Roles for Mitochondrial and Reverse Mode Na⁺/Ca²⁺ Exchange and the Plasmalemma Ca²⁺ ATPase in Post-Tetanic Potentiation at Crayfish Neuromuscular Junctions

Plasmalemmal Na⁺/Ca²⁺exchange does influence PTP

As one probe of plasma membrane Na⁺/Ca²⁺exchange we used the relatively specific blocker KB R7943. This agent is much more effective against the reverse mode of transport, in which Ca²⁺ enters in exchange for Na⁺ efflux (IC₅₀ = 0.3–2 μm cytoplasmic concentration) (Iwamoto et al., 1996; Watano et al., 1996), than against forward transport, in which Ca²⁺ is extruded in exchange for Na⁺ influx (IC₅₀ = 17–30 μm). However, at these higher concentrations KB R7943 (>30 μm) also affected currents through voltage-dependent ion channels, including Ca²⁺ channels (Watano et al., 1996). At 10 μm bath concentration, KB R7943 was without effect on PTP in our preparations. At concentrations of 50 μm and higher, baseline synaptic transmission and tetanic presynaptic [Ca²⁺]_iaccumulation were strongly reduced, probably because of presynaptic Ca²⁺ channel block. At 20 μm, such nonspecific effects on baseline transmission were not observed, but there was a modest reduction in tetanic Ca²⁺ accumulation and tetanic EJP amplitude, and the post-tetanic decay of [Ca²⁺]_i and EJP amplitude were accelerated (Fig. 2, Table1). To confirm inhibition by KB R7943 of Na⁺/Ca²⁺exchange, effects on [Na⁺]_i were observed. KB R7943 (20 μm) had no clear effect on [Na⁺]_iaccumulation during tetanic stimulation, but it did slow post-tetanic [Na⁺]_i decay (Fig.2E,F, Table 1).

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Fig. 2.

Effects of KB R7943 on [Ca²⁺]_i, [Na⁺]_i, and PTP. The first tetanus was given in normal saline and the second one in the presence of 20 μm KB R7943 (reverse mode plasmalemmal Na⁺/Ca²⁺ exchange inhibitor).A, B, Tetanic [Ca²⁺]_i and EJP amplitude were reduced in 20 μm KB R7943. C, D, Running averages of five post-tetanic [Ca²⁺]_i measurements and eight post-tetanic EJP amplitudes in control saline (●) and 20 μm KB R7943 (○). E, F, Tetanic [Na⁺]_i and running average of three post-tetanic [Na⁺]_i measurements in control crayfish saline (●) and 20 μm KB R7943 (○).

The acceleration of post-tetanic [Ca²⁺]_i decay and slowing of [Na⁺]_iremoval are consistent with a block of Na⁺/Ca²⁺exchange occurring in reverse mode. However, the effects on Ca²⁺ accumulation and tetanic EJP amplitude could be attributable to a reduction of Ca²⁺ influx through Ca²⁺ channels. This could result in reduced mitochondrial Ca²⁺ accumulation and therefore a reduction in both PTP and in post-tetanic [Ca²⁺]_i (Tang and Zucker, 1997), but the effect on Na⁺removal is difficult to explain this way and suggests instead an action on Na⁺/Ca²⁺exchange. To distinguish effects on tetanic Ca²⁺ influx from effects on Na⁺/Ca²⁺exchange, KB R7943 was applied at the end of the tetanus. A solution containing 20 μm KB R7943 was washed in during the last minute of tetanic stimulation. The peak EJP amplitude (12.7 mV in the second tetanus vs 11.9 mV in the first tetanus) and [Ca²⁺]_iaccumulation (1.09 μm in the second tetanus vs 1.07 μm in the first tetanus) were not influenced. Nevertheless, KB R7943 still accelerated Ca²⁺ removal (τ_slow = 3.41 min in the second tetanus vs 4.69 min in the first tetanus) and PTP decay (τ = 2.79 min vs the control value of 3.4 min) (Fig. 3). These effects are similar to those occurring when KB R7943 was present throughout the second tetanus (Fig. 2, Table 1).

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Fig. 3.

