Activation of ligand-gated channels is initiated by the binding of small molecules at extracellular sites and culminates with the opening of a membrane-embedded pore. To investigate how perturbations at ligand-binding domains influence the gating reaction, we examined current traces recorded from individual NMDA receptors in the presence of several subunit-specific partial agonists. Here we show that low-efficacy agonists acting at either the GluN1 or the GluN2A subunit had very similar effects on the receptor’s activation reaction, possibly reflecting a high degree of coupling between the two subunit-types during gating. In addition, we demonstrate that partial agonists increased the height of all energy barriers encountered by NMDA receptors during activation. This result stands in sharp contrast to the localized effects observed for pentameric ligand-gated channels and may represent a novel mechanism by which partial agonists reduce receptor activity.

Neurosteroids are potent neuromodulators which act in part by binding to and modifying the activity of neurotransmitter-gated channels. Pregnanolone sulfate (PAS) is an endogenous neurosteroid which inhibits NMDA receptors and is neuroprotective in vivo. To delineate the mechanism of NMDA receptor inhibition by pregnanolone sulfate we used kinetic analyses of equilibrium single-channel currents recorded from individual GluN1/GluN2A receptors. Results show that PAS (0.1 mM) reduces single-channel open probability by 50% solely by increasing ~5-fold the mean time spent by receptors in closed conformations. From these data we derive a kinetic scheme that summarizes the effects of PAS on single channel kinetics, accounts for the PAS effects on macroscopic responses and leads us to propose that PAS inhibits NMDA receptor activity by shifting active receptors into desensitized conformations. These findings highlight the neurosteroid inhibitory site on NMDA receptors as a valuable therapeutic target against excitotoxic pathologies including acute and chronic neurodegeneration.

Glutamate-gated channels mediate fundamental brain processes, yet the mechanisms by which the neurotransmitter controls channel activation are incompletely understood. Structural studies revealed that the agonist has the critical role of bridging the divide between two flexible extracellular lobes and solidified the view that agonist-induced cleft-closure drives further isomerizations, which eventually open the channel. Within the glutamate receptor family, NMDA-sensitive channels are unique in their requirement that both glycine and glutamate bind to homologous regions on GluN1 and GluN2 subunits respectively, before the channel can open. To study the gating reaction in separation from agonist binding and dissociation, we characterized the kinetic mechanism of individual NMDA receptors whose ligand-binding clefts were locked shut by disulfide bridges engineered across lobes. We found that locking GluN1 domains had no observable consequences on receptor activity, whereas locking GluN2A domains increased channel activity without reducing the number of resolvable kinetic states. Based on these results we suggest that glutamate but not glycine activates NMDA receptors with sub-maximal efficacy. Low glutamate efficacy may represent a mechanism by which the neurotransmitter maintains control over receptor kinetics despite sharing with glycine the task of activation.

Background: The C-terminal domains (CTDs) of NMDA receptors are essential for normal brain function. Results: We developed kinetic mechanisms for receptors lacking CTDs using single-channel methods. Conclusion: GluN1 CTDs control primarily unitary conductance and GluN2 CTDs control gating kinetics. Significance: Results afford quantitative insight into how intracellular perturbations can change the time course of NMDA receptor currents.

Epithelial Na؉ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an ␣ 2 ␤␥ or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate site within ␣M1. We performed site-directed mutagenesis at this site and found that, although the mutants reduced channel activity, ENaC protein expression at the plasma membrane was unaffected. To deduce the role of ␣M1 in the pore structure of ENaC, we performed tryptophanscanning mutagenesis throughout ␣M1 (residues 110 -130). We found that mutations within the amino-terminal part of ␣M1 had effects on activity and selectivity with a periodicity consistent with a helical structure but no effect on channel surface expression. We also observed that mutations within the carboxyl-terminal part of ␣M1 had effects on activity and selectivity but with no apparent periodicity. Additionally, these mutants reduced channel surface expression. Our data support a model in which the amino-terminal half of ␣M1 is ␣-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of ␣M1 may be helical or assume a different conformation and may be involved in tertiary interactions essential to proper channel folding or assembly. Together, our data suggest that ␣M1 is divided into two distinct regions.The epithelial Na ϩ channel (ENaC) 3 is expressed in the apical membrane of sodium-absorptive epithelia that line the distal nephron, lung airway and alveoli, and descending colon and is the primary target of the potassium-sparing diuretic, amiloride. Expressed in these tissues, ENaC plays a critical role in maintaining Na ϩ homeostasis and controlling blood pressure and airway fluid volume. ENaC comprises homologous ␣, ␤, and ␥ subunits arranged in either a tetrameric ␣ 2 ␤␥ or higher ordered arrangement (1-5). Each subunit has two membranespanning domains (termed M1 and M2), intracellular amino and carboxyl termini, and large extracellular domains. ENaCs are members of the Degenerin/ENaC family of cation-selective channels that have roles in mechanosensation and nociception.The structure of the ENaC pore has been the subject of several studies. The pore has been shown to be composed of the M2 segments along with the residues immediately extracellular to these segments from all three subunits (6, 7). In the tetrameric ␣ 2 ␤␥ model, the two ␣ subunits are posited to be on opposite sides of the pore (2). Given that the pore-forming components of all three subunits are highly homologous (see Fig. 1), the pore is thought to have 4-fold pseudosymmetry such that all three subunits fold and contribute similarl...