Different relationship of N- and P/Q-type Ca2+ channels to channel-interacting slots in controlling neurotransmission at cultured hippocampal synapses

Abstract

Synaptic transmission at CNS synapses is often mediated by joint actions of multiple Ca(2+) channel subtypes, most prominently, P/Q- and N-type. We have proposed that P/Q-type Ca(2+) channels saturate type-preferring slots at presynaptic terminals, which impose a ceiling on the synaptic efficacy of the channels. To test for analogous interactions for presynaptic N-type Ca(2+) channels, we overexpressed their pore-forming Ca(V)2.2 subunit in cultured mouse hippocampal neurons, recorded excitatory synaptic transmission from transfected cells, and dissected the contributions of N-, P/Q-, and R-type channels with subtype-specific blockers. Overexpression of Ca(V)2.2 did not increase the absolute size of the EPSC even though somatic N-type current was augmented by severalfold. Thus, the strength of neurotransmission is saturated with regard to levels of Ca(2+) channel expression for both N-type and P/Q-type channels. Overexpression of Ca(2+)-impermeable Ca(V)2.2 subunits decreased EPSC size, corroborating competition for channel slots. Striking asymmetries between N- and P/Q-type channels emerged when their relative contributions were compared with channel overexpression. Overexpressed N-type channels could competitively displace P/Q-type channels from P/Q-preferring slots and take over the role of supporting transmission. The converse was not found with overexpression of P/Q-type channels, regardless of their C-terminal domain. We interpret these findings in terms of two different kinds of presynaptic slots at excitatory synapses, one accepting N-type channels but rejecting P/Q-type (N(specific)) and the other preferring P/Q-type but also accepting N-type (PQ(preferring)). The interaction between channels and slots governs the respective contributions of multiple channel types to neurotransmission and, in turn, the ability of transmission to respond to various stimulus patterns and neuromodulators.

Figure 1.

N- and P/Q-type Ca²⁺ channels exhibit different relationship to slots in controlling neurotransmission at cultured hippocampal synapses. A, Schematic representations of the C-terminal cytoplasmic domains of human Ca_V2.2 subunit (N_WT) as well as the short and long splice variants of human Ca_V2.1 subunits (PQ_WT and PQ_WT-long, respectively). The last four amino acids of the C terminus of each subunit are indicated. The lengths of primary sequence are drawn to scale. B, Averaged EPSC traces (5∼7 traces at the end of each condition) elicited by neurons expressing EGFP alone (EGFP), or EGFP with human Ca_V2 subunits (PQ_WT, PQ_WT-long, or N_WT, respectively), as well as the effect of toxins on EPSC amplitude. The N-, P/Q-, or R-type Ca²⁺ channel blockers ω-CTx-GVIA (2 μm), ω-Aga-IVA (1 μm), and SNX482 (0.5 μm) were applied sequentially and cumulatively into the bath solution as indicated. C, Relative contributions of N-, P/Q-, and R-type channels to EPSC amplitude in neurons expressing EGFP alone (EGFP, n = 20), or EGFP with human Ca_V2 subunits (PQ_WT, PQ_WT-long, N_WT, and N_{WT-10 DIV}; n = 16, 7, 23, and 5, respectively). Neurons were transfected at 5 DIV except for the N_{WT-10 DIV} group, which was composed of neurons transfected at 10 DIV. Ca²⁺ channels were blocked as shown in B. Neither the expression of PQ_WT or PQ_WT-long altered the relative contributions of N-, P/Q-, and R-type channels to excitatory transmission. However, overexpression of N_WT (either starting from 5 or 10 DIV) significantly increased the N-type contribution to synaptic transmission (*p < 0.05, compared with EGFP group). Correspondingly, the contribution of PQ to transmission was significantly decreased in N_WT and N_{WT-10 DIV} groups (^# p < 0.05, compared with EGFP group). D, Synaptic functionality of PQ_WT-long channels, tested in hippocampal neurons from Ca_V2.1-knock-out mice. Expression of exogenous PQ_WT-long subunits was capable of restoring the ω-Aga-IVA-sensitive component of synaptic current (n = 3). Data for Ca_V2.1-knock-out (ko) and PQ_WT-transfected neurons from Cao et al. (2004) are included to allow direct comparison. Error bars indicate SEM. E, Overall EPSC amplitude distribution was comparable across all groups (n = 12–70). F, Cumulative distribution of ω-CTx-GVIA-sensitive EPSC amplitude of EGFP, N_WT, and PQ_WT groups of neurons as in C. The N_WT group showed a significantly larger component of N-type-dependent EPSC (p < 0.05 vs EGFP group). No significant difference was found between PQ_WT and EGFP groups (p > 0.4). G, Cumulative distribution of ω-Aga-IVA-sensitive EPSC amplitude of EGFP, N_WT, and PQ_WT groups of neurons as in C. The P/Q-component of the EPSC was significantly smaller in the N_WT group (p < 0.05 vs EGFP group; p < 0.01 vs PQ_WT group). No significant difference was found between PQ_WT and EGFP groups (p > 0.3).

