Background: NMDA receptor (NMDAR) overactivation and the resulting Ca²⁺ overload represent a convergent biophysical pathway implicated in synaptic failure in Alzheimer’s disease (AD) [1]. Memantine is a clinically used uncompetitive NMDAR antagonist that targets the channel pore, but ligand‑site modulation may offer a distinct pharmacological profile.

Methods: Rat brain synaptosomes were isolated by differential centrifugation and loaded with Fluo‑4 AM to monitor cytosolic Ca²⁺ signals. NMDAR‑dependent responses were evoked with glutamate (50 μM) and glycine (50 μM). The polyphenol G‑41 was tested across 10–100 μM and concentration–response relationships were analyzed using the Hill equation (n = 6 independent preparations). To map the target site(s), we assessed interactions with Zn²⁺ and Mg²⁺ (5 μM; additional tests 10–100 μM in the AD condition), and compared G‑41 effects with memantine (50 μM). GABAergic context was probed using GABA (100 μM), picrotoxin (50 μM), diazepam (10–100 μM), and phenobarbital (10–100 μM).

Results: G‑41 produced concentration‑dependent suppression of both glutamate‑ and glycine‑driven Ca²⁺ signals. The apparent IC₅₀ values were 63.9 ± 2.13 μM for the glutamate‑dependent component and 42.93 ± 3.06 μM for the glycine‑dependent component, with steep Hill coefficients (|nH| ≈ 3.35–6.27), consistent with multi‑step receptor gating and/or integrated Ca²⁺ handling. Zn²⁺ and Mg²⁺ did not measurably alter the G‑41 effect, supporting a mechanism not mediated by these allosteric regulators. G‑41 did not overlap with the memantine pore site, suggesting a pharmacological profile distinct from open‑channel blockade.

Conclusions: G‑41 is a synaptosome‑level modulator of NMDAR‑dependent Ca²⁺ dynamics with evidence for dual modulation of glutamate and glycine binding sites and minimal interaction with Zn²⁺/Mg²⁺ allosteric regulation or the memantine pore site. These properties justify follow‑up electrophysiology and binding studies, as well as in vivo validation in AD‑relevant models.

Alzheimer’s disease, NMDA receptor, synaptosome

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