Separate Ca2+ sources are buffered by distinct Ca2+ handling systems in aplysia neuroendocrine cells

Abstract

Although the contribution of Ca(2+) buffering systems can vary between neuronal types and cellular compartments, it is unknown whether distinct Ca(2+) sources within a neuron have different buffers. As individual Ca(2+) sources can have separate functions, we propose that each is handled by unique systems. Using Aplysia californica bag cell neurons, which initiate reproduction through an afterdischarge involving multiple Ca(2+)-dependent processes, we investigated the role of endoplasmic reticulum (ER) and mitochondrial sequestration, as well as extrusion via the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, to the clearance of voltage-gated Ca(2+) influx, Ca(2+)-induced Ca(2+)-release (CICR), and store-operated Ca(2+) influx. Cultured bag cell neurons were filled with the Ca(2+) indicator, fura-PE3, to image Ca(2+) under whole-cell voltage clamp. A 5 Hz, 1 min train of depolarizing voltage steps elicited voltage-gated Ca(2+) influx followed by EGTA-sensitive CICR from the mitochondria. A compartment model of Ca(2+) indicated the effect of EGTA on CICR was due to buffering of released mitochondrial Ca(2+) rather than uptake competition. Removal of voltage-gated Ca(2+) influx was dominated by the mitochondria and PMCA, with no contribution from the Na(+)/Ca(2+) exchanger or sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA). In contrast, CICR recovery was slowed by eliminating the Na(+)/Ca(2+) exchanger and PMCA. Last, store-operated influx, evoked by ER depletion, was removed by the SERCA and depended on the mitochondrial membrane potential. Our results demonstrate that distinct buffering systems are dedicated to particular Ca(2+) sources. In general, this may represent a means to differentially regulate Ca(2+)-dependent processes, and for Aplysia, influence how reproductive behavior is triggered.

Figure 1.

A train of depolarizing stimuli induces a secondary Ca²⁺ rise sensitive to the Ca²⁺ chelator, EGTA. A, Simultaneous measurement of free intracellular Ca²⁺ and membrane current in cultured bag cell neurons using 340/380 fura PE3 fluorescence and whole-cell voltage clamp at −80 mV. A, Inset, A phase contrast image shows the recording pipette, bag cell neuron soma, and its neuritic processes. The bottom image shows the same neuron loaded with fura and the somatic region of interest (ROI) used for data collection. Scale bar applies to both images. A, Top left, Ca²⁺ influx indicated by a change in intensity of the 340/380 fluorescence ratio following a 1 min, 5 Hz train of 75 ms steps from −80 to 0 mV. With 5 mm intracellular EGTA, stimulation causes a large rise in Ca²⁺ followed by a rapid recovery to the prestimulus baseline. Top right, In the absence of intracellular EGTA, there is a prolonged Ca²⁺ plateau subsequent to the initial influx that greatly outlasts the stimulus duration, indicative of CICR. A, Bottom, Traces depict 300 overlaid Ca²⁺ currents from each pulse to 0 mV of the 1 min train stimulus in either 5 or 0 mm intracellular EGTA. The shifting band of traces is due to use-dependent inactivation of Ca²⁺ currents during the train stimulus. Unless stated otherwise, all cells were recorded in a Cs⁺-external and TEA-external (to replace K⁺ and Na⁺, respectively) and a Cs⁺-based intracellular solution (to replace intracellular K⁺). B, Left, The percentage change in 340/380 from baseline to the peak response during the train stimulus is significantly larger in 5 mm EGTA versus 0 mm EGTA (unpaired Student's t test). For this and subsequent bar graphs, data represents the mean ± SE, and the n-value is indicated within the bars. B, Middle and right, Zero mm EGTA significantly increases the total area measured from 1 min after stimulation to 11 min post-train stimulus (Mann–Whitney U test) and the time to reach 75% recovery to baseline Ca²⁺ from the peak of the plateau (Mann–Whitney U test).

Figure 2.

