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Figure 2. Voltage-activated Na+ current in giant oocyte membrane patches. The pipette and the cytoplasmic solutions contain 40 mM Na+. (A) Depolarization from â40 to +40 mV activates an outward current over the course of 10 s, and the current deactivates without generating a significant tail current with signal filtering at 20 Hz. (B) Steady state currentâvoltage relation illustrating the steep voltage dependence of current activation. (C) Currentâvoltage relations under the same conditions, using 50-ms voltage steps as described in the text. After application of ATP (+ATP), the current is roughly doubled, runs down to less than control (after removal of ATP), and can be inhibited further with 20 μM Al3+ in the presence of 10 mM EGTA. The cytoplasmic solutions contained 0.1 mM Fâ and 50 μM vanadate.
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Figure 3. Nucleotide and deduced amino acid sequences of the squid Na+âCa2+ exchanger clone, NCX-SQ1. Sequences have been submitted to GenBank under accession number U93214.
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Figure 4. Amino acid comparison of the squid NCX-SQ1 and the canine NCX1 exchanger. Putative transmembrane segments, predicted by hydropathy analysis, are underlined and numbered. Highlighted in bold lettering are a potential signal peptidase site (SigPase), potential N-linked glycosylation sites (NXS/T), and potential phosphorylation sites (RTIK, protein kinase C; TRKLT, cAMP-dependent kinase and Ca2+/calmodulin-dependent kinase; DEHFY and DDEEEY, tyrosine kinase). The two potential phosphorylation sites marked with an asterisk are unique to NCX-SQ1. The endogenous exchanger inhibitory peptide (XIP) region and Exon A are shaded, and the binding domain for regulatory Ca2+ is boxed. The triple aspartate motifs involved in Ca2+ binding are in bold. Dots in the NCX1 sequence indicate amino acids identical to those of NCX-SQ1.
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Figure 5. (left) Northern blot analysis of NCX-SQ1 RNA. mRNA (1 μg) from squid optical lobe (lane 1) and stellate ganglia (lane 2) was probed with a fragment of the NCX-SQ1 cDNA. (right) Western blot analysis of NCX-SQ1 protein. Protein from squid optical lobe vesicles (lane 1) and oocytes injected with water (lane 2) or cRNA for NCX-SQ1 (lane 3) was probed with an antibody raised against a histidine-tagged fusion protein fragment of NCX-SQ1.
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Figure 6. Functional expression of NCX-SQ1 in Xenopus oocytes. Oocytes injected with cRNA for NCX-SQ1 (A) or control (C), water-injected oocytes were assayed for Na+âCa2+ exchanger activity. 45Ca2+ uptake into Na+ (90 mM)-loaded oocytes was measured in cells diluted into 45Ca2+-containing medium in the presence (extracellular K+) or absence of an outwardly directed Na+ gradient (extracellular Na+). In the middle pair of columns (B), 46 nl of a 100 mM EGTA solution was injected into the oocytes before loading the cells with Na+ to deplete intracellular Ca2+.
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Figure 7. Outward Na+âCa2+ exchange current of NCX-SQ1 in an excised oocyte membrane patch. Cytoplasmic solution with Na+ (40 mM) was applied and removed as indicated, first with 1.0 μM free cytoplasmic Ca2+, then with no cytoplasmic Ca2+, and finally with 5 μM free cytoplasmic Ca2+. The outward current, activated by application of Na+, inactivates partially over 10â50 s. The current is reduced by â¼50% in Ca2+-free solution. In the final sequence, α-chymotrypsin (1 mg/ml) was applied in the absence of cytoplasmic Ca2+. After complete activation by chymotrypsin, the current is insensitive to changes of cytoplasmic free Ca2+ in the micromolar range, and inactivation is abolished.
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Figure 8. Stimulation of NCX1 outward exchange current in an oocyte patch by Mg-ATP. Cytoplasmic Na+-containing solution (40 mM) was applied and removed as indicated in the presence of 0.5 μM free cytoplasmic Ca2+. The solutions contain 0.2 mM Fâ and 50 μM vanadate. (A) Application of 2 mM Mg-AMP-PNP is without effect, whereas 2 mM Mg-ATP stimulates the current to a magnitude somewhat greater than the initial peak current on application of cytoplasmic Na+. The stimulatory effect reverses partially over 2 min. (B) Application of 2 mM Mg-ATPγS has only a small stimulatory effect compared with 2 mM Mg-ATP, applied subsequently. The stimulatory effect reverses by â¼20% over 2 min, and it reverses almost completely in 1 min on application of PIP2 antibody (AB).
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Figure 9. Stimulation of NCX-SQ1 outward exchange current in oocyte patches by Mg-ATP. Cytoplasmic solution with Na+ (40 mM) was applied and removed as indicated in the presence of 0.5 μM free cytoplasmic Ca2+. (A) Stabilization of stimulatory effect of ATP by Fâ and vanadate. First, ATP was applied and removed in the absence of Fâ and vanadate; the stimulatory effect of 2 mM Mg-ATP decays by â¼60% over 1 min after removal of ATP. Next, ATP was applied and removed in the presence of Fâ and vanadate; after removal of ATP, the stimulatory effect is nearly stable for >1 min. (B) Reversal of the ATP effect by PIP2 antibody (PIP2-AB) in the presence of Fâ and vanadate. Exchange current was activated by applying Na+, Mg-ATP was applied for 1 min, ATP was removed for 1 min, and finally PIP2-AB was applied. The stimulatory effect of ATP is stable after removal of ATP, but decays by â¼80% 90 s after application of antibody.