KB R7943 application delayed until the end of tetanic stimulation. A, B, Tetanic [Ca²⁺]_i and EJP amplitude were unchanged when applying 20 μm KB R7943 at the end of a 20 Hz tetanus. C, D, Running averages of three post-tetanic [Ca²⁺]_imeasurements and two post-tetanic EJP amplitudes in control saline (●) and 20 μm KB R7943 (○). E,F, Tetanic [Na⁺]_i and post-tetanic [Na⁺]_i measurements in control crayfish saline (●) and when applying 20 μm KB R7943 at the end of the tetanus (○).

Taken together our results show that plasma membrane Na⁺/Ca²⁺exchange operates in reverse mode (admitting Ca²⁺ into nerve terminals) during and for some minutes after tetanic stimulation, attributable to the presynaptic accumulation of Na⁺, contributing to tetanic [Ca²⁺]_iaccumulation and EJP potentiation and slowing post-tetanic Ca²⁺ removal and PTP decay. Blocking this process then reduces tetanic [Ca²⁺]_iaccumulation and EJP potentiation and speeds Ca²⁺ removal and PTP decay.

Tetanic Na⁺ accumulation reverses plasmalemmal Na⁺/Ca²⁺ exchange and prolongs PTP

The operation of Na⁺/Ca²⁺exchange in reverse mode in PTP is further supported by our measurements of [Na⁺]_i and [Ca²⁺]_i changes during and after tetanic stimulation (Fig.4, Table 1). We have performed such measurements on small individual presynaptic boutons and with greater accuracy on larger preterminal nerve branches. Tetanic stimulation causes [Na⁺]_i to rise from 7 to 80 mm and [Ca²⁺]_i to rise from 0.13 to 1.15 μm. [Ca²⁺]_i recovers to only slightly above resting levels within 2–5 min after the tetanus (Tang and Zucker, 1997), whereas [Na⁺]_i relaxes more slowly to pretetanic levels (τ_Na = 8.52 ± 1.92 min, p < 0.05, vs τ_Ca = 5.71 ± 0.69 min).

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Fig. 4.

Relationship between the membrane potential (V_m) and calculated Na⁺/Ca²⁺ exchange equilibrium potential (E_Na/Ca) before, during, and after 10 min, 20 Hz tetanic stimulation. Top panelshows average [Na⁺]_i (○,n = 7) and [Ca²⁺]_i(●, n = 8) in primary and secondary branches of the exciter axon (left) or in terminal boutons (right). Bottom panel shows the calculated E_Na/Ca (○) and measuredV_m (■, n = 4); it indicates that during the tetanus and PTP phase, the Na⁺/Ca²⁺ exchange operates in reverse mode. Tetanic and PTP periods are indicated by different shading patterns (tetanic period ▨; PTP ▧).

Na⁺/Ca²⁺exchange involves the movement of three Na⁺ ions in one direction in exchange for one Ca²⁺ ion in the opposite direction. Because there is a net transport of charge, the process is not electroneutral and depends on membrane potential as well as the Na⁺ and Ca²⁺gradients across the membrane (Blaustein and Lederer, 1999). The direction of Ca²⁺ flux can be outward (normal mode) or inward (reverse mode), according to whether the difference (Δν) between membrane potential (V_m) and the equilibrium potential for Na⁺/Ca²⁺exchange (E_Na/Ca) is negative or positive: where E_Na/Ca depends on the Nernst potentials for Na⁺(E_Na) and Ca²⁺(E_Ca): From measurements of changes in [Na⁺]_i, and [Ca²⁺]_i during and after stimulation, and with [Na⁺]_o = 205 mm and [Ca²⁺]_o = 13.5 mm, E_Na/Ca can be calculated. When compared with measurements ofV_m (Fig. 4), it can be seen that the Δν is negative at rest (when V_m = −70 mV and E_Na/Ca = −42 mV, Δν = −28 mV), but rapidly goes positive during tetanic stimulation (when V_m = −80 mV andE_Na/Ca = −177 mV, Δν = +93 mV). The hyperpolarization of nerve terminals during tetanic stimulation is caused by operation of the Na⁺/K⁺ pump (Wojtowicz and Atwood, 1985), which extrudes three Na⁺ ions for each two K⁺ ions admitted. Δν remains positive for some 15 min after stimulation, even rising slightly immediately post-tetanically because [Ca²⁺]_i recovers more rapidly than [Na⁺]_i. These data show that the plasma membrane Na⁺/Ca²⁺ exchanger should run in normal mode at rest, extruding Ca²⁺ from cytoplasm, but in reverse mode during and for some time after tetanic stimulation, admitting Ca²⁺ ions from outside, increasing the tetanic rise in [Ca²⁺]_i, potentiating synaptic transmission, slowing the post-tetanic recovery of [Ca²⁺]_i, and prolonging PTP.