Figure 2.

Further tests of the effects of overexpressing N_WT or PQ_WT on excitatory neurotransmission and Ca²⁺ channel currents. A, Pharmacological tests with ω-Aga-IVA applied first. Relative contribution of P/Q-, N-, and R-type channels to EPSC amplitude in neurons expressing EGFP alone (EGFP, n = 19) or EGFP with human Ca_V2 subunits (PQ_WT, n = 8; N_WT, n = 13). The P/Q-, N-, or R-type Ca²⁺ channel blockers ω-Aga-IVA (1 μm), ω-CTx-GVIA (2 μm), and SNX482 (0.5 μm) were introduced sequentially and cumulatively into the bath solution. Overexpression of PQ_WT did not alter the relative contributions of N-, P/Q-, and R-type channels to excitatory transmission. Overexpression of N_WT significantly increased the N-type contribution to synaptic transmission (*p < 0.05, compared with EGFP group). Conversely, a direct comparison of PQ-mediated transmission in EGFP and N_WT groups showed that it was significantly decreased by overexpression of N_WT (p < 0.03). We noted that strict adherence to statistical procedures would preclude such a comparison because one-way ANOVA of the P/Q-mediated transmission in EGFP, PQ_WT, and N_WT groups failed to show significance. B1, Peak whole-cell Ba²⁺ current density through P/Q-type Ca²⁺ channels at +10 mV. P/Q-type component was defined as Ba²⁺ currents sensitive to the blockade of 1 μm ω-Aga-IVA. Hippocampal neurons were transfected with EGFP alone (n = 19), or EGFP with PQ_WT, PQ_WT-long, or N_WT (n = 6, 13, and 10, respectively). Overexpressing PQ_WT or PQ_WT-long caused an increase of whole-cell P/Q current density (***p < 0.001) relative to EGFP group. The level of P/Q current density was similar in PQ_WT and PQ_WT-long groups. In contrast, overexpressing N_WT did not alter P/Q-type current density. B2, Reliability of EGFP expression as a marker of expression of exogenous P/Q-type Ca²⁺ channels. Cumulative distributions of amplitudes of somatodendritic P/Q-type current density. We tested the null hypothesis that P/Q-type current was no different in neurons transfected with EGFP+PQ_WT-long or EGFP+PQ_WT than that in neurons transfected with EGFP alone. This hypothesis can be rejected even for the smallest observed currents in the PQ_WT-long (p < 0.01) or PQ_WT groups (p < 0.001). C1, Peak whole-cell Ba²⁺ current density through N-type Ca²⁺ channels at +10 mV, from the same neurons as in B. N-type component was defined as Ba²⁺ current sensitive to the blockade of 2 μm ω-CTx-GVIA. Overexpression of PQ_WT or PQ_WT-long did not alter whole-cell N-type current density. On the contrary, overexpression of N_WT resulted in a significant increase of N-type current density compared with that of EGFP group (***p < 0.001). Error bars indicate SEM. C2, Reliability of EGFP expression as a marker of expression of exogenous N-type Ca²⁺ channels. Cumulative distributions of amplitudes of somatodendritic N-type current density. We tested the null hypothesis that N-type current density was no different in neurons transfected with EGFP and N_WT than that in neurons transfected with EGFP alone. This hypothesis can be rejected even for the smallest observed currents in the EGFP+N_WT group (p < 0.01).

