one-channel-cell-attached-patch-clamp-recording

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

University at Buffalo, SUNY

NMDA receptors (NRs) are glutamate-gated calcium-permeable channels that are essential for normal synaptic transmssion and contribute to neurodegeneration. Tetrameric proteins consist of two obligatory GluN1 (N1) and two GluN2 (N2) subunits, of which GluN2A (2A) and GluN2B (2B) are prevalent in adult brain. The intracellularly located C-terminal domains (CTDs) make a significant portion of mass of the receptors and are essential for plasticity and excitotoxicity, but their functions are incompletely defined. Recent evidence shows that truncation of the N2 CTD alters channel kinetics; however, the mechanism by which this occurs is unclear. Here we recorded activity from individual NRs lacking the CTDs of N1, 2A, or 2B and determined the gating mechanisms of these receptors. Receptors lacking the N1 CTDs had larger unitary conductance and faster deactivation kinetics, receptors lacking the 2A or 2B CTDs had longer openings and longer desensitized intervals, and the first 100 amino acids of the N2 CTD were essential for these changes. In addition, receptors lacking the CTDs of either 2A or 2B maintained isoform-specific kinetic differences and swapping CTDs between 2A and 2B had no effect on single-channel properties. Based on these results, we suggest that perturbations in the CTD can modify the NR-mediated signal in a subunit-dependent manner, in 2A these effects are most likely mediated by membrane-proximal residues, and the isoform-specific biophysical properties conferred by 2A and 2B are CTD-independent. The kinetic mechanisms we developed afford a quantitative approach to understanding how the intracellular domains of NR subunits can modulate the responses of the receptor.

C-terminal domains of N-methyl-D-aspartic acid receptor modulate unitary channel conductance and gating.

NMDA receptors are glutamate-activated, Ca ( 2+) -permeable ion channels with critical roles in synaptic transmission and plasticity. The shape and size of their current is modulated by several kinase/phosphatase systems, and numerous residues located on the receptors' intracellular C-termini are phosphorylated in vivo. To investigate the mechanisms by which phosphorylation may control channel gating, we examined the single-channel behaviors of receptors carrying the S900A or S929A substitution in their GluN2A subunits and thus were rendered resistant to phosphorylation at those sites. We found that the mutations reduced channel open probability primarily by increasing the frequency of desensitized events. The kinetic models we developed revealed complex but similar changes in mechanism for the two mutants, leading to the view that dephosphorylation at either site may cause receptors to activate slower, deactivate faster and desensitize more frequently. This modulatory mechanism is consistent with the proposed roles for these residues in Ca ( 2+) -dependent desensitization and calcineurin-mediated reduction of current during brain development.

Two serine residues on GluN2A C-terminal tails control NMDA receptor current decay times.

Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca2+ flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered CTDs prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor relative Ca2+ permeability (PCa/PNa) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca2+ conductance, since neither Na+ conductance nor Ca2+-dependent block were altered substantially. Last, we show that both the gating and permeation effects can be repro-duced by changing the phosphorylation state of a single residue: GluN2B S1166 and GluN1 S897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca2+ permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may ac-count for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca2+ permeability.

Article	Year
C-terminal domains of N-methyl-D-aspartic acid receptor modulate unitary channel conductance and gating. The Journal of biological chemistry\| PubMed ID: 22948148	2012
Two serine residues on GluN2A C-terminal tails control NMDA receptor current decay times. Channels (Austin, Tex.)\| PubMed ID: 23503100	2013
Separate intramolecular targets for protein kinase A control of N-methyl-D-aspartate receptor gating and Ca2+ permeability. The Journal of biological chemistry\| PubMed ID: 24847051	2014

Bruce A. Maki

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