The effects of changing Glu-779, located in the fifth transmembrane segment of the Na,K-ATPase ␣ subunit, on the phosphorylation characteristics and ion transport properties of the enzyme were investigated. HeLa cells were transfected with cDNA coding the E779A substitution in an ouabain-resistant sheep ␣1 subunit (RD). Steady state phosphorylation stimulated by Na ؉ concentrations less than 20 mM or by imidazole were similar for RD and E779A enzymes, an indication that phosphorylation and Na ؉ occlusion were not altered by this mutation. With E779A enzyme, higher Na ؉ concentrations reduced the level of phosphoenzyme and stimulated Na-ATPase activity in the absence of K ؉ . These effects were a consequence of Na ؉ increasing the rate of protein dephosphorylation. In voltage-clamped HeLa cells expressing E779A enzyme, a prominent electrogenic Na ؉ -Na ؉ exchange was observed in the absence of extracellular K ؉ . Thus, increased Na-ATPase activity and Na ؉ -dependent dephosphorylation result from Na ؉ acting as a K ؉ congener with low affinity at extracellular binding sites. These data suggest that E779A does not directly participate in ion binding but does affect the connection between extracellular ion binding and intracellular enzyme dephosphorylation. In cells expressing control RD enzyme, Na,K-pump current was dependent on membrane potential and extracellular K ؉ concentration. However, Na,K-pump current in cells expressing E779A enzyme was voltage independent at all extracellular K ؉ tested. These results indicate that Glu-779 may be part of the access channel determining the voltage dependence of ion transport by the Na,K-ATPase.

The involvement of electrogenic reaction steps in K+ transport by the Na+,K+‐ATPase was determined in rat cardiac ventricular myocytes using whole‐cell patch clamp techniques. Under K+–K+ exchange conditions and in the presence of extracellular K+ or Tl+ at concentrations that stimulated submaximal levels of steady‐state Na+,K+‐ATPase activity, ouabain‐sensitive transient currents were observed during (‘on’) and after (‘off’) step changes in membrane potential (Vm). The quantity of charge moved during the transient currents depended, in a saturable manner, on the magnitude of the voltage step. Maximal ouabain‐sensitive ‘on’ and ‘off’ charges were calculated to be 9.6 ± 0.9 and 9.1 ± 0.4 fC pF−1 (n= 4), respectively, with an effective valency of 0.48 ± 0.07 (n= 7). Kinetics of the transient currents were independent of Vm and Tlo+ at positive potentials, but became more rapid at increasingly negative Vm values in an ion concentration‐dependent fashion. These data demonstrate that electrogenic steps participate in K+ transport by the Na+,K+‐ATPase and that the electrogenic step is extracellular ion binding. The temperature‐and Vm‐dependent properties of transient charge movements were compared under K+–K+ and Na+–Na+ exchange conditions. The data suggest that extracellular K+ and Na+ binding occur at different sites in the enzyme or to different enzyme conformations. The sum of the effective valencies, 1.14 ± 0.12, demonstrates that the electrogenicity of extracellular ion binding can explain the Vm dependence of ion transport by the Na+,K+‐ATPase.

L-Arginine (L-Arg) is a basic amino acid that plays a central role in the biosynthesis of nitric oxide, creatine, agmantine, polyamines, proline and glutamate. Most tissues, including myocardium, must import L-Arg from the circulation to ensure adequate intracellular levels of this amino acid. This study reports novel L-Arg-activated inward currents in whole-cell voltage-clamped rat ventricular cardiomyocytes. Ion-substitution experiments identified extracellular L-Arg as the charge-carrying cationic species responsible for these currents, which, thus, represent L-Arg import into cardiac myocytes. This result was independently confirmed by an increase in myocyte nitric oxide production upon extracellular application of L-Arg. The inward movement of Arg molecules was found to be passive and independent of Na + , K + , Ca 2+ and Mg 2+ . The process displayed saturation and membrane potential (V m )-dependent kinetics, with a K 0.5 for L-Arg that increased from 5 mM at hyperpolarizing V m to 20 mM at +40 mV. L-Lysine and L-ornithine but not D-Arg produced currents with characteristics similar to that activated by L-Arg indicating that the transport process is stereospecific for cationic L-amino acids. L-Arg current was fully blocked after brief incubation with 0.2 mM N -ethylmaleimide. These features suggest that the activity of the low-affinity, high-capacity CAT-2A member of the y + family of transporters is responsible for L-Arg currents in acutely isolated cardiomyocytes. Regardless of the mechanism, we hypothesize that a low-affinity arginine transport process in heart, by ensuring substrate availability for sustained NO production, might play a cardio-protective role during catabolic states known to increase Arg plasma levels severalfold.

SYNOPSIS Duchenne muscular dystrophy (DMD) is an incurable, rapidly-worsening neuromuscular degenerative disease caused by the absence of dystrophin. In skeletal muscle, lack of dystrophin disrupts the recruitment of neuronal nitric oxide synthase (nNOS) to the sarcolemma thus affecting nitric oxide (NO) production. Utrophin is a dystrophin homolog which expression is greatly upregulated in the sarcolemma of dystrophin-negative fibers from mdx mice, a mouse model of DMD. Although cardiomyopathy is an important cause of death, little is known about the NO signaling pathway in cardiac muscle of DMD patients. Thus, we used cardiomyocytes and hearts from two month-old mdx and mdx:utrophin (−/−) double knockout mice (mdx:utr) to study key steps in NO signaling: L-arginine transporters, NOS, and soluble guanylyl cyclase (sGC). nNOS did not co-localize with dystrophin or utrophin to the cardiomyocyte membrane. Despite this, nNOS activity was markedly decreased in both mdx and mdx:utr mice while nNOS expression was only decreased in mdx:utr hearts, suggesting that utrophin upregulation in cardiomyocytes maintains nNOS levels but not function. sGC protein levels and activity remained at control levels. Unexpectedly, L-arginine transporter expression and function were significantly increased, suggesting a novel biochemical compensatory mechanism of the NO pathway and a potential entry site for therapeutics.

Cationic L-amino acids enter cardiac-muscle cells through carrier-mediated transport. To study this process in detail, L-[(14)C]lysine uptake experiments were conducted within a 10(3)-fold range of L-lysine concentrations in giant sarcolemmal vesicles prepared from rat cardiac ventricles. Vesicles had a surface-to-volume ratio comparable with that of an epithelial cell, thus representing a suitable system for initial uptake rate studies. Two Na(+)-independent, N-ethylmaleimide-sensitive uptake components were found, one with high apparent affinity (K(m)=222+/-71 microM) and low transport capacity (V(max)=121+/-36 pmol/min per mg of vesicle protein) and the other with low apparent affinity (K(m)=16+/-4 mM) and high capacity (V(max)=4.0+/-0.4 nmol/min per mg of vesicle protein). L-Lysine uptake mediated by both components was stimulated by the presence of intravesicular L-lysine as well as by valinomycin-induced membrane hyperpolarization. Altogether, this behaviour is consistent with the functional properties of the CAT-1 and CAT-2A members of the system y(+) family of cationic amino acid transporters. Furthermore, mRNA transcripts for these two carrier proteins were identified in freshly isolated rat cardiac myocytes, the amount of CAT-1 mRNA, relative to beta-actin, being 33-fold larger than that of CAT-2A. These two transporters appear to function simultaneously as a homoeostatic device that supplies cardiac-muscle cells with cationic amino acids under a variety of metabolic conditions. Analysis of two carriers acting in parallel with such an array of kinetic parameters shows significant activity of the low-affinity component even at amino acid plasma levels far below its K(m).