Conformational Mobility of S1-M1 and S2-M4 Linkers Influence NMDA Receptor Gating

Abstract

NMDA receptors are tetrameric glutamate-gated ion channels pivotal to excitatory neurotransmission in the mammalian central nervous system. Each subunit of the NMDA receptor harbors an extracellular ligand-binding domain (LBD) connected by three linkers to the transmembrane segments that form the ion channel. Activation gating of NMDAR involves the propagation of ligand-induced conformational changes in the LBD into ion channel pore opening via linker peptide segments. The M3-S2 linker, which is attached to the pore-lining M3 transmembrane segment, has been shown to be a critical gating element. Constraining the conformational freedom of M3-S2 relative to the adjacent S2-M4 linker of the same subunit drastically reduced the efficacy of gating with a 30-75-fold reduction in channel open probability. Here we report the gating effects of limiting the relative conformational mobility of the S1-M1 and S2-M4 linkers with crosslinking disulfide bonds. Both intra- and inter- subunit crosslinking strategies in GluN1/GluN2A-containing receptors were used to dissect the linkers’ roles in gating transduction. We found that although the S1-M1 and S2-M4 linkers are connected respectively to the M1 and M4 transmembrane segment located peripheral to the pore, constraining their relative movements significantly hampered gating efficacy, as assayed using single channel recording. These results suggest that all the linkers in a single receptor complex function as an intricate unit in the gating transduction process and have important implications for potential drug targeting as unique noncompetitive antagonists are known to bind to the S1-M1 and S2-M4 linkers of certain glutamate receptor subtypes, although their mechanisms of action are yet undefined.