Matsuoka S et al. (1997), Regulation of cardiac Na(+)-Ca2+ exchanger by t...

XB-ART-16975

J Gen Physiol 1997 Feb 01;1092:273-86. doi: 10.1085/jgp.109.2.273.

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Regulation of cardiac Na(+)-Ca2+ exchanger by the endogenous XIP region.

Matsuoka S , Nicoll DA , He Z , Philipson KD .

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The cardiac sarcolemmal Na(+)-Ca2+ exchanger is modulated by intrinsic regulatory mechanisms. A large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A. Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and K.D. Philipson. 1991. J. Biol. Chem. 266:1014-1020) that a segment of the large intracellular loop, the endogenous XIP region, has an autoregulatory role in exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties. The major alteration was in a regulatory mechanism known as Na(+)-dependent inactivation. This inactivation is manifested as a partial decay in outward Na(+)-Ca2+ exchange current after application of Na+ to the intracellular surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Na+ affinities. Regulation of the exchanger by nontransported, intracellular Ca2+ was also modified by the XIP-region mutations. Binding of Ca2+ to the intracellular loop activates exchange activity and also decreases Na(+)-dependent inactivation. XIP-region mutants were all still regulated by Ca2+. However, the apparent affinity of the group 1 mutants for regulatory Ca2+ was decreased. The responses of all mutant exchangers to Ca2+ application or removal were markedly accelerated. Na(+)-dependent inactivation and regulation by Ca2+ are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement of the exchanger into and out of the Na(+)-induced inactivated state, but that the XIP region is also involved in regulation by Ca2+.

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	Figure 2. Fitting the Na+- dependent inactivation to two exponentials; examples for the wild-type (WT) and mutant F223E exchangers. The slow component (right-most dotted curve) was first fit by the least square method after subtracting the steady state component. The solid line through the data points corresponds to the fitted slow component. The intercept of the fitted line with the ordinate is a measure of Islow. After the slow component was fit, it was subtracted from the current trace, and the remaining current was fit by a second exponential (left-most dotted curve). The solid line through those data points corresponds to the fitted fast component and the intercept of that line with the ordinate corresponds to the magnitude of Ifast.
	Figure 3. Outward Na+-Ca2+ exchange currents at different cytoplasmic Na+ concentrations are shown for the wild type (WT) and representatives of group 1 (Y224T) and group 2 mutants (K229Q) and water-injected cells. Cytoplasmic Na+ concentrations were 6, 12, 25, 50, and 100 mM from the lower trace to the upper trace for all exchangers. The period of Na+ application is indicated by the crosshatched bar. Dotted lines indicate 0 current level.
	Figure 4. Na+ dependence of the Na+-Ca2+ exchanger transport cycle. Currents were normalized at the peak current for the wild type and group 1 mutants and normalized at 2 s after Na+ application for the group 2 mutants. Data (mean ± SD) of the wild type (open circles), a representative group 1 mutant (K225Q, filled circles) and a representative group 2 mutant (229(QQTQ)232, filled squares) are shown as examples. Curves are fits to the Hill equation.
	Figure 5. Na+ dependence of the Na+-dependent inactivation. (A) Na+ concentration-Fss relation for group 1 mutants. Fss values, the ratio of steady-state to peak currents, are plotted versus different Na+ concentrations. See text for detail. (B) Fss values of group 2 mutants. Fss values of group 2 mutants are plotted against different Na+ concentrations. It is notable that Fss is close to 1.0 at all Na+ concentrations studied.
	Figure 6. Na+-dependence of wild-type and group 2 mutants. (A) Peak and steady-state Na+ dependence of the wild-type Na+-Ca2+ exchanger. Normalized peak (open circles) and steady-state (filled circles) currents are plotted as a function of cytoplasmic Na+. Curves are fits to the Hill equation. See text for details. (B) Steady-state Na+ dependence of group 2 mutants. Normalized steady state currents are plotted in a similar manner as for the wild-type exchanger.
	Figure 7. Recording of outward and inward Na+-Ca2+ exchange currents in excised patches from oocytes expressing wild-type and K229Q Na+-Ca2+ exchangers. The pipette solution contained both Na+ (140 mM) and Ca2+ (2 mM). Cytoplasmic Na+ and Ca2+ concentrations are noted under the current traces. (A) Recording of outward (application of Na+) and inward (application of Ca2+ in the absence of Na+) currents from the wild-type exchanger. (B) Recordings of outward and inward currents from mutant exchanger K229Q. Note the amplitude ratio of the outward to the inward currents. Also, for the two exchangers compare the time courses of activation of the inward current by (a) addition of intracellular Ca2+ and (b) removal of intracellular Na+.
	Figure 8. Effect of intracellular Ca2+ on activation of the outward Na+-Ca2+ exchange current in the wild-type (WT), F223E (a group 1 mutant), and K229Q (a group 2 mutant) exchangers. The currents were induced by applying 100 mM cytoplasmic Na+ in the presence of 0, 0.1, 0.5, 1, and 10 μM cytoplasmic Ca2+.
	Figure 9. Cytoplasmic Ca2+ dependence of peak currents from wild-type (WT) and group 1 (Y226T, F223E) exchangers and steady-state currents from a group 2 (229(QQTQ)232) exchanger. Steady-state currents were measured ∼40 s after 100 mM Na+ application. The curves were normalized to the fitted maximal current.
	Figure 10. Cytoplasmic Ca2+ dependence of steady state currents from group 1 mutants (A and B). Curves are fits to the Hill equation. (C) Inhibition of Na+-dependent inactivation by intracellular Ca2+. Fss values are plotted against the Ca2+ concentration. The data at 0.01 μM Ca2+ (WT, Y224T, and F223E) and at 0.1 μM Ca2+ (F223E) were omitted for fitting to the Hill equation.
	Figure 11. Responses of outward Na+-Ca2+ exchange currents in wild type (WT ) and mutant exchangers to removal and reapplication of 1 μM intracellular Ca2+. 100 mM Na+ was present in all cytoplasmic solutions.

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