Pharmacological dissection of calcium channel subtype-related components of strontium inflow in large mossy fiber boutons of mouse hippocampus

Several subtypes of voltage-dependent calcium channels (VDCCs) are present in the presynaptic terminals. In the mammalian hippocampus, P/Q-, N-, and R- but not L-type VDCCs are involved in the fast transmitter release from large mossy fiber (MF) boutons, which are associated with CA3 pyramidal cell dendrites. We investigated whether L-type VDCCs are indeed absent in these large MF boutons. With the use of Sr²⁺ as the Ca²⁺ substitute, the stimulus-evoked Sr²⁺ increment (Δ[Sr²⁺]_pre) was evaluated fluorometrically. Δ[Sr²⁺]_pre appeared to be proportional to Sr²⁺ inflow through VDCCs and was specifically attenuated by conventional VDCC subtype-selective antagonists. The P/Q-type selective ω-agatoxin IVA (AgTx_IVA) blocked Δ[Sr²⁺]_pre with an IC₅₀ of 28 nM and by 30-35% at its maximum effective concentration of 0.5 μM. The N-type selective ω-conotoxin GVIA (CgTx_GVIA) blocked Δ[Sr²⁺]_pre with an IC₅₀ of 15 nM and by 20-25% at its maximum effective concentration of 1 μM. The R-type selective SNX-482 blocked Δ[Sr²⁺]_pre with an IC₅₀ of 79 nM and by 20-25% at its maximum effective concentration of 1 μM. The effects of these toxins did not overlap at their maximum effective concentrations and about 70-80% of Δ[Sr²⁺]_pre was blocked by the simultaneous exposure to these toxins. Δ[Sr²⁺]_pre component that is resistant to AgTx_IVA, CgTx_GVIA, and SNX-482 was significantly potentiated by an L-type agonist, (S)-(-)-Bay K8644, and attenuated by an L-type antagonist, nimodipine, suggesting that L-type VDCCs are present in large MF terminals. The L-type agonist, (±)-Bay K8644, also potentiated Sr²⁺ inflow into individual boutons identified as large MF boutons under confocal microscopy. Almost similar results were observed for Ca²⁺ inflow-dependent fluorescence increments. L-type VDCCs appear to be present in large MF boutons and mediate a substantial Ca²⁺ inflow into presynaptic terminals during action potentials.