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NAADP activates a Ca²⁺ current that is dependent on F-actin cytoskeleton ¹

Laboratory of Cell Biology, Stazione Zoologica "Anton Dohrn," Naples, Italy

²Correspondence: Laboratory of Cell Biology, Stazione Zoologica "Anton Dohrn," Villa Comunale I-80121, Naples, Italy. E-mail: santella{at}szn.it

SPECIFIC AIMS

NAADP is involved in the Ca²⁺ response observed at fertilization in the oocytes of several species, including starfish. In this study, we have used Ca²⁺ imaging and the single-electrode voltage-clamp technique with the aim to investigate whether the NAADP-mediated Ca²⁺ entry in starfish oocytes discovered in our laboratory was underlain by a membrane current and whether the Ca²⁺ response to NAADP required an intact cytoskeleton.

PRINCIPAL FINDINGS

1. NAADP-induced Ca²⁺ wave in starfish oocytes
Starfish oocytes were preinjected with caged NAADP (100 µM in the pipette) and the fluorescent Ca²⁺ indicator Oregon green 488 BAPTA-1 coupled to a 10 kDa dextran (OGBD; 0.05 mg/mL final concentration). The uncaging reaction was performed by exposing the oocytes to UV light for 15 s and the Ca²⁺ response was monitored by a cooled CCD camera. In oocytes bathed in artificial seawater (ASW), photolysis of NAADP triggered a cortical flash, which enveloped the entire cortical region of the oocyte (cortical flash), with a latency of 10.2 ± 1.8 s (n=8). The flash then centripetally spread from the cortex to the whole oocyte at a rate of 153.7 ± 26.4 µm/s (n=7). The Ca²⁺ response reached a peak of 0.63 ± 0.11 arbitrary units (n=8) and decayed in 3–20 min. No increase in [Ca²⁺]_i could be detected upon uncaging NAADP in Ca²⁺-free seawater (CaFSW). However, the intracellular mobilization of Ca²⁺ by NAADP cannot be ruled out, as the Ca²⁺ imaging method we used may not be sensitive enough to detect small and highly localized Ca²⁺ release close to the plasma membrane.

2. NAADP activates a Ca²⁺-mediated membrane current
In the next set of experiments, oocytes were impaled with a single microelectrode 3–15 min after the injection of caged NAADP. The resting membrane potential was measured in the current-clamp mode and its mean value was -69.9 ± 1.2 mV (n=36). In oocytes held at -70 mV, photolysis of NAADP induced the activation of a membrane current with a latency of 2.04 ± 0.06 s (n=9) (Fig. 1 A, upper trace). The current reached the peak in 12.6 ± 4.2 s (n=10) and lasted for 8.28 ± 2.53 min (n=8). The current-voltage (I-V) relation of the NAADP-elicited current was calculated by measuring the peak of the responses in oocytes clamped at -70, -40, -10, +20, and +50 mV (Fig. 1A ). The I-V relation showed a strong inward rectification and reversed at potentials more positive than +50 mV (Fig. 1B ), two features that resemble those of the pure Ca²⁺-mediated currents I_CRAC and I_ARC, but different from those of the cationic current activated by Ca²⁺ release from InsP₃ and cADPr/ryanodine stores in starfish oocytes. The membrane current became smaller and smaller upon subsequent uncaging of NAADP (n=3; Fig. 1C , trace b), suggesting desensitization of the NAADP receptors. The same result was obtained when measuring the Ca²⁺ sweep triggered by NAADP (n=4; data not shown). Removal of external Ca²⁺ abolished the current, while replacement of external Na⁺ with an equimolar amount of choline did not affect it. Finally, the membrane current activated by NAADP was significantly reduced after 15 min of pretreatment of oocytes with the Ca²⁺ channel inhibitor of receptor-operated calcium entry SK&F 96356 (10 µM in the bath) and the L-type calcium channel blocker verapamil (100 µM in the bath).

