Phospholemman inhibition of the cardiac Na+/Ca2+ exchanger. Role of phosphorylation

Abstract

We have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na+/Ca2+ exchanger (NCX1). In addition, protein kinase A phosphorylates serine 68, whereas protein kinase C phosphorylates both serine 63 and serine 68 of PLM. Using human embryonic kidney 293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (I(NaCa)) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in I(NaCa), (ii) attenuation of the increase in I(NaCa) by PMA, and (iii) additional reduction in I(NaCa) in cells treated with forskolin. Mutating serine 63 to alanine (S63A) preserved the sensitivity of PLM to forskolin in terms of suppression of I(NaCa), whereas mutating serine 68 to alanine (S68A) abolished the inhibitory effect of PLM on I(NaCa). Mutating serine 68 to glutamic acid (phosphomimetic) resulted in additional suppression of I(NaCa) as compared with wild-type PLM. These results suggest that PLM phosphorylated at serine 68 inhibited I(NaCa). The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knock-out (KO) mice. When compared with wild-type myocytes, I(NaCa) was significant larger in PLM-KO myocytes. In addition, the PMA-induced increase in I(NaCa) was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on I(NaCa) in wild-type myocytes. We conclude that PLM, when phosphorylated at serine 68, inhibits Na+/Ca2+ exchange in the heart.

Fig. 1. Measurement of Na⁺/Ca²⁺ exchange current (I_NaCa) in transfected HEK293 cells

HEK293 cells were transfected with NCX1. At 48h post-transfection, I_NaCa was measured at 5 mM [Ca²⁺]_o and 30^oC with a descending-ascending voltage ramp protocol (A) described in Experimental Procedures. Free [Ca²⁺] in the Ca²⁺ buffered pipette solution was 205 nM. Holding potential was at the calculated reversal potential of I_NaCa (−73 mV) under our experimental conditions. Ca²⁺, Na⁺-K⁺-ATPase, Cl⁻ and K⁺ currents were blocked by appropriate inhibitors. (B) Membrane currents recorded in a transfected cell during the descending-ascending voltage-ramp from +100 to −120 and back to +100 mV, in the absence and presence of 1 mM Cd²⁺. (C) Derived Cd²⁺-sensitive current in the transfected cell shown in B.

Fig. 2. Effects of PMA on I_NaCa in transfected HEK293 cells

(A). HEK293 cells were transfected with NCX1 (open circles, n=14). At 48h post-transfection, I_NaCa was measured at 30°C using standard solutions and Cd²⁺ as described in Experimental Procedures and Fig. 1. After baseline I_NaCa was obtained, PMA (0.1 μM) was added and I_NaCa was measured 3 to 5 min after drug addition (open squares; n=8). (B). I_NaCa was measured in HEK293 cells transfected with NCX1 under Cl⁻-free conditions (Experimental Procedures), both before (open circles, n=4) and after (open squares, n=4) addition of PMA. Cd²⁺ was used to define I_NaCa. (C). I_NaCa was measured in HEK293 cells transfected with NCX1 under high [Na⁺]_i conditions (Experimental Procedures), both before (open circles, n=6) and after (open squares, n=7) addition of PMA. [Ca²⁺]_o was 0.2 rather than 5.0 mM so that the calculated reversal potential for I_NaCa was −103 mV, as compared to the holding potential of −73 mV used in other experiments. Ni²⁺ was used to define I_NaCa. Error bars are not shown if they fall within boundaries of the symbols.

Fig. 3. Effects of PMA and forskolin on I_NaCa in transfected HEK293 cells

(A). HEK293 cells were transfected with either NCX1 alone (open circles, n=14) or PLM+NCX1 (open diamonds, n=15). At 48h post-transfection, I_NaCa was measured at 5 mM [Ca²⁺]_o and 30°C as described in Fig. 1. After baseline I_NaCa was obtained, PMA (0.1 μM) was added to NCX1 (open squares; n=8) and PLM+NCX1 (filled triangles; n=9) cells. Measurement of I_NaCa was repeated ~3 to 5 min after drug addition. (B). HEK293 cells were transfected with either NCX1 alone (open circles, n=14) or PLM+NCX1 (open diamonds, n=15). At 48h post-transfection, baseline I_NaCa was obtained. Forskolin (1 μM) was then added to NCX1 (open squares; n=4) and PLM+NCX1 (open triangles; n=7) cells. Measurement of I_NaCa was repeated ~3 to 5 min after drug addition. Error bars are not shown if they fall within boundaries of the symbols.

