Marcel P. Goldschen-Ohm

Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels.

Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na+ channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K+ current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

Kinetics and Spontaneous Open Probability Conferred by the Subunit of the GABAA Receptor

GABAA receptors mediate synaptic and extrasynaptic inhibition. Native receptors consist of α and β subunits, which are required for function, and another “modulatory” subunit, for example, γ, δ, or ϵ. Of these, the ϵ subunit has the most restricted distribution, confers resistance to neurosteroid and anesthetic modulation, and causes spontaneous channel opening. Little is known, however, about how ϵ affects receptor kinetics, which in turn shape responses to both ambient and synaptic GABA exposure. Here, we expressed human α2β1, α2β1γ2, or α2β1ϵ subunit combinations in human embryonic kidney 293 cells and used rapid solution exchange to study receptor kinetics in outside-out patches. The ϵ subunit greatly slowed deactivation and recovery after brief GABA pulses. During long, saturating GABA pulses, the rate of desensitization was slower for α2β1ϵ and α2β1γ2 than for α2β1. However, in α2β1ϵ, the final extent of desensitization was large compared with that of α2β1γ2. Responses in α2β1ϵ, but not the others, were often followed by an “overshoot” above the baseline, suggesting that a fraction of channels are spontaneously open and are transiently silenced by receptor activation and subsequent desensitization. The baseline current and associated noise were reduced by picrotoxin, revealing that ϵ-containing channels are open ∼4% of the time in the absence of GABA. These results suggest that, if ϵ-containing receptors are expressed at synapses, the synaptic currents would be long-lasting but may rundown quickly under high-frequency activation. In addition, silencing of spontaneous openings by desensitization raises the possibility that tonic inhibition mediated by ϵ-containing receptors may be regulated by phasic inhibition.

Multiple pore conformations driven by asynchronous movements of voltage sensors in a eukaryotic sodium channel

Voltage-dependent Na+ channels are crucial for electrical signalling in excitable cells. Membrane depolarization initiates asynchronous movements in four non-identical voltage-sensing domains of the Na+ channel. It remains unclear to what extent this structural asymmetry influences pore gating as compared with outwardly rectifying K+ channels, where channel opening results from a final concerted transition of symmetric pore gates. Here we combine single channel recordings, cysteine accessibility and voltage clamp fluorimetry to probe the relationships between voltage sensors and pore conformations in an inactivation deficient Nav1.4 channel. We observe three distinct conductance levels such that DI-III voltage sensor activation is kinetically correlated with formation of a fully open pore, whereas DIV voltage sensor movement underlies formation of a distinct subconducting pore conformation preceding inactivation in wild-type channels. Our experiments reveal that pore gating in sodium channels involves multiple transitions driven by asynchronous movements of voltage sensors. These findings shed new light on the mechanism of coupling between activation and fast inactivation in voltage-gated sodium channels.

Exocytotic fusion pores are composed of both lipids and proteins

During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE proteins catalyze fusion, the structure and composition of fusion pores remain unknown. Here, we exploited the rigid framework and defined size of nanodiscs to interrogate the properties of reconstituted fusion pores, using the neurotransmitter glutamate as a content-mixing marker. Efficient Ca2+-stimulated bilayer fusion, and glutamate release, occurred with approximately two molecules of mouse synaptobrevin 2 reconstituted into ∼6-nm nanodiscs. The transmembrane domains of SNARE proteins assumed distinct roles in lipid mixing versus content release and were exposed to polar solvent during fusion. Additionally, tryptophan substitutions at specific positions in these transmembrane domains decreased glutamate flux. Together, these findings indicate that the fusion pore is a hybrid structure composed of both lipids and proteins.

An Epilepsy-Related Region in the GABA A Receptor Mediates Long-Distance Effects on GABA and Benzodiazepine Binding Sites

The GABAA receptor mutation γ2R43Q causes absence epilepsy in humans. Homology modeling suggests that γ2Arg43, γ2Glu178, and β2Arg117 participate in a salt-bridge network linking the γ2 and β2 subunits. Here we show that several mutations at these locations exert similar long-distance effects on other intersubunit interfaces involved in GABA and benzodiazepine binding. These mutations alter GABA-evoked receptor kinetics by slowing deactivation, enhancing desensitization, or both. Kinetic modeling and nonstationary noise analysis for γ2R43Q reveal that these effects are due to slowed GABA unbinding and slowed recovery from desensitization. Both γ2R43Q and β2R117K also speed diazepam dissociation from the receptor’s benzodiazepine binding interface, as assayed by the rate of decay of diazepam-induced potentiation of GABA-evoked currents. These data demonstrate that γ2Arg43 and β2Arg117 similarly regulate the stability of both the GABA and benzodiazepine binding sites at the distant β/α and α/γ intersubunit interfaces, respectively. A simple explanation for these results is that γ2Arg43 and β2Arg117 participate in interactions between the γ2 and β2 subunits, disruptions of which alter the neighboring intersubunit binding sites in a similar fashion. In addition, γ2Arg43 and γ2Glu178 regulate desensitization, probably mediated within the transmembrane domains near the pore. Therefore, mutations at the γ/β intersubunit interface have specific long-distance effects that are propagated widely throughout the GABAA receptor protein.

