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1Department of Physiology, Queen's University, Kingston, Ontario, Canada; 2Department of Pharmacology, Yale University, New Haven, Connecticut; and 3Department of Physiology and Biophysics, Boston University, Boston, Massachusetts
Submitted 2 February 2006; accepted in final form 24 July 2006
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ABSTRACT |
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INTRODUCTION |
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; Levitan and Kaczmarek 2002). Ca2+ can be released from intracellular stores, which in neuronal somata are principally the endoplasmic reticulum (Berridge 1998; Meldolesi 2001; Verkhratsky 2005), or Ca2+ can enter from the extracellular space through voltage-gated Ca2+ channels (Hille 2001; Reid et al. 2003). Because most nonneuronal/nonexcitable cells cannot employ the voltage sensitivity of Ca2+ channels, they gate Ca2+ influx through an alternative pathway known as capacitative Ca2+ entry or store-operated Ca2+ influx (Casteels and Droogmans 1981; Nilius 2003; Parekh 2003; Putney 1986). Although the mechanism is not fully defined, this pathway is activated when the depletion of Ca2+ from intracellular stores opens specific, Ca2+-permeable channels in the plasma membrane (Nilius 2003; Parekh 2003).
In comparison to nonneuronal cells, the characterization of store-operated Ca2+ influx in neurons is less extensive (Putney 2003). Initially, neurons, neuroendocrine cells, and neuronal cell lines were thought to lack store-operated Ca2+ influx (Friel and Tsien 1992; Jackson et al. 1988; Stauderman and Pruss 1989). However, beginning with a report of Ca2+ entry following store depletion in PC12 cells (Clementi et al. 1992), affirmative evidence began to accumulate (Arakawa et al. 2000; Baba et al. 2003; Bouron 2000; Grudt et al. 1996; Liu and Gylfe 1997; Piper and Lucero 1999; Powis et al. 1996; Prothero et al. 2000; Tozzi et al. 2003; Usachev and Thayer 1999; Villalobos and Garcia-Sancho 1995; Zufall et al. 2000). Nevertheless, reports continue to assert that certain neurons either lack or show limited store-operated Ca2+ influx (Chinopoulos et al. 2004; Emptage et al. 2001; Grimaldi et al. 2001; Koizumi et al. 1999). The ongoing controversy prompted us to explore this pathway in the bag cell neurons of the marine mollusk, Aplysia californica—a preparation in which the regulation of intracellular Ca2+ has been extensively investigated (Fink et al. 1988; Jonas et al. 1997; Knox et al. 1996, 2004; Magoski et al. 2000).
Brief synaptic input to the bag cell neurons triggers an 
30-min afterdischarge, resulting in neuropeptide secretion and the initiation of egg-laying behavior (Conn and Kaczmarek 1989; Kupfermann 1967; Kupfermann and Kandel 1970; Pinsker and Dudek 1977; Rothman et al. 1983). Previously, Knox et al. (1996) used a Ca2+-sensitive vibrating extracellular probe to shown that the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (Thastrup et al. 1990), may initiate Ca2+ entry in bag cell neurons. In the present study, we employ intracellular Ca2+ imaging to demonstrate that this indeed is a store-operated Ca2+ influx pathway. During an afterdischarge, Ca2+ enters through voltage-gated Ca2+ channels and is released from intracellular stores, apparently in an IP3-dependent manner (Fink et al. 1988; Fisher et al. 1994). The bag cell neurons may require store-operated Ca2+ influx to replete these intracellular stores or may use this source of Ca2+ entry to elicit neuropeptide secretion itself.
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METHODS |
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Adult A. californica weighing 100–250 g were obtained from Marine Specimens Unlimited (San Francisco, CA) or Marinus (Long Beach, CA). Animals were housed in an 300l aquarium containing continuously circulating, aerated artificial sea water (Instant Ocean, Aquarium Systems, Mentor, OH, or Kent sea salt, Kent Marine, Acworth, GA) at 14–16°C on an 12/12 h light/dark cycle and fed Romaine lettuce three to five times a week.
