Distinct Mechanisms of Sodium–Calcium Exchanger NCX1.4 Regulation by Ca2+, ATP, and PIP2

Figures

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  Figure 1. Overall structural and functional features of NCX1.4. A schematic illustration of the inactivation (a) and deactivation (b) assemblies, derived from cryo-EM structures of NCX1.1 [10] and NCX1.3 [11]. Structural elements not resolved by cryo-EM are shown with dashed lines. Cyan and white cylinders in panels A and B represent the ion-transporting helices (TM2, TM3, TM7, and TM8) and the two-helix sliding bundle (TM1/TM6), respectively. Red ellipses in A and B depict assembly and disassembly of the inhibitory complex (for more details, see the text). (c) Representative traces of NCX1.4 outward current triggered by 1 mM extracellular calcium for 10 s at the indicated fixed concentrations of cytosolic free Ca2+ (0, 0.1, 1.0, and 10 µM) in the whole-cell configuration of the patch-clamp technique (holding potential 0 mV). (d) Representative traces of NCX1.4-mediated outward currents obtained after multiple stimulations of the same cell with 1 µM cytosolic free Ca2+. The first (red) and second (green) sweep recordings indicate a change in the time-to-peak ratio. (e) The NCX1.4 outward current (black trace) is completely suppressed by 10 µM NCX1 inhibitor, SEA0400 (red trace). (f)…

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  Figure 2. ATP or PIP2 reduces Ipeak and Iss, but not Iss/Ipeak, when both CBDs are Ca2+-unoccupied at 0 µM cytosolic free Ca2+. NCX1.4 was activated with 1 mM extracellular Ca2+ for 10 s. (a) To ensure that free intracellular Ca2+ was tightly and rapidly controlled to near zero, we used 5 mM BAPTA instead of 5 mM EGTA. (Left) Without intracellular ATP or PIP2, NCX1.4 has an activity of 4.1 ± 0.6 pA/pF (n = 6). However, 1 mM ATP or 10 μM PIP2 significantly decreases the NCX1.4 activity to 0.82 ± 0.51 pA/pF (n = 5) and to 0.68 ± 0.43 pA/pF (n = 5), respectively; Kruskal–Wallis test, * p < 0.05. (Middle) Without intracellular ATP and PIP2, NCX1.4 has a steady-state current of 1.39 ± 0.29 pA/pF (n = 6), while in the presence of 1 mM intracellular ATP or PIP2, this decreases significantly to 0.27 ± 0.16 pA/pF (n = 5) and 0.22 ± 0.14 (n = 5), respectively; Kruskal–Wallis, * p < 0.05. (Right) Fractional activity of NCX1.4 steady-state currents without intracellular ATP and PIP2 (0.39 ± 0.12, n = 6), with 1 mM intracellular ATP (0.14 ± 0.83, n = 5) or with 10 μM PIP2 (0.13 ± 0.08, n = 5); Kruskal–Wallis test, p > 0.05. (b) NCX1.4 was activated by perfusion of 1 mM extracellular calcium for 10 s.…

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  Figure 3. Effect of PIP2 or ATP on NCX1.4 outward currents at 0.1 μM cytosolic free Ca2+. NCX1.4 outward currents were elicited by rapid perfusion with 1 mM extracellular Ca2+ for 10 s. In contrast to conditions at 0 µM cytosolic Ca2+, at 0.1 µM cytosolic Ca2+, neither 1 mM ATP, 10 µM PIP2, nor 1 mM ATP + 10 µM PIP2 suppressed the outward NCX1.4 currents. At 0.1 µM cytosolic Ca2+, peak currents of 7.7 ± 1.8 pA/pF (a) and steady-state currents of 2.0 ± 0.5 pA/pF (b) were observed in the absence of ATP or PIP2 (n = 9). Neither ATP, PIP2, nor ATP+PIP2 had a significant effect on the Ipeak (a), Iss (b) or Iss/Ipeak (c) parameters of NCX1.4-mediated outward currents. (d) Representative traces of NCX1.4 outward currents in the absence or presence of ATP or PIP2.