Mitochondria remove voltage-gated Ca²⁺ influx and clear Ca²⁺ from repeated stimuli. A, Neurons voltage-clamped to −80 mV with 5 mm intracellular EGTA to allow for isolated measurement of voltage-gated Ca²⁺ influx and removal. A, Left, In DMSO, cytosolic Ca²⁺ transients evoked by the 5 Hz, 1 min train stimulus are followed by rapid recovery to baseline Ca²⁺. A, Right, Pretreatment with 20 μm FCCP, a protonophore that collapses the mitochondrial membrane potential and prevents Ca²⁺ sequestration, slows the recovery of Ca²⁺ following stimulation. A, Inset, The exponential decay time constant (τ) of the Ca²⁺ transient recovery phase is significantly larger in FCCP-treated neurons (unpaired Student's t test). B, Top, Relative Ca²⁺ clearance rate (R), calculated from the decay phase of Ca²⁺ transients shown in A, as a function of 340/380 ratio (rates normalized to the maximal value of the 340/380 range). Second-order polynomial fitted lines are plotted overtop of the data points. The difference between the control (R_total) and FCCP (R_FCCP) fits produce the estimated mitochondrial uptake (R_mit), represented by the light dashed line. B, Bottom, Second-order polynomial fit lines for R_control, R_FCCP, and R_mit representing averaged removal rates from multiple neurons. Sample sizes are different from the decay time constants shown in A due to quality of fit criteria required for rate functions (see Materials and Methods). C, Ca²⁺ influx from a train stimulus loads mitochondria with Ca²⁺. C, Left, FCCP (20 μm) elevates Ca²⁺ in neurons under voltage clamp at −80 mV with 5 mm EGTA in the pipette and 100 μm TPP to prevent potential release of mitochondrial Ca²⁺. C, Right, FCCP-induced Ca²⁺ release after a large influx of Ca²⁺ from a 5 Hz, 1 min train stimulus is increased. D, Train stimulation, before FCCP application, significantly enhances the peak percentage change upon FCCP-induced Ca²⁺ liberation (unpaired Student's t test). E, Mitochondrial Ca²⁺ clearance is necessary for recovery from repeated stimuli. E, Left, After a train stimulus in FCCP, a second stimulus produces a Ca²⁺ load that is largely unremoved. E, Right, To replicate the slow Ca²⁺ recovery in FCCP, a control cell is given a train stimulus, but then subsequently held at −30 mV to allow for a small persistent Ca²⁺ influx. Ca²⁺ levels are quickly restored following a second train stimulus when the cell is clamped at −80 mV. F, The ratio between the first and second percentage recovery at 5 min is significantly larger in FCCP-treated neurons (unpaired Student's unpaired t test).

Figure 3.

Removal of voltage-gated Ca²⁺ influx is slowed by inhibition of the PMCA, but is not influenced by acidic stores, ER, or the Na⁺/Ca²⁺-exchanger. A, Left, Blocking PMCA function by the addition of 2 mm extracellular La³⁺ on the last pulse of the 1 min, 5 Hz train stimulus, hinders Ca²⁺ removal (dark trace), whereas H₂O (control) (light trace) applied in the same manner has no effect. A, Middle left, In contrast, the inclusion of Na⁺ (dark trace) rather than TEA (light trace) in the extracellular saline, to permit Na⁺/Ca²⁺ exchanger activity, has no effect. Similarly, treatment with 20 μm CPA (middle right), to inhibit SERCA, or 100 nm bafilomycin A (baf) (right), to prevent uptake by acidic stores, does not alter Ca²⁺ removal after a train stimulus. B, Neurons treated with 20 μm FCCP present significantly larger exponential decay time constants (τ) relative to controls (reproduced from Fig. 2A), whereas cells in the presence of extracellular Na⁺, exposed to CPA, or baf do not have significantly different τ values (all comparisons using unpaired Student's t test). C, FCCP (Mann–Whitney U test) and La³⁺ significantly (Welch corrected unpaired Student's t test) reduce the percentage recovery at 5 min post-train stimulus whereas extracellular Na⁺, CPA, and baf have no effect (all comparisons using unpaired Student's t test).

Figure 4.