For primary cultures of isolated bag cell neurons, animals were anesthetized by an injection of isotonic MgCl2 (50% of body weight), the abdominal ganglion removed and treated for 18 h at 20–22°C with neutral protease (13.33 mg/ml; 165859, Roche Diagnositics, Indianapolis, IN) dissolved in tissue culture artificial sea water (tcASW; composition in mM: 460 NaCl, 10.4 KCl, 11 CaCl2, 55 MgCl2, 15 HEPES, 1 mg/ml glucose, 100 U/ml penicillin, and 0.1 mg/ml streptomycin, pH 7.8 with NaOH). The ganglion was then transferred to fresh tcASW and the bag cell neuron clusters dissected from their surrounding connective tissue. Using a fire-polished Pasteur pipette and gentle trituration, neurons were dispersed in tcASW onto regular 35 x 10-mm polystyrene tissue culture dishes (25000, Corning, Corning, NY) or glass coverslips (No. 1; 48366045, VWR, West Chester, PA) that were coated with poly-D-lysine (1 µg/ml, 70,000–150,000 MW; P0899, Sigma-Aldrich, Oakville, ON, Canada, or St. Louis, MO) and glued to drilled out tissue culture dishes. Cultures were maintained in tcASW for 1–3 days in a 14°C incubator. Salts were obtained from Fisher (Ottawa, ON, Canada), ICN (Aurora, OH), or Sigma-Aldrich.
Intracellular Ca2+ measurements
Somatic intracellular Ca2+ was measured by ratiometric imaging of the dye, fura PE3 (K+ salt; 0110, Teflabs, Austin, TX) (Vorndran et al. 1995). Fura-PE3 was pressure injected via sharp electrodes using either a Picospritzer II (General Valve) or a PMI-100 pressure microinjector (Dagan, Minneapolis, MN) while simultaneously monitoring membrane potential with an Axoclamp 2B amplifier (Axon Instruments, Union City, CA). Microelectrodes were pulled from 1.2-mm internal diameter, borosilicate glass capillaries (1B120F-4, World Precision Instruments, Sarasota, FL) and had a resistance of 30–50 M
when the tip was filled with 10 mM fura-PE3 then backfilled with 3 M KCl. Injections usually required 10–15 300- to 900-ms pulses at 30–60 kPa to fill the neurons with an optimal amount of dye—estimated to be 50–100 µM. All neurons used for subsequent imaging showed resting potentials of –50 to –60 mV and displayed action potentials that overshot 0 mV after depolarizing current injection (0.5–1 nA, directly from the amplifier). After dye injection, neurons were allowed to equilibrate for 
30 min. Ca2+ imaging was performed using a Nikon Diaphot inverted microscope (Nikon, Mississauga, ON, Canada) equipped with a Nikon Fluor 10X objective (numerical aperture (NA) = 0.5) or Nikon TS100-F inverted microscope equipped with a Nikon Plan Fluor 10X objective (NA = 0.3). The light source was a 75 W Xenon arc lamp and a multi-wavelength DeltaRAM V monochromatic illuminator (Photon Technology International, London, ON, Canada) coupled to the microscope with a UV-grade liquid-light guide. Between acquisition episodes, the excitation illumination was blocked by a shutter, which along with the excitation wavelength, was controlled by a IBM-compatible personal computer, a Photon Technology International computer interface, and ImageMaster Pro software (version 1.49, Photon Technology International). The emitted light passed through a 510/40 nm barrier filter prior to being detected by either a Hamamatsu C2400 (Hamamatsu, Bridgewater, NJ) or Photon Technology International IC200 intensified charge coupled device camera. The camera intensifier voltage was set based on the initial fluorescence intensity of the cells at the beginning of each experiment and maintained constant thereafter. The camera black level was set prior to an experiment using the camera controller such that at a gain of 1 there was a 50:50 distribution of blue and black pixels on the image display with no light going to the camera. The ratioed image of the fluorescence intensities (converted to pixel values) from 340 and 380 nm excitation wavelengths was derived and, if necessary, averaged four to eight frames per acquisition, resulting in a single full-frame (520 x 480 pixels) acquisition time of 0.5–4 s. A sample of the fluorescence intensities ratio was taken typically at 1 min intervals using regions of interests (ROIs) defined over the neuronal somata prior to the start of the experiment. The ratio was then either recorded simply as 340/380 to reflect free intracellular Ca2+ or used to calculate the actual free intracellular Ca2+ from the relationship, [Ca2+] = Kd · Q(R – Rmin)/Rmax – R (Grynkiewicz et al. 1985). Rmin, Rmax, and Q were determined in intact bag cell neurons by applying 1–10 µM digitonin (D-8449, Molecular Probes, Eugene, OR) under Ca2+-free conditions followed by perfusion with saline containing a saturating amount of Ca2+ (11 mM). The constant Q was determined from the ratio of 380 nm evoked fura PE3 fluorescence in Ca2+-free ASW and 11 mM Ca2+-containing normal ASW (nASW). Values for Rmin, Rmax, and Q ranged from 0.11 to 0.33, 5.1–7.5, and 42.6–50, respectively, whereas the Kd was 204 nM (from Vorndran et al. 1995). The black level determination, image acquisition, frame averaging, emitted light ROI sampling, and ratio calculations were carried out using the ImageMaster Pro software. Ratio calculations were saved for subsequent analysis (see following text). Imaging was carried out at room temperature (20–22°C) and performed in both nASW (composition as per tcASW but with the glucose, penicillin, and streptomycin omitted) and Ca2+-free ASW (composition as per nASW but with CaCl2 omitted and 0.5 mM EGTA added).
