Modulating the Intrinsic Disorder in the Cytoplasmic Domain Alters the Biological Activity of the N-Methyl-d-aspartate-sensitive Glutamate Receptor

Ucheor B Choi,Rashek Kazi,Natalie Stenzoski,Lonnie P Wollmuth,Vladimir N Uversky,Mark E Bowen

doi:10.1074/jbc.m113.477810

Abstract

The NMDA-sensitive glutamate receptor is a ligand-gated ion channel that mediates excitatory synaptic transmission in the nervous system. Extracellular zinc allosterically regulates the NMDA receptor by binding to the extracellular N-terminal domain, which inhibits channel gating. Phosphorylation of the intrinsically disordered intracellular C-terminal domain alleviates inhibition by extracellular zinc. The mechanism for this functional effect is largely unknown. Proline is a hallmark of intrinsic disorder, so we used proline mutagenesis to modulate disorder in the cytoplasmic domain. Proline depletion selectively uncoupled zinc inhibition with little effect on receptor biogenesis, surface trafficking, or ligand-activated gating. Proline depletion also reduced the affinity for a PDZ domain involved in synaptic trafficking and affected small molecule binding. To understand the origin of these phenomena, we used single molecule fluorescence and ensemble biophysical methods to characterize the structural effects of proline mutagenesis. Proline depletion did not eliminate intrinsic disorder, but the underlying conformational dynamics were changed. Thus, we altered the form of intrinsic disorder, which appears sufficient to affect the biological activity. These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating.

Highlights

The NMDA-sensitive glutamate receptors contain disordered cytoplasmic domains that support isoformspecific signaling
These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating
To address whether the conformational dynamics of C-terminal domain 2 (CTD2) contribute to channel gating, we introduced proline depletions into the full-length GluN2B subunit and recorded membrane currents in NMDA receptors using patch clamp electrophysiology

Summary

Background

The NMDA-sensitive glutamate receptors contain disordered cytoplasmic domains that support isoformspecific signaling. We altered the form of intrinsic disorder, which appears sufficient to affect the biological activity These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating. The GluN2B CTD directs synaptic targeting through interactions with scaffold proteins such as PSD-95 and modulates ion channel gating and sensitivity to extracellular Zn2ϩ [1]. By altering the conformation of the disordered CTD with proline depletion, we were able to uncouple physiological regulation of the NMDA receptor by extracellular Zn2ϩ while preserving basic functions associated with gating, biogenesis, and trafficking. The same mutations affected the biological activity of the intact receptor These findings suggest that the underlying dynamics of the disordered cytoplasmic domain mediate the allosteric regulation of ionotropic glutamate receptor gating

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION