Effects of External and Internal Ca2+ on Unitary NMDA Receptor Properties

Abstract

N-Methyl-D-aspartate (NMDA) receptors are glutamate- and glycine-gated channels widely expressed throughout the central nervous system. When active, they generate electric and biochemical signals by fluxing Na+ and Ca2+ into the post-synaptic cell. The NMDA receptor-mediated Ca2+ transient initiates internal cascades that result in synaptic plasticity and excitotoxicity. In turn, Ca2+ regulates channel activity directly and indirectly through allosteric and 2nd messenger mechanisms that are poorly understood. We investigated the effects of external and internal Ca2+ concentrations on recombinant GluN1/GluN2A (N1/2A) receptors using single-channel current recordings, statistical analysis, and kinetic modeling. Increasing concentrations of extracellular Ca2+ reduced the unitary channel conductance from 75.9±1.4 pS in 0 mM [Ca2+]e, to 55.2±1.6 pS in 1.8 mM [Ca2+]e, and to 11.8±0.7 pS in 75 mM [Ca2+]e. Importantly, receptors lacking the intracellular C-terminal domain (CTD) of the N1 subunit (N1Δ/2A) but not the 2A subunit (N1/2AΔ) exhibited higher unitary conductance in the absence of external Ca2+ (N1Δ/2A γ = 83.3±1.0 pS, N1/2AΔ γ = 79.2±1.4 pS). This is consistent with a role for the N1 CTD in setting channel conductance for Na+. However, in the presence of physiological external Ca2+ (1.8 mM), both exhibited N1Δ/2A (γ = 50.4±1.2 pS, p<0.05) and N1/2AΔ (γ = 60.8±2.0 pS, p<0.05) channels had a lower conductance relative to wild-type. Based on these novel results, we asked whether this Ca2+-dependent regulation of conductance depends on calmodulin binding to either the C0 or C1 cassettes of N1. Furthermore, we asked whether local Ca2+ influx through the NMDA receptor pore is sufficient for this regulation. Our results help to further unravel the Ca2+-dependent processes that control the properties of individual NMDA receptor channels.