View larger version (19K): [in this window] [in a new window]   Figure 1. NAADP activates a membrane current in starfish oocytes. A) Membrane currents evoked by NAADP (100 µM) at different holding potentials. Each trace was obtained on the same day by different oocytes extracted by the same animal. The UV flash was given at the time indicated by the arrow. B) I-V relation of the peak current induced by NAADP (n=3–4 for each potential). C) Trace a: current was elicited when oocytes were exposed to UV without previous injection of NAADP; trace b: the membrane current became smaller and smaller upon subsequent uncaging of NAADP. The UV flash was given at the time indicated by the arrow.

3. Heparin and 8-NH₂-cADPr do not affect the membrane current
It has been shown that the Ca²⁺ increase evoked by NAADP is amplified by InsP₃- and cADPr/ryanodine receptors through a CICR process. However, preinjection of heparin (0.5 mg/mL final concentration), an inhibitor of InsP₃ receptors, 8-NH₂-cADPr (4 µM final concentration), an antagonist of cADPr, or both did not significantly affect the membrane current elicited by NAADP. The concentrations of heparin and 8-NH₂-cADPr we used have previously been shown to block the response to InsP₃ and cADPr, respectively.

4. Role of intracellular Ca²⁺ in the stimulation of the membrane current evoked by NAADP
Preincubation of oocytes with thapsigargin (5 µM) significantly reduced both the amplitude and duration of the NAADP-induced current (n=3), while preinjection of the Ca²⁺ chelator BAPTA (20 µM final concentration) prevented its onset (n=5). To investigate whether the activation of the current required Ca²⁺ release from intracellular Ca²⁺ stores sensitive to NAADP but not to InsP₃ and cADPr, [Ca²⁺]_i was increased by photoliberating Ca²⁺ from NP-EGTA (10 µM final concentration) for 8–10 min. In 3 of 11 oocytes, the intracellular Ca²⁺ elevation induced a transient inward current, which was clearly different from that triggered by NAADP, but similar to that activated by Ca²⁺ release from InsP₃ and cADPr/ryanodine-sensitive stores. To explain these findings, we suggest that NAADP triggers a highly localized cortical Ca²⁺ increase from a thapsigargin-sensitive store that is tightly coupled to the NAADP-dependent membrane channels. The activation of the latter would then require both the submembrane Ca²⁺ pulse and NAADP itself.

5. Impairment of actin cytoskeleton reduces the Ca²⁺ response to NAADP
As maintenance of the filamentous actin cytoskeleton is required for the activation of several Ca²⁺-permeable ionic channels, oocytes were first pretreated with latrunculin A (3 µM), which depolymerizes F-actin bundles. The photoliberation of NAADP in treated oocytes activated a cortical Ca²⁺ flash with a latency of 8.46 ± 1.2 s (n=5), which was not significantly different from that observed in control cells (6.60 ± 0.54 s, n=7). However, the peak of the Ca²⁺ response to NAADP in latrunculin A-treated oocytes was significantly smaller than in control cells, the values being 0.018 ± 0.03 (n=5) and 0.41 ± 0.05 arbitrary units (n=5), respectively. Moreover, the Ca²⁺ wave propagated at a significantly slower speed (256.53 ± 58.30 µm/s, n=7) than in control oocytes (334.1 ± 76.63 µm/s, n=5). In accordance with these observations, the NAADP-activated current was inhibited by the addition of latrunculin-A. Indeed, the ttp, amplitude, and duration of the current in control cells were equal to 3.38 ± 0.37 s (n=5), -0.75 ± 0.13 nA (n=5), and 7.18 ± 1.02 min (n=5), respectively, while in latrunculin-A-treated cells their values were 5.36 ± 2.40 s (n=5), -0.13 ± 0.05 nA (n=5), and 1.32 ± 0.61 min (n=5). In a subsequent set of experiments, jasplakinolide was used to stabilize F-actin. Incubation of oocytes with jasplakinolide (12 µM) did not delay the onset of the cortical flash, but reduced both the amplitude of the global Ca²⁺ elevation (0.19 ± 0.06 arbitrary units, n=4) and the velocity of the Ca²⁺ wave (130.50 ± 23.52 µm/s, n=4). In accordance with this result, jasplakinolide (12 µM) significantly reduced the ttp, the amplitude and the duration of the membrane current elicited by NAADP (Fig. 2 A, C). Their values in jasplakinolide-treated cells were 6.99 ± 2.49 s (n=5), -0.17 ± 0.05 nA (n=5), and 0.5 ± 0.21 min (n=5), respectively.