Membrane potential recordings
An Axoclamp 2B amplifier (Axon Instruments) in bridge mode was used to measure membrane potential. Current-clamp recordings were made using sharp microelectrodes (glass as per dye injection) with a resistance of 5–10 M when filled with 2 M K-acetate (supplemented with 100 mM KCl and 10 mM HEPES; pH = 7.3 with KOH). Voltage was low-pass filtered at 3 kHz using the Axoclamp 2B Bessel filter and acquired at a sampling rate of 100 Hz with an IBM-compatible personal computer, a Digidata 1300 A/D converter (Axon Instruments), and the Clampex acquisition program of pCLAMP (version 8.0; Axon Instruments).
Drug application and reagents
Drug application or solution exchanges were accomplished by manual perfusion using a plastic transfer pipette to exchange the bath (tissue culture dish) solution. Complete exchange of the bath could be achieved in <30 s. In some cases, drugs were introduced directly into the bath by pipetting a small volume (<10 µl) of concentrated stock solution or a larger volume of saline (500 µl) that was initially removed from the bath, mixed with the stock solution, and then reintroduced. Care was taken to perform all pipetting near the side of the dish and as far away as possible from the neurons. 2-aminoethyldiphenyl borate (2-APB; 100065, Calbiochem, San Diego, CA), bafilomycin A (B1793, Sigma-Aldrich), BTP2 (203890, Calbiochem), 4-chloro-3-ethylphenol (CEP; 279552, Sigma-Aldrich), cyclopiazonic acid (CPA; C1530, Sigma-Aldrich or 239805, Calbiochem), 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF 96365; 567310, Calbiochem or S7809, Sigma-Aldrich), thapsigargin (T3250, Sigma-Aldrich), and xestospongin C (CA409, Cedarlane, Hornby, ON, Canada) all required DMSO (BP231, Fisher) as a vehicle, whereas jasplakinolide (420107, Calbiochem) required methanol (A412, Fisher). The maximal final concentration of DMSO and methanol was 0.01 µM and 0.01%, respectively, which in control experiments had no effect on intracellular Ca2+. LaCl3 (L4131, Sigma-Aldrich) and NiCl2 (N6136, Sigma-Aldrich) were dissolved as a stock in water or to the final desired concentration in saline.
Analysis
Origin (version 7, OriginLab Corporation, Northampton, MA) was used to import and plot ImageMaster Pro files as line graphs. For display, intracellular Ca2+ concentrations or the 340/380 ratios were averaged and presented as the means ± SE versus time. Analysis used steady-state changes acquired by taking the average value, by eye or with adjacent-averaging, from regions that had reached steady state for 5–10 min in plots of individual experiments. Statistics were performed on percent change values using Instat (version 3.0, GraphPad Software, San Diego, CA). The Kolmogorov-Smirnov method was used to test data sets for normality. A standard ANOVA with the Dunnett's multiple comparisons test was used to test for differences between multiple means. In a few cases, Student's paired or unpaired t-test was used to test for differences between two means. Data were considered significantly different if the two-tailed P value was <0.05.