Fig. 4. Effects of serine⁶⁸ mutants of PLM on I_NaCa in transfected HEK293 cells

(A). HEK293 cells were transfected with either NCX1 alone (open circles, n=14) or S68A+NCX1 (open squares, n=7). At 48 h post-transfection, I_NaCa was measured at 5 mM [Ca²⁺]_o and 30°C as described in Fig. 1. After baseline I_NaCa was obtained, PMA (0.1 μM) was added to S68A+NCX1 cells (open triangles; n=7) and I_NaCa was again measured. (B). HEK293 cells were transfected with NCX1 alone (open circles, n=10), PLM+NCX1 (open diamonds, n=8), or S68E+NCX1 (open squares, n=6). I_NaCa was measured 48h post-transfection. In S68E+NCX1 cells, I_NaCa was measured both before (open squares) and after (open triangles) addition of PMA (0.1 μM). Error bars are not shown if they fall within boundaries of the symbols.

Fig. 5. Effects of serine⁶³ mutants of PLM on I_NaCa in transfected HEK293 cells

(A). HEK293 cells were transfected with either NCX1 alone (open circles, n=10) or S63A+NCX1 (open squares, n=5). At 48 h post-transfection, I_NaCa was measured at 5 mM [Ca²⁺]_o and 30°C as described in Fig. 1. After baseline I_NaCa was obtained, forskolin (1 μM) was added to S63A+NCX1 cells (open triangles; n=5) and I_NaCa was again measured. (B). HEK293 cells were transfected with S63A+NCX1 (open squares, n=6). I_NaCa was measured 48h post-transfection, both before (open squares) and after (open triangles) addition of PMA (0.1 μM). Error bars are not shown if they fall within boundaries of the symbols.

Fig. 6. Immunoblots of Na⁺/Ca²⁺ exchanger (NCX1), calsequestrin and phospholemman (PLM) from murine hearts

Left ventricular homogenates were prepared from wild-type and PLM-KO mice of congenic C57BL/6 background, as described in Experimental Procedures. Proteins were separated by gel electrophoresis under non-reducing conditions for NCX1 (50 μg/lane) and calsequestrin (100 μg/lane), and reducing conditions for PLM (5 μg/lane). After transfer to PVDF membranes, immunoblotting were performed as described in Experimental Procedures. Numbers on the left refer to apparent molecular mass.

Fig. 7. Measurement of Na⁺/Ca²⁺ exchange current (I_NaCa) in murine cardiac myocytes

I_NaCa was measured in ventricular myocytes isolated from adult mouse hearts at 5 mM [Ca²⁺]_o and 30°C with a descending-ascending voltage ramp protocol (A) as described in Experimental Procedures. Free Ca²⁺ in the Ca²⁺-buffered pipette solution was 205 nM. Holding potential was at the calculated reversal potential of I_NaCa (−73 mV) under our experimental conditions. Ca²⁺, Na⁺-K⁺-ATPase, Cl⁻ and K⁺ currents were blocked by appropriate inhibitors. (B) Membrane currents recorded in a wild -type myocyte during the descending-ascending voltage-ramp from +100 to −120 and back to +100 mV, in the absence and presence of 1 mM Cd²⁺. (C) Derived Cd²⁺-sensitive current in the wild-type myocyte shown in B.

Fig. 8. I_NaCa is larger in PLM-KO when compared to wild-type cardiac myocytes

I_NaCa was measured in ventricular myocytes isolated from wild-type and PLM-KO mouse hearts at 5 mM [Ca²⁺]_o and 30°C as described in Fig. 7. Shown are current density-voltage relationships of I_NaCa (means ± SE) from wild-type (open circles; n=20) and PLM-KO (filled circles; n=23) myocytes. Error bars are not shown if they fall within the boundaries of symbols.

Fig. 9. Effects of PMA and forskolin on I_NaCa in wild-type and PLM-KO cardiac myocytes

(A). I_NaCa was measured in a second group of ventricular myocytes isolated from wild-type (diamonds, n=7) and PLM-KO (circles, n=7) mouse hearts at 5 mM [Ca²⁺]_o and 30°C as described in Fig. 6. After baseline I_NaCa was obtained, PMA (1 μM) was added to both wild-type (squares, n=7) and PLM-KO (triangles, n=7) and I_NaCa measurement was repeated. (B). I_NaCa was measured in a third group of wild-type myocytes, both before (diamonds, n=6) and after (squares, n=6) addition of forskolin (1 μM). Similarly, I_NaCa was measured in PLM-KO myocytes. For clarity of presentation, only data from PLM-KO myocytes treated with forskolin (triangles, n=6) are shown. Error bars are not shown if they fall within boundaries of the symbols.