Polyamines and the N-Methyl-D-Aspartate receptor: functional studies of receptor modulation in Xenopus oocytes and rat hippocampal slices.

Abstract

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is involved in various forms of synaptic plasticity and fundamental neuropathology in the CNS. In order to develop treatment strategies that target inappropriate NMDA receptor activity with minimal disruption of normal function, an understanding of the details of NMDAR activation, modulatory mechanisms and the consequences of such modulation on specific receptor functions is essential. We set out with two aims: to study the mechanisms of NMDA receptor modulation by spermine and ifenprodil; and to study the effects of ifenprodil on simple evoked and epileptiform events in the rat hippocampus. For Aim 1, we used single amino acid mutations in the pre-M1 and M3 regions of NR1 subunit and M3 region of NR2B subunit that generate constitutively active NMDA receptors to characterize ligand-gated currents, constitutive currents and proton inhibition. Some mutations in both subunits abolished spermine potentiation and ifenprodil inhibition indicating that both subunits are involved in binding/activity of spermine and ifenprodil. All mutations cause a reduction in receptor sensitivity to protons and differential effects on both ifenprodil inhibition and spermine potentiation including no effect at all in some cases. This implies proton inhibition alone cannot completely account for potentiation of receptors by spermine or inhibition by ifenprodil. The fact that mutations in either subunit had similar effects on receptor modulation by spermine, ifenprodil and protons indicates that there must either be multiple binding sites for spermine, ifenprodil and protons or some form of subunit cooperativity. For Aim 2, we studied the effects of partial blockade of NMDA receptors by ifenprodil on monosynaptic and epileptiform activities in the hippocampus. Most NMDA receptors in the adult central nervous system contain combinations of NR1 and NR2 subunits with the electrophysiological and pharmacological profile of the NMDA receptor channels largely determined by the NR2 subunit. NR1//NR2A and NR1/NR2B are the major NMDAR receptor subtypes are expressed in adult hippocampus and ifenprodil has been shown to have an inhibitory effect at NMDA receptors containing the NR2B subunit. Our hypothesis was that in regions or lamellae within a region of the hippocampus in which NR2B subtype of NMDA receptors are expressed, we will see a reduction in EPSPs by ifenprodil whereas where the NR2B subtype is absent we will not see an effect with ifenprodil. Using electrophysiological recordings of simple and complex synaptic events, we compared broad spectrum NMDA receptor blockade by AP5 with actions of ifenprodil in adult brain slices. Our data show that ifenprodil depresses simple excitatory synaptic activity in stratum oriens and stratum moleculare and potentiates this activity in stratum pyramidale and radiatum of area CA1 but does not affect epileptiform events in the various lamellae suggesting that simple evoked and epileptiform differ in their underlying mechanisms of action. Results of Aim 1 provide insights on the details of subunit interactions in NMDA receptors, gating mechanisms, and the sites and mechanism of action of spermine and ifenprodil at NMDA receptors. Those of Aim 2 help us understand NMDA receptor subunit involvement in hippocampal synaptic events. Taken together these data give useful insights into the NMDA receptor activity that are useful in the development of treatment strategies for NMDA receptor-mediated nervous system disorders that would work by selectively targeting NMDA receptor activity without undermining the functioning of other glutamate receptors.