In the absence of mitochondrial function, PMCA, but neither SERCA nor the Na⁺/Ca²⁺-exchanger, influence the removal of voltage-gated Ca²⁺ influx. A, Left, Post-stimulus addition of the PMCA inhibitor, La³⁺ (2 mm) (dark trace), in the presence of 20 μm FCCP, strongly attenuates the subsequent recovery relative to FCCP alone (light trace). The inclusion of extracellular Na⁺ (middle) or pretreatment with 20 μm CPA (right) does not influence Ca²⁺ removal in the presence of FCCP. B, For neurons pretreated with FCCP, the mean post-train stimulus recovery τ values are not significantly altered by the inclusion of extracellular Na⁺ or exposure to CPA (unpaired Student's t test and Mann–Whitney U test, respectively). C, Summary of mean percentage recovery at 5 min post-train stimulus. In the presence of FCCP, La³⁺ significantly increases the percentage recovery at 5 min, whereas the application CPA (Mann–Whitney U test) or the inclusion of extracellular Na⁺ remains ineffective (all other comparisons using unpaired Student's t tests).

Figure 5.

The EGTA-sensitive Ca²⁺ plateau is caused by mitochondrial Ca²⁺ release. A, A post-stimulus Ca²⁺ plateau is evoked by a 5 Hz, 1 min train of depolarizing steps under voltage clamp. Intracellular EGTA is absent (0 mm) to allow for CICR. The prolonged Ca²⁺ plateau in control conditions (light trace) is substantially smaller in 100 μm the mitochondrial Ca²⁺ exchange blocker, TPP (dark trace). B, Treatment with CPA, to inhibit SERCA and prevent ER Ca²⁺ release, does not affect the post-train stimulus Ca²⁺ plateau (dark trace) when compared with DMSO-treated neurons (light trace). C, D, TPP but not CPA, significantly reduces the 1–11 min area relative to controls (unpaired Student's t test in both cases). TPP also significantly reduces the time to reach 75% recovery of baseline Ca²⁺ from peak CICR (Welch corrected, unpaired Student's t test). In contrast, CPA has no significant effect on the recovery time (unpaired Student's t test). Sample sizes for the time to 75% recovery in CPA are smaller than those used for post-stimulus area measurements as some neurons did not achieve 75% recovery.

Figure 6.

Mitochondrial Ca²⁺ release is removed by the Na⁺/Ca²⁺ exchanger and the PMCA. A, Left, In the absence of extracellular Na⁺ (TEA, light trace), the mitochondrial Ca²⁺ plateau evoked by a 1 min, 5 Hz train stimulus from −80 mV is prolonged compared with cells bathed in extracellular Na⁺ (dark trace). A, Inset, The presence of extracellular Na⁺ significantly accelerated the time to 75% recovery for CICR (Mann–Whitney U test). A, Middle, In Na⁺-free extracellular saline (TEA), the application of La³⁺ (dark trace) during the onset of CICR occludes recovery. In these experiments, cells are voltage-clamped at −80 mV following the stimulus. A, Right, As a control, a 1 min 5 Hz train stimulus from −80 mV, followed by a prolonged step depolarization evokes a Ca²⁺ plateau similar to CICR (at break, a portion of the Ca²⁺ influx is omitted for clarity to emphasize the Ca²⁺ plateau). The recovery of this Ca²⁺-influx plateau is insensitive to the replacement of extracellular Na⁺ (dark trace) for TEA (light trace). For this experiment, neurons are pretreated with 100 μm TPP to prevent any influence of mitochondrial Ca²⁺ release and are recorded with 0 mm intracellular EGTA. B, The post-train stimulus area from 1 to 11 min during mitochondrial Ca²⁺ release is not significantly different between Na⁺ and TEA conditions (unpaired Student's t test). However, the area from 11 to 21 min is significantly larger in TEA-containing extracellular saline (Mann–Whitney U test). C, In the absence of Na⁺/Ca²⁺ exchanger activity, the application of 2 mm La³⁺ during CICR does not alter the post-train stimulus area from 1 to 11 min (Mann–Whitney U test), but significantly increases the area from 11 to 21 min (Welch corrected Student's t test, respectively). D, Area summary data for the Ca²⁺ plateau evoked by post-train stimulus depolarization at (ranging from −10 to −20 mV). Replacing extracellular Na⁺ with TEA does not significantly alter the post-train stimulus area from time 1–11 min or 11–21 (Student's t test for both).

Figure 7.