Figure 3.

Characterization of nonpermeable human Ca_V2.2 subunits (N_imperm). A, Topology of human Ca_V2.2 subunit. The locations of four concerted glutamate-to-alanine mutations in the pore-forming region of N_imperm are indicated. B, Representative AP traces from hippocampal neurons expressing EGFP alone or EGFP with N_imperm. Somatic APs were evoked by a depolarizing current injection (0.5 ms, 1 nA). Expression of N_imperm did not change AP waveform (N_imperm vs EGFP group). Furthermore, blocking both endogenous N-type Ca²⁺ channels and exogenous N_imperm channels with 2 μm ω-CTx-GVIA did not significantly alter the AP shape (N_imperm+GVIA vs other groups). See Table 1 for comparisons of AP properties. C, The current–voltage relationships (I–V curves) of N_WT and N_imperm in HEK293 cells (n = 5 in each group). The N_imperm group did not show any inward Ba²⁺ current but displayed an outward current when depolarized beyond 0 mV. D, The I–V curves of the N-type (ω-CTx-GVIA-sensitive) Ba²⁺ current in hippocampal neurons expressing EGFP alone (EGFP, n = 6), EGFP with N_imperm (N_imperm, n = 5), or EGFP with both N_imperm and PQ_WT subunits (N_imperm+PQ_WT, n = 8). Transfected neurons were voltage clamped at −80 mV. I–V curves were generated by applying a ramp depolarization from −80 to +100 mV at 1.8 mV/ms. The ω-CTx-GVIA-sensitive current densities were obtained by subtraction of I–V relationships in the absence and presence of toxin. Inward currents were consistently seen over the voltage range between −20 and +20 mV. At more depolarizing voltages, I–V curves from N_imperm-expressing neurons exhibited a negative shift of reversal potential and an increase of outward current compared with the I–V curve of EGFP-expressing neurons, as expected for a combination of endogenous N-type channels and exogenous N_imperm channels operating in parallel. E, Peak whole-cell Ba²⁺ current density through P/Q-type Ca²⁺ channels at +10 mV from hippocampal neurons expressing EGFP alone (EGFP, n = 12), EGFP with N_imperm (N_imperm, n = 8), or EGFP with both N_imperm and PQ_WT Ca_V2 subunits (N_imperm+PQ_WT, n = 7). The expression of N_imperm channels did not alter the current density from endogenous P/Q-type channels (N_imperm vs EGFP groups), nor did it affect the increase of P/Q-current density as a result of overexpression of PQ_WT (***p < 0.001, N_imperm+PQ_WT vs other groups). F, Peak whole-cell Ba²⁺ current density through N-type Ca²⁺ channels at −10 mV from the same neurons in D. At this voltage, the N_imperm channels did not exhibit inward or outward current. The expression of N_imperm (and further addition of PQ_WT) did not alter the current density from the endogenous N-type Ca²⁺ channels. Error bars indicate SEM.

Figure 4.