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RESULTS |
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To determine if Ca2+ store depletion can initiate a Ca2+ influx pathway, cultured bag cell neurons were bathed in Ca2+-free ASW and exposed to agents that liberate intracellular Ca2+. The smooth endoplasmic reticulum Ca2+ pump inhibitor, CPA (10–50 µM) (Seidler et al. 1989), depleted intracellular stores and caused Ca2+ to rise quickly (Fig. 1A; n = 12). Despite the continued presence of CPA, Ca2+ levels recovered to near-control levels, most likely attributable to active and passive removal of Ca2+ from the intracellular to the extracellular compartment (Clapham 1995; Knox et al. 1996; Meldolesi 2001; Verkhratsky 2005). In separate experiments, the subsequent addition of extracellular Ca2+ by exchanging the Ca2+-free ASW for nASW initiated a marked and rapid rise in intracellular Ca2+ but only in those neurons depleted with CPA and not those merely exposed to Ca2+-free ASW alone (Fig. 1B; n = 44 versus 11). This suggested that depletion of intracellular Ca2+ stores activates a plasma membrane Ca2+ entry pathway. Although this pathway is presumably open during depletion in Ca2+-free conditions, it cannot be detected until extracellular Ca2+ is added and Ca2+ begins to flow back into the neurons. Similar results were achieved with 2–3 µM of the irreversible, smooth endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin (Thastrup et al. 1990) (Fig. 1C; n = 15). On average, addition of extracellular Ca2+ after depletion with CPA resulted in an 47% increase in intracellular Ca2+ that was statistically different from the 25% increase observed following thapsigargin-induced depletion (Fig. 6; 2nd vs. 1st bar).
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; Knox et al. 1996; A. Y. Hung and N. S. Magoski, unpublished data). Activating ryanodine receptors does not initiate the store-operated Ca2+ influx pathway
Although CPA and thapsigargin liberate Ca2+ by blocking Ca2+ pumps on the endoplasmic reticulum, an alternate means is to open the Ca2+-permeable channels within the store. In Ca2+-free ASW, 100 µM of the ryanodine receptor agonist, CEP (Zorzato et al. 1993), rapidly elevated intracellular Ca2+, which then returned to near baseline levels even with maintained exposure to the agonist (Fig. 2A; n = 15). Subsequent delivery of extracellular Ca2+ caused, on average, intracellular Ca2+ to rise by only 5%, which was significantly different from the influx that occurred with CPA (Fig. 6; 3rd vs. 1st bar). When CPA was applied after CEP in Ca2+-free ASW, there was a small but detectable Ca2+ elevation as well as obvious store-operated Ca2+ influx on return to Ca2+-containing nASW (Fig. 2B; n = 8). Overall, addition of extracellular Ca2+ to neurons treated with CEP followed by CPA resulted in an 55% increase in intracellular Ca2+ that was not significantly different from that seen after CPA alone (Fig. 6; 4th vs. 1st bar). CEP was chosen as a ryanodine receptor agonist over caffeine (Rousseau et al. 1988; Weber 1968) or ryanodine (Meissner 1985) because we have found that the former has a number of nonspecific effects on bag cell neuron ion channels, whereas the latter is not an efficacious agonist of the bag cell neuron Ca2+ release channel—perhaps because of the high salt conditions (N. S. Magoski, R. J. Knox, and L. K. Kaczmarek, unpublished observation).
Ca2+ liberated from acidic stores replenishes stores depleted with CPA
The acidic compartment is a bag cell neuron Ca2+ store that has yet to be investigated. Bafilomycin A, a vacuolar H+-ATPase inhibitor (Bowman et al. 1988), caused bag cell neuron intracellular Ca2+ to slowly rise in Ca2+-free ASW when applied at 50 nM (Fig. 3A; n = 6). The source was designated an acidic store because Ca2+ was liberated after the bafilomycin-A-induced collapse of its H+ gradient, which is presumably required for maintaining Ca2+ uptake (Christensen et al. 2002; Dunn et al. 1994; Goncalves et al. 1999; Ohsumi and Anraku 1983). The time to onset of the bafilomycin-A-induced elevation in intracellular Ca2+ was shorter if the neurons were first exposed to CPA (Fig. 3B; n = 15); however, the mean change for bafilomycin A alone (39.7 ± 4.65% increase) versus CPA then bafilomycin A (56.6 ± 4.8% increase) only approached significance (P < 0.06; Student's unpaired t-test). Wash of the bafilomycin A + CPA resulted in intracellular Ca2+ recovering to near CPA-depleted levels, whereas subsequent exchange to Ca2+-containing nASW initiated some store-operated Ca2+ influx, although it was very muted (Fig. 3, B and C). Compared with responses after depletion in CPA alone, the amplitude of the response was small and the time to onset was markedly slower (compare Figs. 1B with 3C). At only an 8% increase, the average Ca2+ influx after introduction of extracellular Ca2+ to bafilomycin A + CPA treated neurons was significantly different from that after CPA alone (Fig. 6; 5th vs. 1st bar).