A model of bag cell neuron Ca²⁺ dynamics demonstrates the sensitivity of mitochondrial Ca²⁺ release to the Ca²⁺ chelating agent, EGTA. A, The inset illustrates the components involved in the 3 compartment model of Ca²⁺. Ca²⁺ influx is represented by J_influx, cytosolic Ca²⁺ removal is mediated by mitochondrial uptake (J_uptake) or plasma membrane efflux (J_efflux), and mitochondrial Ca²⁺ release is denoted as J_release. Collectively, these variables determine the cytosolic and mitochondrial Ca²⁺concentrations (see Materials and Methods). Raw data from a bag cell neuron (light trace) presenting stimulus-induced Ca²⁺ influx, CICR, and recovery is fit by the model (dark trace) to estimate values for free intracellular Ca²⁺ fluxes. Train stimulus-induced Ca²⁺ influx (bottom) is simulated in the model by transiently increasing the Ca²⁺ influx rate constant (k_influx). Directly after reducing k_influx there is a rapid recovery leading to a prolonged Ca²⁺ plateau that readily fits the raw data (root mean square error: 2.3 × 10⁻⁸). Parameters for this fit are (± 95% confidence interval): V_{max, efflux}: 6.2 ± 0.26 nm/s, k_{max, uptake}: 4.45 ± 0.82 (s⁻¹), V_{max, release}: 6.2 ± 1.14 nm/s. Unless otherwise stated, all subsequent model presentations were derived using the average values presented in Table 1. The values in Table 1 reflect parameters measured from changes to free Ca²⁺, before converting to rates of total Ca²⁺. B, For this and subsequent model graphs, Ca²⁺ levels reflect total, rather than free intracellular Ca²⁺ to correct for the presence of exogenous (fura and/or EGTA) and endogenous Ca²⁺ buffers present when estimating rates (see Materials and Methods). Serially reducing the rate constant of mitochondrial Ca²⁺ uptake (k_{max, uptake}) from 10 to 3.3 then 0 (s⁻¹) attenuates the degree of mitochondrial Ca²⁺ uptake (right, light traces), slows the post-stimulus removal, and reduces CICR (left, light traces). C, Left inset, to include EGTA (0.5 mm), the bag cell neuron model of Ca²⁺ dynamics is increased to four components by adding Ca²⁺ removal by an exogenous Ca²⁺ binding agent. The clearance of Ca²⁺ by EGTA is determined by its forward (k_on) and reverse (k_off) rate constants, respectively. In the absence of EGTA, evoking Ca²⁺ influx causes a rise in Ca²⁺ and a subsequent Ca²⁺ plateau (left, dark trace). Under these conditions, mitochondrial Ca²⁺ increases then falls as Ca²⁺ is released into the cytosol (right, dark trace). The cytosolic Ca²⁺ response in the presence of EGTA slightly reduces peak Ca²⁺ influx magnitude and eliminates mitochondrial CICR (left, light traces). EGTA also partially attenuates the magnitude of mitochondrial Ca²⁺ influx after stimulation (right, light traces). D, In the presence of an EGTA component (0.5 mm), increasing the mitochondrial uptake rate constant (k_{max, uptake}) from 10 to 40 then 160 (s⁻¹), potentiates the degree of mitochondrial Ca²⁺ loading (right, light traces), speeds the rate of cytosolic Ca²⁺ recovery after influx, and produces very limited CICR (left, light traces).

Figure 8.