Evolutionarily conserved intracellular gate of voltage-dependent sodium channels

Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

Three arginines in the GABAA receptor binding pocket have distinct roles in the formation and stability of agonist- versus antagonist-bound complexes.

Binding of the agonist GABA to the GABAA receptor causes channel gating, whereas competitive antagonists that bind at the same site do not. The details of ligand binding are not well understood, including which residues interact directly with ligands, maintain the structure of the binding pocket, or transduce the action of binding into opening of the ion channel gate. Recent work suggests that the amine group of the GABA molecule may form a cation-π bond with residues in a highly conserved “aromatic box” within the binding pocket. Although interactions with the carboxyl group of GABA remain unknown, three positively charged arginines (α1Arg67, α1Arg132, and β2Arg207) just outside of the aromatic box are likely candidates. To explore their roles in ligand binding, we individually mutated these arginines to alanine and measured the effects on microscopic ligand binding/unbinding rates and channel gating. The mutations α1R67A or β2R207A slowed agonist binding and sped unbinding with little effect on gating, demonstrating that these arginines are critical for both formation and stability of the agonist-bound complex. In addition, α1R67A sped binding of the antagonist 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide (SR-95531), indicating that this arginine poses a barrier to formation of the antagonist-bound complex. In contrast, β2R207A and α1R132A sped antagonist unbinding, indicating that these arginines stabilize the antagonist-bound state. α1R132A also conferred a new long-lived open state, indicating that this arginine influences the channel gate. Thus, each of these arginines plays a unique role in determining interactions with agonists versus antagonists and with the channel gate.

A nonequilibrium binary elements-based kinetic model for benzodiazepine regulation of GABAA receptors

A nonequilibrium kinetic model that explicitly treats the energetics of interactions between structural domains is used to describe positive modulation of the GABAA receptor by benzodiazepines.

Observing Single‐Molecule Dynamics at Millimolar Concentrations

Single-molecule fluorescence microscopy is a powerful tool for revealing chemical dynamics and molecular association mechanisms, but has been limited to low concentrations of fluorescent species and is only suitable for studying high affinity reactions. Here, we combine nanophotonic zero-mode waveguides (ZMWs) with fluorescence resonance energy transfer (FRET) to resolve single-molecule association dynamics at up to millimolar concentrations of fluorescent species. This approach extends the resolution of molecular dynamics to >100-fold higher concentrations, enabling observations at concentrations relevant to biological and chemical processes, and thus making single-molecule techniques applicable to a tremendous range of previously inaccessible molecular targets. We deploy this approach to show that the binding of cGMP to pacemaking ion channels is weakened by a slower internal conformational change.

The intrinsically liganded cyclic nucleotide–binding homology domain promotes KCNH channel activation

Channels in the ether-à-go-go or KCNH family of potassium channels are characterized by a conserved, C-terminal domain with homology to cyclic nucleotide–binding homology domains (CNBhDs). Instead of cyclic nucleotides, two amino acid residues, Y699 and L701, occupy the binding pocket, forming an “intrinsic ligand.” The role of the CNBhD in KCNH channel gating is still unclear, however, and a detailed characterization of the intrinsic ligand is lacking. In this study, we show that mutating both Y699 and L701 to alanine, serine, aspartate, or glycine impairs human EAG1 channel function. These mutants slow channel activation and shift the conductance–voltage (G–V) relation to more depolarized potentials. The mutations affect activation and the G-V relation progressively, indicating that the gating machinery is sensitive to multiple conformations of the CNBhD. Substitution with glycine at both sites (GG), which eliminates the side chains that interact with the binding pocket, also reduces the ability of voltage prepulses to populate more preactivated states along the activation pathway (i.e., the Cole–Moore effect), as if stabilizing the voltage sensor in deep resting states. Notably, deletion of the entire CNBhD (577–708, &Dgr;CNBhD) phenocopies the GG mutant, suggesting that GG is a loss-of-function mutation and the CNBhD requires an intrinsic ligand to exert its functional effects. We developed a kinetic model for both wild-type and &Dgr;CNBhD mutant channels that describes all our observations on activation kinetics, the Cole–Moore shift, and G-V relations. These findings support a model in which the CNBhD both promotes voltage sensor activation and stabilizes the open pore. The intrinsic ligand is critical for these functional effects.