Ca2+-regulated expression of high affinity methylaminoisobutryic acid transport in hippocampal neurons inhibited by riluzole and novel neuroprotective aminothiazoles

Abstract

High affinity methylaminoisobutyric acid(MeAIB)/glutamine(Gln) transport activity regulated by neuronal firing occurs at the plasma membrane in mature rat hippocampal neuron-enriched cultures. Spontaneous Ca²⁺-regulated transport activity was similarly inhibited by riluzole, a benzothiazole anticonvulsant agent, and by novel naphthalenyl substituted aminothiazole derivatives such as SKA-378. Here, we report that spontaneous transport activity is stimulated by 4-aminopyridine (4-AP) and that phorbol-myristate acetate (PMA) increases high K⁺ stimulated transport activity that is inhibited by staurosporine. 4-AP-stimulated spontaneous and PMA-stimulated high K⁺-induced transport is not present at 7 days in vitro (DIV) and is maximal by DIV∼21. The relative affinity for MeAIB is similar for spontaneous and high K⁺-stimulated transport (K_m ∼ 50 μM) suggesting that a single transporter is involved. While riluzole and SKA-378 inhibit spontaneous transport with equal potency (IC₅₀ ∼ 1 μM), they exhibit decreased (∼3-5 X) potency for 4-AP-stimulated spontaneous transport. Interestingly, high K⁺-stimulated MeAIB transport displays lower and differential sensitivity to the two compounds. SKA-378-related halogenated derivatives of SKA-75 (SKA-219, SKA-377 and SKA-375) preferentially inhibit high K⁺-induced expression of MeAIB transport activity at the plasma membrane (IC₅₀ < 25 μM), compared to SKA-75 and riluzole (IC₅₀ > 100 μM). Ca²⁺-dependent spontaneous and high K⁺-stimulated MeAIB transport activity is blocked by ω-conotoxin MVIIC, ω-agatoxin IVA, ω-agatoxin TK (IC₅₀ ∼ 500 nM) or cadmium ion (IC₅₀ ∼ 20 μM) demonstrating that P/Q-type Ca_V channels that are required for activity-regulated presynaptic vesicular glutamate (Glu) release are also required for high-affinity MeAIB transport expression at the plasma membrane. We suggest that neural activity driven and Ca²⁺ dependent trafficking of the high affinity MeAIB transporter to the plasma membrane is a unique target to understand mechanisms of Glu/Gln recycling in synapses and acute neuroprotection against excitotoxic presynaptic Glu induced neural injury.