Store-operated Ca2+ influx pathway is sensitive to La3+, Ni2+, SKF 96365, and BTP2
Depleting intracellular stores clearly initiates Ca2+ influx in bag cell neurons. However, to better compare this route of Ca2+ entry with similar pathways in other neuronal and nonneuronal cells, it was important to learn more about its pharmacological properties. Store-operated Ca2+ influx is often blocked by tri- or divalent metals such as La3+ and Ni2+ (Gore et al. 2004; Hoth and Penner 1993; Ross and Cahalan 1995). With 100 µM La3+ in the bath, addition of extracellular Ca2+ to CPA-depleted bag cell neurons brought about a rise in intracellular Ca2+ that was more modest in comparison to control (Fig. 4A; n = 23). Although this block was by no means complete, the difference between the mean change in intracellular Ca2+ in La3+ (23% increase) was significantly different from that in control (47% increase; Fig. 6; 6th vs. 1st bar). When the same experiment was performed using 100 µM Ni2+, no block of the Ca2+ influx pathway was seen (Fig. 4B; n = 10), although increasing the concentration of Ni2+ to 10 mM quite effectively inhibited the increase in intracellular Ca2+ normally observed with the addition of Ca2+-containing nASW (Fig. 4C; n = 13). The average change in intracellular Ca2+ after the introduction of extracellular Ca2+ to neurons treated with CPA +100 µM Ni2+ was an 40% increase, which was not significantly different from CPA alone (Fig. 6; 7th vs. 1st bar). Conversely, there was only a mean 10% increase in intracellular Ca2+ for CPA +10 mM Ni2+, and this value readily reached the level of significance when compared with neurons treated with just CPA (Fig. 6; 8th vs. 1st bar).
In addition to tri- or divalent metals, an imidazole known as SKF-96365 is commonly used to block store-operated Ca2+ influx (Cabello and Schilling 1993; Daly et al. 1995, Doi et al. 2000; Fasolato et al. 1990; Merritt et al. 1990). The Ca2+ entry route initiated by store depletion in bag cell neurons was antagonized by 20 µM SKF 96365 (Fig. 4D; n = 15). The mean change in intracellular Ca2+ after exchange to Ca2+-containing nASW for those neurons exposed to CPA + SKF 96365 was an 8% increase, and this was significantly different from the Ca2+ elevation elicited in neurons subjected to only CPA (Fig. 6; 9th vs. 1st bar). Recently, a pyrazole derivative designated as BTP2 was found to block both store-operated Ca2+ influx and depletion-activated current in Jurkat T-lymphocytes (Ishikawa et al. 2003; Zitt et al. 2004). For bag cell neurons, 1 µM BTP2 inhibited store-operated Ca2+ influx (Fig. 4E; n = 12). In neurons exposed to CPA + BTP2 there was an 25% elevation in intracellular Ca2+ after exchange to nASW, which was significantly different from the approximately 47% increase seen in neurons given only CPA (Fig. 6; 10th vs. 1st bar).
Neither IP3 receptors nor microfilaments likely play a role in store-operated Ca2+ influx
Ma et al. (2000) reported that inhibition of IP3 receptors by the membrane-permeable antagonist, 2-APB (Maruyama et al. 1997), prevents store-operated Ca2+ influx. However, subsequent work has suggested that 2-APB is in fact a direct inhibitor of store-operated channels (Baba et al. 2003; Braun et al. 2001; Cordova et al. 2003; Prakriya and Lewis 2001; Tozzi et al. 2003). Therefore, we felt it necessary to test the ability of this agent to block Ca2+ entry after store depletion in bag cell neurons. For bag cell neurons that had been depleted with CPA in Ca2+-free ASW, the application of 100 µM 2-APB itself produced an elevation in intracellular Ca2+; moreover, 2-APB quite effectively prevented store-operated Ca2+ influx when extracellular Ca2+ was introduced with nASW (Fig. 5A; n = 17). On average, there was only an 9% increase in intracellular Ca2+ when Ca2+-containing nASW was delivered to neurons treated with CPA +2-APB, which was significantly different from the 47% increase seen in the CPA alone treated neurons (Fig. 6; 11th vs. 1st bar). Perhaps on account of its antagonistic actions or because it simply is not a true depleting agent, store-operated Ca2+ influx was not initiated following release of intracellular Ca2+ by 2-APB alone (Fig. 6; 12th vs. 1st bar). The release of intracellular Ca2+ by 2-APB was not altogether unexpected. In their initial report that 2-APB could inhibit IP3 receptors with an IC50 of 42 µM, Maruyama et al. (1997) noted that >90 µM, it also caused Ca2+ release. Because 2-APB is known to inhibit both IP3 receptors and store-operated channels, we tested xestospongin C, an alkaloid IP3 receptor antagonist that is thought to attenuate store-operated Ca2+ influx by interfering with the coupling between depletion and activation of the pathway (Gafni et al. 1997; Kiselyov et al. 1998; Ma et al. 2000). Despite the effectiveness of 2-APB, 15 µM xestospongin C did not alter store-operated influx in CPA-depleted bag cell neurons (Fig. 5B; n = 17). Overall, intracellular Ca2+ went up by 57% when Ca2+-containing nASW was introduced to neurons bathed in CPA + xestospongin C. This elevation was not significantly different from that observed in neurons bathed in CPA alone (Fig. 6; 13th vs. 1st bar).
