Zinc inhibits NMDA receptor function through both voltage-dependent and voltage-independent mechanisms. In this report we have investigated the role that the NR1 subunit plays in voltage-independent Zn²⁺ inhibition. Our data show that inclusion of exon 5 into the NR1 subunit increases the IC₅₀ for voltage-independent Zn²⁺inhibition from 3-fold to 10-fold when full length exon 22 is also spliced into the mature NR1 transcript and the NMDA receptor complex contains the NR2A or NR2B subunits; exon 5 has little effect on Zn²⁺ inhibition of receptors that contain NR2C and NR2D. Mutagenesis within exon 5 indicates that the same residues that control proton inhibition, including Lys²¹¹, also control the effects of exon 5 on Zn²⁺inhibition. Amino acid exchanges within the NR1 subunit but outside exon 5 (E181Q, E339Q, E342Q, N616R, N616Q, D669N, D669E, C744A, and C798A) that are known to decrease the pH sensitivity also decrease the Zn²⁺ sensitivity, and concentrations of spermine that relieve tonic proton inhibition also relieve Zn²⁺ inhibition. In summary, our results define the subunit composition of Zn²⁺-sensitive NMDA receptors and provide evidence for structural convergence of three allosteric regulators of receptor function: protons, polyamines, and Zn²⁺.

Modulation of the N- methyl- d -aspartate (NMDA)-selective glutamate receptors by extracellular protons and Zn ²⁺ may play important roles during ischemia in the brain and during seizures. Recombinant NR1/NR2A receptors exhibit a much higher apparent affinity for voltage-independent Zn ²⁺ inhibition than receptors with other subunit combinations. Here, we show that the mechanism of this apparent high-affinity, voltage-independent Zn ²⁺ inhibition for NR2A-containing receptors results from the enhancement of proton inhibition. We also show that the N-terminal leucine/isoleucine/valine binding protein (LIVBP)-like domain of the NR2A subunit contains critical determinants of the apparent high-affinity, voltage-independent Zn ²⁺ inhibition. Mutations H42A, H44G, or H128A greatly increase the Zn ²⁺ IC ₅₀ (by up to ≈700-fold) with no effect on the potencies of glutamate and glycine or on voltage-dependent block by Mg ²⁺ . Furthermore, the amino acid residue substitution H128A, which mediates the largest effect on the apparent high-affinity Zn ²⁺ inhibition among all histidine substitutions we tested, is also critical to the pH-dependency of Zn ²⁺ inhibition. Our data revealed a unique interaction between two important extracellular modulators of NMDA receptors.

Seizures are the manifestation of highly synchronized burst firing of a large population of cortical neurons. Epileptiform bursts with an underlying plateau potential in neurons are a cellular correlate of seizures. Emerging evidence suggests that the plateau potential is mediated by neuronal canonical transient receptor potential (TRPC) channels composed of members of the TRPC1/4/5 subgroup. We previously showed that TRPC1/4 double-knockout (DKO) mice lack epileptiform bursting in lateral septal neurons and exhibit reduced seizure-induced neuronal cell death, but surprisingly have unaltered pilocarpine-induced seizures. Here, we report that TRPC5 knockout (KO) mice exhibit both significantly reduced seizures and minimal seizure-induced neuronal cell death in the hippocampus. Interestingly, epileptiform bursting induced by agonists for metabotropic glutamate receptors in the hippocampal CA1 area is unaltered in TRPC5 KO mice, but is abolished in TRPC1 KO and TRPC1/4 DKO mice. In contrast, long-term potentiation is greatly reduced in TRPC5 KO mice, but is normal in TRPC1 KO and TRPC1/4 DKO mice. The distinct changes from these knockouts suggest that TRPC5 and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms. Furthermore, the reduced seizure and excitotoxicity and normal spatial learning exhibited in TRPC5 KO mice suggest that TRPC5 is a promising novel molecular target for new therapy.

Previous studies suggest that selective antagonists of specific subtypes of muscarinic acetylcholine receptors (mAChRs) may provide a novel approach for the treatment of certain central nervous system (CNS) disorders, including epileptic disorders, Parkinson9s disease, and dystonia. Unfortunately, previously reported antagonists are not highly selective for specific mAChR subtypes, making it difficult to definitively establish the functional roles and therapeutic potential for individual subtypes of this receptor subfamily. The M₁ mAChR is of particular interest as a potential target for treatment of CNS disorders. We now report the discovery of a novel selective antagonist of M₁ mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl)piperazin-1-yl)propyl)-benzo[c][1,2,5]thiadiazole-4 sulfonamide]. Equilibrium radioligand binding and functional studies demonstrate a greater than 75-fold selectivity of VU0255035 for M₁ mAChRs relative to M₂-M₅. Molecular pharmacology and mutagenesis studies indicate that VU0255035 is a competitive orthosteric antagonist of M₁ mAChRs, a surprising finding given the high level of M₁ mAChR selectivity relative to other orthosteric antagonists. Whole-cell patch-clamp recordings demonstrate that VU0255035 inhibits potentiation of N-methyl-d-aspartate receptor currents by the muscarinic agonist carbachol in hippocampal pyramidal cells. VU0255035 has excellent brain penetration in vivo and is efficacious in reducing pilocarpine-induced seizures in mice. We were surprised to find that doses of VU0255035 that reduce pilocarpine-induced seizures do not induce deficits in contextual freezing, a measure of hippocampus-dependent learning that is disrupted by nonselective mAChR antagonists. Taken together, these data suggest that selective antagonists of M₁ mAChRs do not induce the severe cognitive deficits seen with nonselective mAChR antagonists and could provide a novel approach for the treatment certain of CNS disorders.