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dc.contributor.authorMaki, Bruce Edward
dc.date.accessioned2016-04-05T19:59:54Z
dc.date.available2016-04-05T19:59:54Z
dc.date.issued2015
dc.identifier.isbn9781321922196
dc.identifier.other1700219314
dc.identifier.urihttp://hdl.handle.net/10477/51682
dc.description.abstractThe development, maintenance, and plasticity of central excitatory synapses depend critically on the Ca 2+ fluxes produced by the activation of NMDA receptors. In turn, the amplitude and time course of these Ca 2+ fluxes depend on the receptor's gating kinetics, unitary conductance, and Ca 2+ -permeability. NMDA receptors are transmembrane glutamate-gated excitatory channels with characteristically slow gating and high Ca 2+ permeability. They are expressed throughout the brain and spinal cord as heterotetramers of two obligatory GluN1 subunits and two regionally and developmentally regulated GluN2(A-D) subunits. Three regions of the protein control the characteristically high Ca 2+ permeability of GluN1/GluN2A receptors: several charged residues within the DRPEER sequence located on the GluN1 subunit at the extracellular mouth of the pore; an asparagine residue (the N 0 -site) located on the GluN1 subunit at the narrowest region of the pore; and residues on the receptor's intracellular carboxy-terminal domain (CTD). In this dissertation, I describe experiments and results that illuminate how residues in these three regions modulate NMDA receptor gating kinetics, unitary conductance, and Ca 2+ permeability. To delineate the biophysical mechanisms that control NMDA receptor Ca 2+ permeability, I used an integrated approach that included mutagenesis, electrophysiology, modeling of single-molecule kinetics, and calcium imaging. Recordings obtained from single-molecules embedded in cell-attached membrane patches showed that increasing extracellular Ca 2+ concentrations reduced both channel conductance and channel open probability, with a half-maximal effect in the physiologic range: IC 50 , ~2 mM. Importantly, this effect was independent of intracellular domains, and was also independent of residues in the pore. Instead, the electrical charge of the first two residues of the DRPEER sequence was critical for these effects. Based on these results, I propose that these two residues form an external Ca 2+ -binding site that is required for the receptor's high Ca 2+ permeability. The effects on unitary conductance and gating appeared highly coupled, and also correlated with increased permeability for larger organic cations, thus suggesting a concerted increase in pore size. Notably, truncating the side-chain of the N 0 -site with a glycine substitution ablated these effects. Based on these results, I propose that that Ca 2+ binding to DR side-chains at the mouth of the pore reduces channel conductance by constricting the pore diameter through an allosteric effect. Last, I probed the C-terminal tails of GluN1 and GluN2A subunits to investigate their effects on channel properties. I found that truncating the GluN1 CTD produced channels with increased unitary conductance, and truncating the GluN2 CTD produced channels with lower activity. This is potentially important physiologically because these domains contain multiple residues that are modified by phosphorylation/dephosphorylation cycles. Consistent with this hypothesis, I found that phosphomimetic (S/D) substitutions at two known PKC phosphorylation sites on the GluN1 subunit replicated the increase in unitary conductance observed with receptors lacking GluN1 CTD, while phosphodeficient substitutions (S/A) at two known PKA phosphorylation sites on the GluN2 replicated the lower activity observed with receptors lacking GluN2 CTD. Together, these results demonstrate that NMDA receptor conductance and gating may be controlled separately by targeting phosphorylation sites on GluN1 and GluN2 residues, respectively. Overall, the results described here provide mechanistic insight into the complex relationship between NMDA receptor conductance, gating, and Ca 2+ permeability. My novel hypothesis that Ca 2+ binding to external charged residues controls pore dimensions, together with my discovery that separate intracellular domains on GluN1 and GluN2 subunits control channel conductance and gating, respectively, represent substantial advances toward understanding how specific structural features of the NMDA receptor protein control the receptor's biophysical properties, and ultimately its role in the physiology and pathology of the central nervous system. This new knowledge will facilitate further inquiries into how dynamic signaling in mammalian brain can control the NMDA receptor mediated signal in both physiological and pathological conditions.
dc.languageEnglish
dc.sourceDissertations & Theses @ SUNY Buffalo,ProQuest Dissertations & Theses Global
dc.subjectPure sciences
dc.subjectBiological sciences
dc.subjectCalcium signaling
dc.subjectGating
dc.subjectIon channel
dc.subjectNMDA receptor
dc.subjectPermeation
dc.subjectPhosphorylation
dc.titleLinking NMDA Receptor Conductance, Gating and Calcium Permeability
dc.typeDissertation/Thesis


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