Inactivation of outward Na(+)‐Ca2+ exchange current in guinea‐pig ventricular myocytes.

Abstract

4. With 100 mm cytoplasmic Na+ and no extracellular Na+ (replaced with 140mm Li+), application of extracellular Ca2+ was predicted to reorient exchanger binding sites from the extracellular side to the cytoplasmic side and thereby favour inactivation. During such protocols, the outward exchange current decayed by 60-80 % when activated by extracellular Ca2+. The current decayed similarly when extracellular Ca2+ and Na+ were applied together, whereby current magnitudes were about 3-fold smaller. 5. The decay of outward exchange current usually followed a biexponential time course (5-8 + 3-5 and 27-3 + 16-3 s, means + S.D., n = 11). Intracellular application of 0'5-2 mg ml' trypsin attenuated the fast component more than the slow component, suggesting that the fast component reflects an inactivation process. 6. Current-voltage (I-V) relations of the outward exchange current became less steep during the inactivation protocols, but this flattening could not be correlated with inactivation. 7. Replacement of extracellular Li+ with N-methyl-D-glucamine (NMG), tetraethylammonium (TEA), sucrose or Cs+ resulted in a flattening of I-V relations and a decrease of the outward exchange current amplitude by approximately 3-fold, but the kinetics and extent of inactivation were not remarkably changed. Thus, the mechanism of inactivation appears to be independent of the mechanism(s) of activation by extracellular monovalent cations. 8. Preapplication of extracellular Na+ was predicted to orient binding sites to the cytoplasmic side and thereby induce inactivation in the absence of extracellular Ca2+. As predicted, exchange currents subsequently activated by extracellular Ca2+ were small and showed a small recovery phase, rather than current decay.9. A consecutive model of the exchange cycle with inactivation taking place from fully Na+-loaded binding sites with cytoplasmic orientation described well most of the results on inactivation in whole myocytes.10. It is concluded that, as in giant cardiac membrane patches, Na+-Ca2+ exchange function in intact myocytes is modulated by secondary inactivation reactions.

Sodium/Calcium Exchanger (NCX1) Macromolecular Complex

“…Several studies were designed to resolve this problem. Hilgemann and colleagues (11,12,27,28) investigated the question by measuring NCX1 currents in giant excised patches. The work used two preparations, NCX1-expressing Xenopus oocytes (27) or cardiac myocytes expressing native NCX1 (11,12,28).…”

“…Hilgemann and colleagues (11,12,27,28) investigated the question by measuring NCX1 currents in giant excised patches. The work used two preparations, NCX1-expressing Xenopus oocytes (27) or cardiac myocytes expressing native NCX1 (11,12,28). These investigations found no functional change in cardiac NCX1 activity following application of PKA or protein kinase C (PKC) catalytic subunits to the intracellular side of the giant patch.…”

Increased O2 consumption in excitation–contraction coupling in hypertrophied rat heart slices related to increased Na+–Ca2+ exchange activity

“…Our study actually demonstrated that the increase in active NCX, which was caused by the attenuation of inactivation, is the major cause of the increase, although the inactivation (I 1 and I 2 inactivation) [28,36,37] could not be distinguished by the present protocol. Concomitant changes in other proteins, such as PLM, might also be related to the attenuation, but the underlying mechanisms need to be further clarified.…”

“…6a). The intrinsic inactivation mechanisms of NCX1 [28,36,37] was assessed in a manner similar to a previous study [28] in Fig 6b. Slower inactivation of I NCX was observed in myocytes from HYP, suggesting the attenuation of inactivation mechanisms. The mean time constant of I NCX inactivation in HYP myocytes (n = 7) was significantly larger than that in CONT myocytes (n = 4) (11.4 ± 1.6 s vs. 8.4 ± 1.2 s) (Fig.…”

“…I NCX was induced by applying 2 mM Ca 2? for 5 or 30 s at intervals of 30 or 120 s and determined as the difference current in the same manner as described in our previous studies [27,28]. I NKA was induced similarly by applying 10 mM K ?…”