The store-operated Ca²⁺ influx pathway is cleared by the SERCA to replete the ER. A, Addition of 20 μm CPA depletes ER Ca²⁺ in a cultured bag cell neuron pressure-injected with fura-PE3. Because neurons are not recorded under voltage clamp, a normal Na⁺-containing and K⁺-containing external solution is used. After the first depletion, CPA is washed out using bath exchange (at break); upon recording resumption, the addition of extracellular Ca²⁺ results in a rapid elevation of intracellular Ca²⁺. Delivering CPA a second time again evokes a Ca²⁺ rise, albeit smaller than the first, indicating repletion of the CPA-sensitive store. B, The magnitude of Ca²⁺ release to the second CPA exposure, after store-operated influx, is significantly smaller than the response elicited during the first CPA-induced depletion (unpaired Student's t test). C, Left, Washout of CPA, before addition of extracellular Ca²⁺, speeds the recovery of influx back to baseline. The removal of store-operated Ca²⁺ recovers at a slower rate in the presence of CPA (dark trace). Both neurons previously depleted in Ca²⁺-free ASW with 20 μm CPA. C, Right, Ca²⁺ influx, similar in size to that evoked by store-operated influx, caused by a short 5 Hz train of depolarizing stimuli from −80 to 0 mV in the presence (dark trace) and absence (light trace) of CPA. CPA has no effect on the speed of recovery from the short stimulus. D, Left, Mean percentage change in 340/380 (left) indicates that the increase in cytosolic Ca²⁺ during store-operated Ca²⁺ influx and the short train stimulus Ca²⁺ influx are not significantly different within and between conditions (ANOVA). D, Middle, CPA significantly increases the mean store-operated Ca²⁺ influx decay time constant (Welch corrected unpaired Student's t test) although having no effect on the mean τ of the similarly sized train stimulus-induced Ca²⁺ influx (unpaired Student's t test). D, Right, The percentage recovery at 5 min (right) after the application of extracellular Ca²⁺ is significantly shorter with prior CPA washout (Welch corrected unpaired Student's t test). However, the percentage recovery at 5 min after the short train stimulus Ca²⁺ influx is not significantly altered by CPA.

Figure 9.

Store-operated Ca²⁺ influx is reduced in magnitude by FCCP but is not influenced by acidic stores, the PMCA, or the Na⁺/Ca²⁺ exchanger. A, Left, Neurons exposed to 20 μm FCCP before the addition of extracellular Ca²⁺ (dark trace) causes a reduction in store-operated influx relative to control (light trace). A, Right, Exposure to 100 nm bafilomycin A (baf) before the addition of extracellular Ca²⁺ (dark trace) does not influence the size or recovery of store-operated Ca²⁺ influx. Neurons were previously depleted in Ca²⁺-free ASW with 20 μm CPA. B, Treatment with FCCP significantly reduces the peak Ca²⁺ influx after the addition of extracellular Ca²⁺ (left), whereas bafilomycin has no effect (both comparisons using Mann–Whitney U test). C, Summary data demonstrating that the mean decay time constant (τ) is significantly larger in CPA (Welch corrected unpaired Student's t test), but not in bafilomycin (unpaired Student's t test), 20 μm carboxyeosin (CE) (Welch corrected Student's t test), or where extracellular Na⁺ is replaced with NMDG (Mann–Whitney U test). D, CPA pretreatment significantly reduces the percentage recovery from peak Ca²⁺, whereas incubation with bafilomycin, carboxyeosin, or extracellular NMDG does not (all comparisons using unpaired Student's t test).

Figure 10.

Ca²⁺ from multiple sources is cleared by distinct sets of Ca²⁺ removal systems in the bag cell neurons. A conceptual model of Ca²⁺ dynamics during an afterdischarge based on the current study and prior work by our laboratory and others (Fink et al., 1988; Fisher et al., 1994; Michel and Wayne, 2002; Kachoei et al., 2006; Geiger and Magoski, 2008). Top, right inset, A sample trace of intracellular Ca²⁺ presumed to reflect afterdischarge dynamics in vivo. Numbers correspond to a series of chronological events depicted in the main illustration: 1, During the fast phase of the afterdischarge there is a large Ca²⁺ influx through action potential-evoked voltage-gated Ca²⁺ channels (VGCC). 2, This early Ca²⁺ influx is predominantly removed by rapid mitochondrial uptake, with ancillary assistance from the PMCA. 3, Ca²⁺ accumulates in the mitochondria, after which it is slowly extruded into the cytosol through a TPP-sensitive Ca²⁺-exchanger. Additionally, second messenger cascades are activated that initiate Ca²⁺ liberation from the ER through IP₃ and ryanodine receptors. 4, The release of Ca²⁺ from intracellular stores causes a prolonged rise in cytosolic Ca²⁺ that is removed by the Na⁺/Ca²⁺ exchanger and the PMCA. 5, Last, sustained Ca²⁺ release during the afterdischarge depletes the ER and initiates store-operated Ca²⁺ influx: a distinct Ca²⁺ source that is removed by the SERCA into the ER lumen.