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Figure 10. Stimulation of outward NCX-SQ1 Na+âCa2+ exchange current by Mg-ATP-γ-S. (A) After activation of exchange current by applying Na+-containing solution, 2 mM Mg-AMP-PNP was applied for 1 min, resulting in no stimulatory effect. Then, 2 mM Mg-ATP-γ-S was applied, resulting in stimulation of the exchange current to above the initial peak obtained on applying Na+. The effect reversed on removal of nucleotide over â¼3 min. (B) After activating exchange current by applying 40 mM Na+-containing solution, the current was stimulated by applying 2 mM Mg-ATP-γ-S. On removal of the nucleotide, current is stable for 30 s. The stimulatory effect reverses in â¼1 min on application of PIP2 antibody (AB).
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Figure 11. Stimulation of outward NCX-SQ1 Na+âCa2+ exchange current by PIP2. (A) Current was activated by cytoplasmic Na+ and was then allowed to run down to <5 pA. PIP2 was applied and the outward current increased over 5 min to a magnitude more than twofold greater than the peak current on application of Na+. Currentâvoltage relations were taken just after application of PIP2 (1), after the maximum stimulatory effect was obtained (2), and after exchange current was turned off by removing cytoplasmic Na+ (3). (B) Currentâvoltage relation of the exchange current. The exchange current is defined by subtracting records before PIP2 application from those with PIP2 (2-1, â¢) and those after removing Na+ from those with Na+ plus PIP2 (2-3, â). (C) Comparison of current transients obtained on activating exchange current before (control) and after (+PIP2) applying PIP2. The time constant (t) of inactivation increases from 3.9 to 8.2 s. Results are from a different patch.
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Figure 12. Lack of effect of phosphoarginine (p-ARG) on NCX-SQ1 exchange current in an excised oocyte patch. After activation of the exchange current by applying Na+-containing solution, 5 mM phosphoarginine was applied with 3 mM Mg2+ (pH 7.0). There is no stimulatory effect, whereas application of 50 μM PIP2 strongly stimulates the exchange current to a magnitude more than twofold greater than the peak obtained on applying Na+ initially.
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Figure 13. Outward NCX-SQ1 Na+âCa2+ exchange current in a chymotrypsin-treated patch. The pipette solution contains 4 mM Ca2+ and no Na+; the cytoplasmic solution contains 10 mM EGTA and no Ca2+. (A) Currentâvoltage relations at the given Na+ concentrations from 5 to 90 mM. Data points for descending and ascending voltage steps show no hysteresis. (B) Cytoplasmic Na+ dependence of outward exchange current at +60 and â60 mV. The data points are fit to a Hill equation; the slope is 1.2 at +60 and 1.7 at â60 mV; the K50 is 27 mM at +60 mV and 24 mM at â60 mV.
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Figure 14. Comparison of inward NCX1 and NCX-SQ1 Na+â Ca2+ exchange currents in chymotrypsin-treated patches. The pipette solution contains (mM): 120 Na+, 10 EGTA, 20 Cs+, 20 HEPES, 4 Mg2+, and no Ca2+ (pH 7.0 with NMG); the cytoplasmic solution contains 10 mM EGTA and no Na+. The inward currentâ voltage relations are defined by subtracting records with Ca2+ from records without Ca2+. In descending order, the currentâvoltage relations are with 0.2, 2, 10, and 300 μM Ca2+. (A) Currentâvoltage relations for NCX1. (B) Currentâvoltage relations for NCX-SQ1. Same batch of oocytes as in A. (C) Cytoplasmic Ca2+ dependence of the inward NCX1 exchange current at â150 and â30 mV. The K50, indicated by an arrow, is 2.5 μM at â30 mV and 4.2 μM at â150 mV. (D) Cytoplasmic Ca2+ dependence of the inward NCX-SQ1 exchange current at â150 and â30 mV. The K50 is 3.5 μM at â30 mV and 7.2 μM at â150 mV.
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Figure 15. Identification of electrogenic reactions of NCX1 and NCX-SQ1 using concentration jumps. (A) Current transients recorded from NCX1-expressing patch when 40 mM cytoplasmic Na+ is applied and removed in the presence of 20 mM extracellular Na+. (B) Typical lack of current transients recorded from NCX-SQ1-expressing patch when 40 mM cytoplasmic Na+ is applied and removed in the presence of 20 mM extracellular Na+. (C) Inward NCX1 current activated when a solution with 5 μM free Ca2+ is applied as in A. (D) Inward NCX-SQ1 current activated when a solution with 5 μM free Ca2+ is applied as in B. (E) Typical lack of current transients for a Ca2+ jump to 5 μM free Ca2+ in NCX1- expressing patch; 50 μM extracellular Ca2+. (F) Current transients recorded from NCX-SQ1-expressing patch when a solution with 5 μM free Ca2+ is applied and removed in the presence of 50 μM extracellular Ca2+. (G) Outward current activated by applying 40 mM Na+ to an NCX1 patch with 50 μM extracellular Ca2+. (H) Outward current activated by applying 40 mM Na+ to an NCX1 patch with 50 μM extracellular Ca2+. See text for details.
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Figure 16. Charge movements related to Ca2+ transport by NCX-SQ1 Na+âCa2+ exchanger. (A) In the presence of 4 mM extracellular Ca2+, charge records were taken with and without 2 μM cytoplasmic free Ca2+. The holding potential was â40 mV, and potential was stepped for 1 ms to different values in 40-mV steps. Membrane current is the first derivative of these records. Signals were essentially flat at the same amplification in control oocyte patches. (B) Voltage dependence of the Ca2+-dependent charge movements recorded in A. The Boltzmann slope (q) of the fitted Boltzmann function was 0.46. (C) Voltage dependence of rates of the charge movements, determined by fitting the charge transients to single exponential functions. The Boltzmann slope (q) of the fitted sum of two exponentials is 0.59. See text for details.
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