Аннотация
The activity of the cardiac Na$^+$-Ca$^2+$ exchanger (NCX1.1)
is allosterically regulated by Ca$^2+$, which binds to two acidic
regions in the cytosolically disposed central hydrophilic domain
of the NCX protein. A mutation in one of the regulatory Ca$^2+$
binding regions (D447V) increases the half-activation constant (K(h))
for allosteric Ca$^2+$ activation from approximately 0.3 to >
1.8 microm. Chinese hamster ovary cells expressing the D447V exchanger
showed little or no activity under physiological ionic conditions
unless cytosolic Ca$^2+$ was elevated to > 1 microm. However,
when cytosolic Na$^+$ was increased to 20 mm or more (using
ouabain-induced inhibition of the Na$^+$,K$^+$-ATPase or
the ionophore gramicidin), cells expressing the D447V mutant rapidly
accumulated Ca$^2+$ or Ba$^2+$ when the reverse (Ca$^2+$
influx) mode of NCX activity was initiated, although initial cytosolic
Ca$^2+$ was < 100 nm. Importantly, the time course of Ca$^2+$
uptake did not display the lag phase that reflects allosteric Ca$^2+$
activation of NCX activity in the wild-type NCX1.1; indeed, at elevated
Na$^+$, the D447V mutant behaved similarly to the constitutively
active deletion mutant Delta(241-680), which lacks the regulatory
Ca$^2+$ binding sites. In cells expressing wild-type NCX1.1,
increasing concentrations of cytosolic Na$^+$ led to a progressive
shortening of the lag phase for Ca$^2+$ uptake. The effects of
elevated Na$^+$ developed rapidly and were fully reversible.
The activity of the D447V mutant was markedly inhibited when phosphatidylinositol
4,5-bisphosphate (PIP2) levels were reduced. We conclude that when
PIP2 levels are high, elevated cytosolic Na$^+$ induces a mode
of exchange activity that does not require allosteric Ca$^2+$
activation.
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