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Transport and regulation of the cardiac Na(+)-Ca2+ exchanger, NCX1. Comparison between Ca2+ and Ba2+.
Trac M
,
Dyck C
,
Hnatowich M
,
Omelchenko A
,
Hryshko LV
.
???displayArticle.abstract??? Cardiac muscle fails to relax upon replacement of extracellular Ca2+ with Ba2+. Among the manifold consequences of this intervention, one major possibility is that Na(+)-Ba2+ exchange is inadequate to support normal relaxation. This could occur due to reduced transport rates of Na(+)-Ba2+ exchange and/or by failure of Ba2+ to activate the exchanger molecule at the high affinity regulatory Ca2+ binding site. In this study, we examined transport and regulatory properties for Na(+)-Ca2+ and Na(+)-Ba2+ exchange. Inward and outward Na(+)-Ca2+ or Na(+)-Ba2+ exchange currents were examined at 30 degrees C in giant membrane patches excised from Xenopus oocytes expressing the cloned cardiac Na(+)-Ca2+ exchanger, NCX1. When excised patches were exposed to either cytoplasmic Ca2+ or Ba2+, robust inward Na(+)-Ca2+ exchange currents were observed, whereas Na(+)-Ba2+ currents were absent or barely detectable. Similarly, outward currents were greatly reduced when pipette solutions contained Ba2+ rather than Ca2+. However, when solution temperature was elevated from 30 degrees C to 37 degrees C, a substantial increase in outward Na(+)-Ba2+ exchange currents was observed, but not so for inward currents. We also compared the relative abilities of Ca2+ and Ba2+ to activate outward Na(+)-Ca2+ exchange currents at the high affinity regulatory Ca2+ binding site. While Ba2+ was capable of activating the exchanger, it did so with a much lower affinity (KD approximately 10 microM) compared with Ca2+ (KD approximately 0.3 microM). Moreover, the efficiency of Ba2+ regulation of Na(+)-Ca2+ exchange is also diminished relative to Ca2+, supporting approximately 60% of maximal currents obtainable with Ca2+. Ba2+ is also much less effective at alleviating Na+i-induced inactivation of NCX1. These results indicate that the reduced ability of NCX1 to adequately exchange Na+ and Ba2+ contributes to failure of the relaxation process in the cardiac muscle.
Figure 2. The effects of replacing 1 mM extracellular Ca2+ with 1 mM Ba2+ on electrically stimulated shortening for a canine ventricular myocyte are shown. Upon substituting Ba2+ for Ca2+, shortening rapidly fails, and a sustained contracture develops. Restoration of extracellular Ca2+ leads to near full recovery of resting length and shortening.
Figure 3. (A) Illustrates typical inward currents activated by the application of cytoplasmic Ca2+ or Ba2+. The pipette solution contained 100 mM Na+. Pooled results from three to nine patches (normalized to the current value obtained at 3 μM Ca2+i in nine patches) (means ± SE) are shown in B.
Figure 5. The effects of different concentrations of regulatory Ca2+ or Ba2+ on outward Na+-Ca2+ exchange currents are shown for a single patch in A and B. The pipette solution contained 8 mM Ca2+. The different concentrations of regulatory Ca2+ (A) or Ba2+ (B) were present before and during the application of 100 mM Na+ to activate the current. Typical concentration dependencies of peak and steady-state (SS) outward currents are shown for regulation by Ca2+ (C) and Ba2+ (D) from two separate patches. Note the difference in concentration range between these graphs.
Figure 4. Typical outward Na+-Ba2+ (A and B) and Na+-Ca2+ (C and D) exchange currents examined at 30°C (left) and 37°C (right). The pipette solution contained 8 mM Ba2+ or Ca2+ and currents were activated by the application of 100 mM Na+ at the indicated concentrations of regulatory Ca2+i. Data were obtained by making measurements at 30°C followed by increasing bath temperature and repeating measurements at 37°C (5–7 min later).
Figure 6. Regulation of outward Na+-Ca2+ exchange currents by Ba2+ and Ca2+ (inset). Pooled results (mean ± SD) from three to nine determinations in nine separate patches. Currents were normalized to the value of current obtained at 3 μM regulatory Ca2+i (in all 9 patches).
Figure 7. The relationship between peak and steady-state outward Na+-Ca2+ exchange currents regulated by Ca2+i (filled squares) and Ba2+i (open squares) is shown for pooled results from three to nine patches (mean ± SD). For Ca2+i regulation, steady-state current approaches peak current levels reflecting the progressive reduction in I1 inactivation. In contrast, I1 inactivation is not attenuated by regulatory Ba2+i.
Figure 8. Typical deregulated outward Na+-Ca2+ exchange currents from a single patch. The pipette contained 8 mM Ca2+ and currents were activated by the application of 100 mM Na+. Both I1 and I2 regulation are absent after deregulation and the inhibitory effects of different concentrations of Ca2+ and Ba2+ are evident. Pooled results from four patches (mean ± SD) are shown in B. Currents were normalized to the value of current obtained in the absence of regulatory divalent cations.
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