Both Yao et al. (1999) and Patterson et al. (1999) provided evidence that store-operated Ca2+ influx in Xenpous oocytes or mammalian cell lines was initiated by secretion or trafficking of a signal from the endoplasmic reticulum to the plasma membrane. In the case of cell lines, influx was blocked following microfilament stabilization with the membrane-permeable actin polymerizer, jasplakinolide (Bubb et al. 1994; Patterson et al. 1999). However, when 1 µM jasplakinolide was applied to bag cell neurons, store-operated Ca2+ influx was not prevented (Fig. 5C; n = 17). There was no significant difference between the 35% mean change in intracellular Ca2+ after introduction of extracellular Ca2+ to neurons exposed to CPA + jasplakinolide, and the 47% increase for neurons treated with only CPA (Fig. 6; 14th vs. 1st bar).
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DISCUSSION |
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; Thastrup et al. 1990). Ca2+ then leaks out of the organelle and is removed to the extracellular space by plasma membrane exchangers or pumps (Clapham 1995; Meldolesi 2001; Verkhratsky 2005). It is unlikely that Ca2+-free saline itself triggers influx given that without prior depletion, no change in intracellular Ca2+ is observed on delivery of extracellular Ca2+. These results extend the findings of Knox et al. (1996), who detected Ca2+ entry into bag cell neurons with a Ca2+-sensitive vibrating extracellular probe after application of thapsigargin in the presence of extracellular Ca2+. However, they did not determine if store depletion itself initiates Ca2+ entry nor did they demonstrate sensitivity to store-operated channel antagonists.
Both neuronal and nonneuronal store-operated channels are sensitive to certain di- and trivalent metals as well as SKF 96365, BTP2, and 2-APB. La3+ is the most potent metal, producing near-complete block at 0.1–1 mM (Baba et al. 2003; Bouron 2000; Gore et al. 2004; Grudt et al. 1996; Hoth and Penner 1993; Lepple-Wienhues and Cahalan 1996; Liu and Gylfe 1997; Prothero et al. 2000; Ross and Cahalan 1995). Ni2+, the other metal used here, is less effective and requires 5–10 mM for substantial inhibition (Arakawa et al. 2000; Hoth and Penner 1993; Prothero et al. 2000; Trepakova et al. 2001; Usachev and Thayer 1999; Villalobos and Garcia-Sancho 1995). Regarding SKF 96365, Merritt et al. (1990) first proposed it as a blocker of store-operated Ca2+ influx, and although not absolutely specific, inhibition by 10–100 µM remains a key indicator of this pathway (Arakawa et al. 2000; Cabello and Schilling 1993; Daly et al. 1995; Grudt et al. 1996; Prakriya and Lewis 2003). The later developed BTP2 will completely antagonize store-operated influx after short-term incubation at 10 µM, whereas 1 µM produces a less potent reduction (Ishikawa et al. 2003). Finally, 10–100 µM 2-APB eliminates store-operated influx (Baba et al. 2003; Braun et al. 2001; Cordova et al. 2003; Prakriya and Lewis 2001; Tozzi et al. 2003). Accordingly, the profile of the bag cell neuron Ca2+ elevation, i.e., partial block by 100 µM La3+ and 1 µM BTP2, as well as near-complete block by 10 mM Ni2+, 20 µM SKF 96365, or 100 µM 2-APB, is consistent with store-operated Ca2+ influx channels.