Effects of N_imperm channels on synaptic transmission. A, Averaged EPSC traces (5∼7 traces at the end of each condition) elicited by neurons expressing EGFP alone (EGFP), or EGFP with human Ca_V2 subunits (N_imperm, N_imperm+PQ_WT, and PQ_WT, respectively) as well as the effect of toxins on EPSC amplitude. Neurons were exposed to the N-, P/Q-, or R-type Ca²⁺ channel blockers ω-CTx-GVIA (2 μm), ω-Aga-IVA (1 μm), and SNX482 (0.5 μm) sequentially and cumulatively as indicated. B, Relative contributions of N-, P/Q-, and R-type channels to EPSC amplitude in neurons expressing EGFP alone (EGFP, n = 20), or EGFP with human Ca_V2 subunits (N_imperm, N_imperm+PQ_WT, and PQ_WT; n = 10, 10, and 18, respectively). Ca²⁺ channels were blocked as shown in A. Overexpression of N_imperm did not alter the relative contributions of N-, P/Q-, and R-type channels to excitatory transmission. However, coexpression of PQ_WT and N_imperm resulted in a significant reduction of N-type contribution to synaptic transmission (*p < 0.05, N_imperm+PQ_WT vs EGFP group,). Correspondingly, the contribution of P/Q-type channels to transmission was significantly increased in N_imperm+PQ_WT group (^# p < 0.05, compared with EGFP group). C, Cumulative distribution of overall EPSC amplitude at synapses expressing EGFP alone (n = 70), or EGFP with human Ca_V2 subunits (N_imperm, n = 22; N_imperm+PQ_WT, n = 18; PQ_WT, n = 26). Presynaptic expression of N_imperm channels alone significantly reduced the size of EPSCs (p < 0.05, N_imperm vs EGFP group). In contrast, the synaptic strength was well maintained when PQ_WT and N_imperm were coexpressed at presynaptic terminals (p > 0.17, compared with EGFP group). D, Ensemble average of overall EPSC amplitude from the same groups in C. E, PPR at synapses expressing EGFP alone or EGFP with various human Ca_V2 subunits (n = 12–70). Pairs of EPSCs were elicited by stimuli 50 ms apart, and the ratio of the EPSC amplitudes was determined. PPR was significantly increased at synapses expressing N_imperm compared with that at synapses expressing EGFP alone (*p < 0.05, one-way ANOVA post hoc Bonferroni). PPR values were not altered in other experimental groups. Error bars indicate SEM.

Figure 5.

N-type-specific slots are filled to capacity by endogenous N-type channels. A, Relative contributions of N-, P/Q-, and R-type channels to EPSC amplitude in neurons expressing EGFP alone (EGFP, n = 20), or EGFP with human Ca_V2 subunits (N_WT, n = 23; N_WT+PQ_WT, n = 11). The N-, P/Q-, or R-type Ca²⁺ channel blockers ω-CTx-GVIA (2 μm), ω-Aga-IVA (1 μm), and SNX482 (0.5 μm) were applied sequentially and cumulatively into the bath solution. In the presence of extra PQ_WT, overexpression of N_WT could no longer increase the N contribution to synaptic transmission, indicating that N- and P/Q-type channels compete for a common subset of slots (*p < 0.05, N_WT vs EGFP group; no significant difference between N_WT+PQ_WT and EGFP groups). B, Cumulative distribution of overall EPSC amplitude at synapses expressing EGFP alone (n = 70), or EGFP with human Ca_V2 subunits (N_WT, n = 61; N_WT+PQ_WT, n = 18). The synaptic strength was comparable across all groups (p > 0.33, Kolmogorov–Smirnov test). C, Peak whole-cell Ba²⁺ current density through P/Q-type Ca²⁺ channels at +10 mV from hippocampal neurons expressing EGFP alone (EGFP, n = 19), or EGFP with human Ca_V2 subunits (N_WT, n = 10; N_WT+PQ_WT, n = 7). Overexpression of PQ_WT, either alone or with N_WT, resulted in significant increase of whole-cell P/Q current density (***p < 0.001, relative to EGFP group). The magnitude of P/Q-current density was not significantly different between N_WT+PQ_WT (50.1 ± 3.8 pA/pF) and the PQ_WT groups (58.0 ± 13.3 pA/pF) (Fig. 2 B) (p > 0.5). Overexpressing N_WT in neurons did not alter P/Q-type current density. D, Peak whole-cell Ba²⁺ current density through N-type Ca²⁺ channels at +10 mV from the same neurons in C. Overexpression of N_WT, either alone or with PQ_WT, significant increased the whole-cell N-type current density (**p < 0.01, relative to EGFP group). The magnitude of N-type current density was comparable between the N_WT (32.8 ± 3.3 pA/pF) and the N_WT+PQ_WT (35.1 ± 4.7 pA/pF; p > 0.6) groups. Error bars indicate SEM. E, Different relationship of N- and P/Q-type Ca²⁺ channels to slots in controlling neurotransmission at cultured hippocampal synapses. Our data indicate that there are at least two different kinds of presynaptic slots at excitatory hippocampal synapses. The N _specific slots accept N-type channels selectively but reject P/Q-type Ca²⁺ channels. The PQ _preferring slots prefer P/Q-type Ca²⁺ channels but also accept N-type when P/Q-type channels are missing or N-type are overexpressed. Note that terminology for slots does not consider R-type channels.