Mitochondrial Na⁺/Ca²⁺exchange does not appear to participate in PTP

In addition to the plasmalemmal Na⁺/Ca²⁺exchanger, mitochondrial Na⁺/Ca²⁺exchange might also play a role in PTP. We probed this possibility with CGP 37157, a specific inhibitor of mitochondrial Ca²⁺ efflux (IC₅₀ = 0.36 μm cytoplasmic concentration) (Cox et al., 1993). If mitochondrial Na⁺/Ca²⁺exchange regulates [Ca²⁺]_i in PTP, then with sufficient tetanic Na⁺accumulation, mitochondrial Na⁺/Ca²⁺exchange might transport Ca²⁺ into the cytoplasm, increasing [Ca²⁺]_iaccumulation and EJP potentiation and retarding the post-tetanic decay of [Ca²⁺]_i and PTP. Block of this process would then reduce tetanic cytoplasmic [Ca²⁺]_iaccumulation and synaptic transmission, and [Ca²⁺]_i and PTP would decay more rapidly. Contrary to this scenario, we found that 25 μm CGP 37157 applied 20 min before a tetanus and left on until at least 20 min after the end of the tetanus was completely without effect on the pretetanic, tetanic, and post-tetanic [Ca²⁺]_i changes and baseline transmission or PTP (Fig. 5, Table 1). Because the EJP during tetanic stimulation reflects facilitation and augmentation as well as potentiation (Zucker, 1989), these processes were all apparently unaffected.

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Fig. 5.

Effects of mitochondrial Na⁺/Ca²⁺ transport inhibitor CGP 37157 on [Ca²⁺]_i and PTP. The first tetanus was given in normal saline and the second one in the presence of 25 μm CGP 37157. In 25 μm CGP 37157, [Ca²⁺]_i (A) and EJP amplitude (B) were unchanged.

Plasmalemma Ca²⁺ ATPase does influence PTP

Transport inhibitory peptide (XIP) is another blocker of Na⁺/Ca²⁺exchange (Li et al., 1991), which binds to a cytoplasmic autoinhibitory calmodulin-binding domain of the exchanger with IC₅₀ = 1.7 μm (Xu et al., 1997). This agent must be injected into presynaptic nerve terminals, so it is impractical to measure [Ca²⁺]_i and synaptic transmission in the same preparations before and after drug administration. We therefore compared [Ca²⁺]_i responses with tetanic stimulation in two groups of experiments (different from those used to record effects on EJPs), in one of which XIP had been injected to an estimated concentration of ∼5–10 μm. If Na⁺/Ca²⁺exchange operates in reverse mode in a tetanus, we would expect block of this process to reduce [Ca²⁺]_iaccumulation and EJP potentiation and speed post-tetanic Ca²⁺ removal and PTP decay. In fact, we observed just the opposite effects (Fig. 6A, Table2).

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Fig. 6.

XIP and C28R2 increase [Ca²⁺]_i accumulation and potentiation and retard [Ca²⁺]_i decay in PTP.A, B, EJP amplitudes during the first control tetanus and the second tetanus after presynaptic injection of inhibitors of plasma membrane Na⁺/Ca²⁺ exchange and Ca²⁺ ATPase: XIP (A) or C28R2 (B). C, D, Running averages of five (C) or four (D) post-tetanic EJP amplitudes in control saline (●) and in the presence of inhibitory peptide (○).E, F, [Ca²⁺]_i, averages with SEs in controls (●; with no presynaptic peptide injection;n = 7), XIP injections (○ in E;n = 4), or C28R2 injections (○ inF; n = 4).