There are two categories of store-operated Ca2+ influx channels. One, Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective, very-small-conductance (estimated 20 fS) channels (Hoth and Penner 1993; Prakriya and Lewis 2003; Zweifach and Lewis 1993). Two, Ca2+-permeable, larger-conductance (tens of pS), nonselective cation channels (Albert and Large 2002; Curtis and Scholfield 2001; Su et al. 2001; Trepakova et al. 2001). Some of the latter belong to the transient receptor potential channel family originally cloned from Drosophila (Beech et al. 2003; Kiselyov et al. 1998; Ma et al. 2000; Nilius 2003; Petersen et al. 1995; Philipp et al. 1996; Tozzi et al. 2003; Zitt et al. 1996). Recently, a membrane-spanning protein, termed Orai1 or CRACM1, has been identified as an fundamental constituent or regulator of store-operated Ca2+ influx channels (Feske et al. 2006; Vig et al. 2006). The channel-type mediating bag cell neuron store-operated Ca2+ influx is currently unknown, although the modest depolarization seen with extracellular Ca2+ following depletion may be due to its opening. Alternatively, store-operated Ca2+ influx could cause depolarization by triggering one of the bag cell neuron Ca2+-activated cation channels (Knox et al. 1996; Lupinsky and Magoski 2006). Nevertheless, the source of Ca2+ during influx appears to be solely from the store-operated pathway, given that the depolarization is too small to activate voltage-gated Ca2+ current (–30 to –25 mV threshold) (Kaczmarek and Strumwasser 1984; Knox et al. 1996; A. Y. Hung and N. S. Magoski, unpublished data).
Models for depletion-activation of store-operated channels include diffusional coupling of an unknown small-molecule released from stores to stimulate channels (Randriamampita and Tsien 1993), trafficking or secretion coupling of channels or channel regulators, such a STIM1, to the plasma membrane (Patterson et al. 1999; Spassova et al. 2006Yao et al. 1999; Zhang et al. 2005), and conformational coupling of IP3 receptors through a physical link with channels (Kiselyov et al. 1998; Ma et al. 2000). In smooth muscle cells and platelets, secretion coupling of store-operated Ca2+ influx is prevented by jasplakinolide-induced microfilament stabilization (Patterson et al. 1999; Rosado et al. 2004). However, this is not the case for epithelia or thyroid cells (Abeele et al. 2004; Gratschev et al. 2004) or, as shown in the current study, the bag cell neurons. The ineffectiveness of jasplakinolide cannot be explained by species differences in pharmacology because jasplakinolide and its membrane-impermeant analogue, phalloidin, are effective cytoskeletal stabilizers in Aplysia and Drosophila (Forscher et al. 1992; Tilney et al. 2004). Thus, trafficking or secretion coupling are unlikely to contribute to store-operated Ca2+ influx in bag cell neurons. For conformational coupling, a hallmark is the block of store-operated Ca2+ influx by IP3 receptor antagonists, such as 2-APB and xestospongin C (Kiselyov et al. 1998; Ma et al. 2000; Maruyama et al. 1997). For the present study, 2-APB inhibited store-operated Ca2+ influx, yet xestospongin C was ineffective. Given that 2-APB also blocks store-operated channels directly (Prakriya and Lewis 2001), the parsimonious conclusion would be that diffusional coupling, rather than conformational coupling via IP3 receptors, is the mechanism in bag cell neurons.