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  Figure 4. ATP and PIP2 do not affect NCX1.4 outward currents when Ca2+ binds to CBD1. NCX-mediated outward currents were measured at 1 µM free cytosolic Ca2+ in the presence or absence of 1 mM cytosolic ATP or 10 µM PIP2. (a,c): At 1 µM cytosolic Ca2+ and in the absence of ATP and PIP2, peak currents were significantly larger than those at 0 µM cytosolic Ca2+ (9.3 ± 1.2 pA/pF [n = 11] versus 6.2 ± 0.9 pA/pF [n = 16]; Mann–Whitney test * p = 0.0460). (b,d): Although steady-state currents at 1 µM Ca2+ were slightly larger than those at 0 µM Ca2+, these differences were not significant (2.28 ± 0.41 pA/pF [n = 11] versus 1.19 ± 0.17 pA/pF [n = 16]; Mann–Whitney test p = 0.0515). At 1 µM Ca2+, neither ATP, PIP2, nor ATP+PIP2 affected the peak (a) and steady-state currents (b) or the fraction (e) of steady-state currents (Iss/Ipeak) of the NCX1.4-mediated reverse (Ca2+-entry) mode. (f): Representative traces of NCX1.4-mediated currents in the absence or presence of ATP or PIP2.

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  Figure 5. ATP and PIP2 do not affect NCX1.4 outward currents when Ca2+ occupies both CBDs. NCX1.4-mediated outward currents were measured at 10 µM free cytosolic Ca2+, under conditions in which Ca2+ occupies CBD1 and CBD2. (a,c) At 10 µM cytosolic Ca2+ and in the absence of ATP and PIP2, peak currents were significantly smaller than those at 1 µM cytosolic Ca2+ (3.8 ± 1.8 pA/pF [n = 8] versus 9.3 ± 1.2 pA/pF [n = 12]; Mann–Whitney test * p = 0.0159). (b,d) Although the steady-state currents recorded at 10 µM Ca2+ were somewhat lower than those at 1 µM cytosolic Ca2+, this difference was not significant (1.08 ± 0.26 pA/pF [n = 8] versus 2.28 ± 0.41 pA/pF [n = 11]; Mann–Whitney test p = 0.0620). At 10 µM cytosolic Ca2+, neither ATP nor PIP2 had a significant effect on the peak (a) or steady-state (b) currents. (e) At 10 µM Ca2+, the fraction of the steady-state current (Iss/Ipeak) was significantly larger than that observed at 1 µM Ca2+. (f) Representative traces of NCX1.4 outward currents in the absence or presence of ATP or PIP2.

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  Figure 6. Proposed mechanism of Ca2+- ATP- and PIP2 regulatory effects on NCX1.4-mediated outward current. A schematic illustration of the proposed conformational changes associated with the sequential occupation of CBD1 and CBD2 at 0 μM (a), 1 μM (b), and 10 μM (c) cytosolic Ca2+ in the presence or absence of ATP and PIP2. Based on recorded NCX1.4-mediated outward currents, it is proposed that at zero cytosolic Ca2+ (b), NCX1.4 retains substantial ion-exchange activity (perhaps due to cytosolic loop–lipid membrane interactions). Adding ATP or PIP2 in the absence of Ca2+ suppresses the NCX1.4 peak current, establishing baseline activity (possibly due to destabilization of cytosolic loop–lipid membrane interactions). At 1 μM Ca2+ (b), Ca2+ binding to CBD1 increases the NCX1.4 peak current (since the cytosolic loop–lipid membrane interactions may become more stable). Neither ATP nor PIP2 has a significant effect on the NCX1.4 outward current at 1 μM Ca2+. (c) At 10 μM Ca2+, Ca2+ binding to CBD2 significantly decreases NCX1.4 peak currents while increasing the fraction of steady-state current (Iss/Ipeak), suggesting that a probable occupancy of CBD2 by Ca2+ destabilizes the inactivation…