View larger version (20K): [in this window] [in a new window]   Figure 2. Latrunculin-A and jasplakinolide reduced the membrane current activated by NAADP. Membrane currents elicited by NAADP (100 µM) in control oocytes (A) and oocytes incubated in presence of latrunculin-A (3 µM; B) and jasplakinolide (12 µM; C). The UV flash was given at the time indicated by the arrow. Note the reduction in both amplitude and duration of the current exerted by latrunculin A and jasplakinolide.

CONCLUSIONS AND SIGNIFICANCE

This study has shown for the first time that the Ca²⁺ wave triggered by NAADP in starfish oocytes is due to the activation of a Ca²⁺-mediated membrane current, whose biophysical features strongly resemble those of I_CRAC and I_ARC but are strikingly different from those of the nonselective cationic current triggered by InsP₃- and cADPr-dependent Ca²⁺ release in starfish oocytes. Therefore, we suggest that the NAADP-stimulated current represents a novel member in the family of the second messengers-activated Ca²⁺-selective currents. Besides the cortical flash observed in starfish oocytes, this Ca²⁺ current could also underlie the Ca²⁺ entry induced by NAADP in T lymphocytes and the cortical flash recorded in sea urchin eggs. In sea urchin and other cell types such as pancreatic human ß cells and ascidian oocytes, however, the main source for the Ca²⁺ response is an intracellular store, which has been located to the lysosomes or in the secretory vesicles. The reduction or the inhibition of the NAADP-induced current exerted by thapsigargin and BAPTA suggests the involvement of a highly localized Ca²⁺ pulse, produced by NAADP acting on thapsigargin-sensitive Ca²⁺ stores close to the plasma membrane, in the onset of the current in starfish oocytes, too. It is likely that such a Ca²⁺ release was too small and/or localized to be detected by our CCD camera. Ca²⁺ mobilized by NAADP as well as NAADP itself would then be required to trigger the Ca²⁺ current.

The initial Ca²⁺ influx induced by NAADP is intracellularly amplified by InsP₃- and cADPr/ryanodine receptors by a CICR mechanism, as also shown in pancreatic acinar cells and sea urchin eggs. This feature is suggested by the inhibition exerted by heparin and 8-NH₂-cADPr on the Ca²⁺ wave, but not on the membrane current.

The Ca²⁺ current and the ensuing Ca²⁺ wave induced by NAADP were reduced after depolymerization and stabilization of F-actin cytoskeleton. This inhibition demonstrates for the first time that the dynamic turnover of actin filaments is required for the onset of the response to NAADP (see Fig. 3 ). The suggested coupling between both the NAADP-dependent cortical stores and the plasma membrane channels would likely be affected by the impairment of actin turnover, thus explaining the inhibition exerted by latrunculin-A and jasplakinolide on the Ca²⁺ response.

View larger version (30K): [in this window] [in a new window]   Figure 3. Model suggesting the interaction between NAADP-sensitive channels and actin cytoskeleton. A Ca2+-permeable channel allowing Ca2+ entry into the cells is activated following NAADP uncaging. NAADP induces a localized Ca2+ increase from a cortical Ca2+ store. The mobilized Ca2+ and NAADP itself then trigger the Ca2+ current and the ensuing Ca2+ wave. Coupling between the cortical store and the Ca2+-permeable channel requires an intact actin cytoskeleton.

The activation of NAADP receptors triggers the Ca²⁺ wave observed at fertilization of Asterina pectinifera oocytes and sea urchin eggs. It is therefore likely that the Ca²⁺-mediated current activated by NAADP is involved in the fertilization potential, the cortical flash, and the Ca²⁺ wave observed at fertilization.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0178fje doi: 10.1096/fj.03-0178fje

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This Article Full Text (PDF) All Versions of this Article: 17/13/1907 03-0178fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MOCCIA, F. Articles by SANTELLA, L. Search for Related Content PubMed PubMed Citation Articles by MOCCIA, F. Articles by SANTELLA, L.