The overlap between stores with CPA-sensitive Ca2+ pumps and stores with CEP-sensitive ryanodine receptors influences bag cell neuron store-operated Ca2+ influx. Although, store-operated Ca2+ influx can be initiated by ryanodine receptors in DRG neurons and smooth muscle (Curtis and Scholfield 2001; Usachev and Thayer 1999), CEP fails to activate this pathway in bag cell neurons despite causing Ca2+ release. The failure to trigger influx may be due to CEP not preventing Ca2+ uptake, which leaves the Ca2+ in the stores at a lowered but not depleted level. Interaction also occurs between acidic stores and the endoplasmic reticulum. Acidic stores sequester Ca2+ via a Ca2+/H+ exchanger driven by the H+ gradient (Christensen et al. 2002; Dunn et al. 1994; Goncalves et al. 1999; Ohsumi and Anraku 1983). Inhibition of bag cell neuron vacuolar H+-ATPases with bafilomycin A (Bowman et al. 1988) initiates a slow rise in intracellular Ca2+. Because this rise is accelerated by CPA, the endoplasmic reticulum presumably takes up some of the Ca2+ released by bafilomycin A. Moreover, the Ca2+ liberated from acidic stores appears to replenish the CPA-sensitive stores, given that store-operated influx is attenuated on return to Ca2+-containing saline. Although mitochondrial Ca2+ uptake is known to promote CRAC channel activation (Gilabert and Parekh 2000; Glitsch et al. 2002), this is the first report, to our knowledge, of acidic stores regulating store-operated Ca2+ influx. Vacuolar H+-ATPases are not localized to mitochondria but are found on a number of organelles, including vesicles (Calakos and Scheller 1996; Nelson 1992). Vesicles have low pH with high Ca2+, and if the H+-pump is blocked, Ca2+ will leak into the cytosol (Floor et al. 1990; Goncalves et al. 1999; Grohovaz et al. 1996; Scheenen et al. 1998; Thirion et al. 1995). Jonas et al. (1997) demonstrated that insulin triggers bag cell neuron peptide secretion by releasing Ca2+ from a nonendoplasmic reticulum/nonmitochondrial store that likely represents vesicles or granules.
Store-operated Ca2+ influx is universal in nonneuronal cells (Prakriya and Lewis 2003; Putney 2003), but for neurons it clearly varies between species and cell type. Although not detected in bullfrog sympathetic neurons or rat hippocampal and cortical neuron cultures (Chinopoulos et al. 2004; Friel and Tsien 1992; Koizumi et al. 1999), store-operated Ca2+ influx is observed in neurons from squid olfactory receptors, rat DRG, substantia nigra, and a number of cortical area-derived cultures (Arakawa et al. 2000; Baba et al. 2003; Bouron 2000; Piper and Lucero 1999; Prothero et al. 2000; Tozzi et al. 2003; Usachev and Thayer 1999). For neuroendocrine cells, there is evidence for and against store-operated Ca2+ influx in bovine chromaffin cells (Powis et al. 1996; Stauderman and Pruss 1989) as well as proof positive in murine pancreatic 
cells (Liu and Gylfe 1997).
Given the presence of voltage-gated Ca2+ channels, the role of neuronal store-operated Ca2+ influx appears somewhat enigmatic. That said, store-operated channels do initiate secretion in chromaffin cells at membrane potentials too negative for voltage-gated Ca2+ channel activation (Fomina and Nowycky 1999). Depleting stores in the hippocampus can also enhance spontaneous transmitter release or induce long-term potentiation (Emptage et al. 2001; Ris et al. 2003). The bag cell neuron afterdischarge is an 30-min barrage of action potentials that triggers neuropeptide secretion and egg-laying behavior (Conn and Kaczmarek 1989; Kupfermann 1967; Kupfermann and Kandel 1970; Pinsker and Dudek 1977; Rothman et al. 1983). Along with voltage-gated Ca2+ channels, store-operated Ca2+ influx could promote neuropeptide secretion, particularly late in the afterdischarge when release is maintained despite lowered firing frequency (Loechner et al. 1990; Michel and Wayne 2002). The afterdischarge is also associated with IP3-dependent release of Ca2+ from intracellular stores (Fink et al. 1988; Fisher et al. 1994). After the afterdischarge, the bag cells neurons enter a prolonged term of inactivity, known as the refractory period (Kaczmarek and Kauer 1983; Kaczmarek et al. 1982). The lack of voltage-gated Ca2+ channel opening during the quiescence of the refractory period would hinder Ca2+ store repletion; however, this could be circumvented by store-operated Ca2+ influx, which would readily replenish Ca2+ stores in the absence of activity.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present addresses: R. J. Knox, Dept. of Lead Discovery and Profiling, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT, 06492; D. Uthuza, VA Medical Center, Cardiology Div., University of California, San Francisco, CA, 94121.
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. S. Magoski, Dept. of Physiology, Queen's University, 4th Floor, Botterell Hall, 18 Stuart St., Kingston, ON, K7L 3N6, Canada (E-mail: magoski{at}post.queensu.ca)
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ABSTRACT
INTRODUCTION
), results in a rapid elevation of intracellular Ca2+ in neurons depleted with CPA (n = 11; representative of 44 in total) but not in neurons simply maintained in Ca2+-free ASW (n = 11). The CPA-treated neurons were exposed to the drug for 
