SJ. Functional interactions of phospholemman (PLM) (FXYD1) with Na,K-ATPase. Purification of alpha1/beta1/PLM complexes expressed in Pichia pastoris. J Biol Chem281: 15790–15799, 2006 [[PubMed]
- 13. Lindemann JP. Alpha-adrenergic stimulation of sarcolemmal protein phosphorylation and slow responses in intact myocardium. J Biol Chem261: 4860–4867, 1986 [[PubMed]
- 14. Mahmmoud YA, Cramb G, Maunsbach AB, Cutler CP, Meischke L, Cornelius F. Regulation of Na,K-ATPase by PLMS, the phospholemman-like protein from shark: molecular cloning, sequence, expression, cellular distribution, and functional effects of PLMS. J Biol Chem278: 37427–37438, 2003 [[PubMed]
- 15. Mahmmoud YA, Vorum H, Cornelius F. Identification of a phospholemman-like protein from shark rectal glands. Evidence for indirect regulation of Na,K-ATPase by protein kinase c via a novel member of the FXYDY family. J Biol Chem275: 35969–35977, 2000 [[PubMed]
- 16. Nestor NB, Lane LK, Blostein R. Effects of protein kinase modulators on the sodium pump activities of HeLa cells transfected with distinct alpha isoforms of Na,K-ATPase. Ann NY Acad Sci834: 579–581, 1997 [[PubMed]
- 17. Palmer CJ, Scott BT, Jones LR. Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. J Biol Chem266: 11126–11130, 1991 [[PubMed]
- 18. Presti CF, Jones LR, Lindemann JP. Isoproterenol-induced phosphorylation of a 15-kilodalton sarcolemmal protein in intact myocardium. J Biol Chem260: 3860–3867, 1985 [[PubMed]
- 19. Price EM, Rice DA, Lingrel JB. Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1–H2 extracellular domain of the alpha subunit. J Biol Chem265: 6638–6641, 1990 [[PubMed]
- 20. Silverman BZ, Fuller W, Eaton P, Deng J, Moorman JR, Cheung JY, James AF, Shattock MJ. Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase. Cardiovasc Res65: 93–103, 2005 [[PubMed]
- 21. Sweadner KJ, Rael E. The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression. Genomics68: 41–56, 2000 [[PubMed]