One explanation of these results is that XIP also blocks the plasma membrane Ca²⁺ ATPase at similar levels (2.5 μm) to those blocking Na⁺/Ca²⁺exchange (Enyedi and Penniston, 1993). Block of Ca²⁺ efflux by this route should increase [Ca²⁺]_iaccumulation and EJP potentiation and retard post-tetanic Ca²⁺ removal and PTP decay, as we observed. To test this explanation, we used a different inhibitory peptide, C28R2, an agent that is somewhat more effective in blocking the plasmalemma Ca²⁺ ATPase (IC₅₀ = 1 μm) (Enyedi and Penniston, 1993) than Na⁺/Ca²⁺exchange (IC₅₀ = 6.2 μm) (Xu et al., 1997). Presynaptic injection of this inhibitor to ∼1–5 μm had effects identical to those of XIP (Fig.6B, Table 2), suggesting that both act mainly on the Ca²⁺ ATPase to increase [Ca²⁺]_iaccumulation and EJP potentiation and retard post-tetanic Ca²⁺ removal and PTP decay.

Increasing [Na⁺]_i with ouabain acts on plasmalemmal Na⁺/Ca²⁺exchange

The results of Figures 2-4 and Table 1 suggest that tetanic Na⁺ accumulation activates the Na⁺/Ca²⁺exchanger in reverse mode, enhancing tetanic Ca²⁺ accumulation and prolonging its post-tetanic decay. Previous results also implicated Na⁺/Ca²⁺exchange as a target for Na⁺ action in PTP, but they did not distinguish mitochondrial from plasmalemmal Na⁺/Ca²⁺exchange (Mulkey and Zucker, 1992). It was shown, for example, that elevation of presynaptic [Na⁺]_i by block of the Na⁺/K⁺ATPase with ouabain (Delaney and Tank, 1994) led to an increase in [Ca²⁺]_i and EJP amplitude. We have now used the mitochondrial Na⁺/Ca²⁺exchange inhibitor CGP 37157 and the plasmalemmal Na⁺/Ca²⁺exchange inhibitors KB R7943 and XIP to identify the target of Na⁺ action on [Ca²⁺]_i when ouabain is used to elevate [Na⁺]_i (Fig.7).

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Fig. 7.

Effects of transport inhibitors on ouabain-induced [Ca²⁺]_i accumulation and enhancement of synaptic transmission. A, Effects of 0.5 mm ouabain on presynaptic [Ca²⁺]_i and EJP amplitude (n = 6). The preparations were stimulated at 2 Hz during the experiment, and [Ca²⁺]_i was measured every 3 min. Arrows indicate ouabain application (black arrow) and subsequent washout (gray arrow). B, C, Response of [Ca²⁺]_i and EJP amplitude to 25 μm CGP 37157 (●, n = 6), 20 μm KB R7943 (▪, n = 5), or after presynaptic injection of C28R2 (■, n = 5) or XIP (▵, n = 4), compared with controls (○, n = 6). Drugs blocked the [Ca²⁺]_i elevation and EJP enhancement in proportion to their efficacy in blocking plasmalemmal Na⁺/Ca²⁺ exchange.

The Na⁺-dependent increases in [Ca²⁺]_i and EJP amplitude were blocked almost completely by inhibitors of the plasma membrane exchanger but were affected very little by inhibition of mitochondrial Na⁺/Ca²⁺exchange. We also tested the effect of C28R2 on Na⁺-dependent [Ca²⁺]_i elevation and EJP potentiation and found it to have a modest blocking effect (Fig. 7). C28R2 is most effective in blocking the plasmalemma Ca²⁺ ATPase, but it also inhibits plasmalemmal Na⁺/Ca²⁺exchange to some extent, as indicated above. In ouabain, with no tetanic stimulation, the only source for a rise in [Ca²⁺]_i is by reverse mode Na⁺/Ca²⁺exchange, and its inhibition would prevent a rise in [Ca²⁺]_i. Under these circumstances, with Ca²⁺accumulation reduced, inhibition of the Ca²⁺ ATPase should have little effect, and this accounts for our observation of reduced Ca²⁺ accumulation in ouabain in the presence of C28R2. This is the opposite of the effect of C28R2 on Ca²⁺ accumulation and EJP potentiation in a tetanus, where its major action is to reduce the Ca²⁺ ATPase-dependent extrusion of Ca²⁺ that has entered through voltage-dependent Ca²⁺ channels in the tetanus (Fig. 6).