Transport and Regulation of the Cardiac Na 1 - Ca 2 1 Exchanger , NCX 1

Cardiac muscle fails to relax upon replacement of extracellular Ca 2 1 with Ba 2 1 . Among the manifold consequences of this intervention, one major possibility is that Na 1 -Ba 2 1 exchange is inadequate to support normal relaxation. This could occur due to reduced transport rates of Na 1 -Ba 2 1 exchange and/or by failure of Ba 2 1 to activate the exchanger molecule at the high affinity regulatory Ca 2 1 binding site. In this study, we examined transport and regulatory properties for Na 1 -Ca 2 1 and Na 1 -Ba 2 1 exchange. Inward and outward Na 1 -Ca 2 1 or Na 1 Ba 2 1 exchange currents were examined at 30 8 C in giant membrane patches excised from Xenopus oocytes expressing the cloned cardiac Na 1 -Ca 2 1 exchanger, NCX1. When excised patches were exposed to either cytoplasmic Ca 2 1 or Ba 2 1 , robust inward Na 1 -Ca 2 1 exchange currents were observed, whereas Na 1 -Ba 2 1 currents were absent or barely detectable. Similarly, outward currents were greatly reduced when pipette solutions contained Ba 2 1 rather than Ca 2 1 . However, when solution temperature was elevated from 30 8 C to 37 8 C, a substantial increase in outward Na 1 -Ba 2 1 exchange currents was observed, but not so for inward currents. We also compared the relative abilities of Ca 2 1 and Ba 2 1 to activate outward Na 1 -Ca 2 1 exchange currents at the high affinity regulatory Ca 2 1 binding site. While Ba 2 1 was capable of activating the exchanger, it did so with a much lower affinity ( K D z 10 m M) compared with Ca 2 1 ( K D z 0.3 m M). Moreover, the efficiency of Ba 2 1 regulation of Na 1 -Ca 2 1 exchange is also diminished relative to Ca 2 1 , supporting z 60% of maximal currents obtainable with Ca 2 1 . Ba 2 1 is also much less effective at alleviating Na 1 i -induced inactivation of NCX1. These results indicate that the reduced ability of NCX1 to adequately exchange Na 1 and Ba 2 1 contributes to failure of the relaxation process in cardiac muscle. key words: sodium-calcium exchange • transport • regulation • calcium • barium i n t r o d u c t i o n Na 1 -Ca 2 1 exchange plays a major role in Ca 2 1 homeostasis in cardiac muscle. Removal of myoplasmic Ca 2 1 by this mechanism is essential for physiological cardiac relaxation (Bers, 1991). In general, removal of Ca 2 1 by Na 1 -Ca 2 1 exchange is equivalent to Ca 2 1 entry through L-type Ca 2 1 channels on a beat-to-beat basis (Bridge et al., 1990). Na 1 -Ca 2 1 exchange may also serve an important role as a Ca 2 1 entry mechanism during cardiac excitation. Several studies have demonstrated that this “reverse mode” of Na 1 -Ca 2 1 exchange can trigger sarcoplasmic reticulum Ca 2 1 release (LeBlanc and Hume, 1990; Kohmoto et al., 1994; Levi et al., 1994; Vornanen et al., 1994; Wasserstrom and Vites, 1996). Consequently, alterations in Na 1 -Ca 2 1 exchange function, exemplified by digitalis treatment (Lee and Dagastino, 1982) or alterations in the intracellular Na 1 concentration (Harrison and Boyett, 1995), produce major effects on cardiac contractility. Recently, several regulatory properties have been characterized for the cardiac Na 1 -Ca 2 1 exchanger, NCX1. Detailed characterization of these regulatory mechanisms has been accomplished for the native and cloned cardiac Na 1 -Ca 2 1 exchanger using the giant excised patch technique. Regarding ionic regulation, both Na 1 and Ca 2 1 regulate exchange activity in addition to serving as the transport substrates (Hilgemann, 1990). Examination of outward (reverse) Na 1 -Ca 2 1 exchange currents reveals a complex waveform. The application of Na 1 i induces an outward current which undergoes a time-dependent inactivation. The extent of this inactivation is governed by both cytoplasmic Na 1 and Ca 2 1 levels. However, in the presence of a constant level of cytoplasmic Ca 2 1 (e.g., 1 m M), both outward currents and the extent of inactivation increase as Na 1 i levels are increased. This behavior is referred to as Na 1 i -induced or I 1 inactivation (Hilgemann et al., 1992 b ). Cytoplasmic Ca 2 1 levels regulate exchange activity by influencing the extent of Na 1 i -induced inactivation and through an apparent direct activation of the exchange molecule (Hilgemann et al., 1992 a , b ). This direct pathway is referred to as I 2 inactivation where removal of cytosolic Ca 2 1 favors entry into an inactive (I 2 ) state. Both forward and reverse modes of Na 1 -Ca 2 1 exchange are Address correspondence to Dr. Larry V. Hryshko, Institute of Cardiovascular Sciences, University of Manitoba, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada, R2H 2A6. Fax: 204-233-6723; E-mail: lhryshko@sbrc. umanitoba.ca on A ril 5, 2017 D ow nladed fom Published March 1, 1997