Divergent mechanisms of steroid inhibition in the human ρ1 GABA receptor.

ρ-type γ-aminobutyric acid-A (GABA) receptors are widely distributed in the retina and brain, and are potential drug targets for the treatment of visual, sleep and cognitive disorders. Endogenous neuroactive steroids including β-estradiol and pregnenolone sulfate negatively modulate the function of ρ1 GABA receptors, but their inhibitory mechanisms are not clear. By combining five cryo-EM structures with electrophysiology and molecular dynamics simulations, we characterize binding sites and negative modulation mechanisms of β-estradiol and pregnenolone sulfate at the human ρ1 GABA receptor.

Structural basis for hyperpolarization-dependent opening of human HCN1 channel.

Hyperpolarization and cyclic nucleotide (HCN) activated ion channels are critical for the automaticity of action potentials in pacemaking and rhythmic electrical circuits in the human body. Unlike most voltage-gated ion channels, the HCN and related plant ion channels activate upon membrane hyperpolarization. Although functional studies have identified residues in the interface between the voltage-sensing and pore domain as crucial for inverted electromechanical coupling, the structural mechanisms for this unusual voltage-dependence remain unclear.

Mapping the contribution of the C-linker domain to gating polarity in CNBD channels.

Ion channels of the cyclic nucleotide-binding domain (CNBD) family play a crucial role in the regulation of key biological processes, such as photoreception and pacemaking activity in the heart. These channels exhibit high sequence and structural similarity but differ greatly in their functional responses to membrane potential. The CNBD family includes hyperpolarization-activated ion channels and depolarization-activated ether-à-go-go channels.

Structure and dynamics of differential ligand binding in the human ρ-type GABA receptor.

The neurotransmitter γ-aminobutyric acid (GABA) drives critical inhibitory processes in and beyond the nervous system, partly via ionotropic type-A receptors (GABARs). Pharmacological properties of ρ-type GABARs are particularly distinctive, yet the structural basis for their specialization remains unclear. Here, we present cryo-EM structures of a lipid-embedded human ρ1 GABAR, including a partial intracellular domain, under apo, inhibited, and desensitized conditions.

Structural Basis for Hyperpolarization-dependent Opening of the Human HCN1 Channel.

Hyperpolarization and cyclic-nucleotide (HCN) activated ion channels play a critical role in generating self-propagating action potentials in pacemaking and rhythmic electrical circuits in the human body. Unlike most voltage-gated ion channels, the HCN channels activate upon membrane hyperpolarization, but the structural mechanisms underlying this gating behavior remain unclear. Here, we present cryo-electron microscopy structures of human HCN1 in Closed, Intermediate, and Open states.

Interplay between VSD, pore, and membrane lipids in electromechanical coupling in HCN channels.

Hyperpolarized-activated and cyclic nucleotide-gated (HCN) channels are the only members of the voltage-gated ion channel superfamily in mammals that open upon hyperpolarization, conferring them pacemaker properties that are instrumental for rhythmic firing of cardiac and neuronal cells. Activation of their voltage-sensor domains (VSD) upon hyperpolarization occurs through a downward movement of the S4 helix bearing the gating charges, which triggers a break in the alpha-helical hydrogen bonding pattern at the level of a conserved Serine residue. Previous structural and molecular simulation studies had however failed to capture pore opening that should be triggered by VSD activation, presumably because of a low VSD/pore electromechanical coupling efficiency and the limited timescales accessible to such techniques.

About hysteresis in Shaker: Response to note by Villalba-Galea.

In this issue, Villalba-Galea (2023. J. Gen.

Charge-voltage curves of Shaker potassium channel are not hysteretic at steady state.

Charge-voltage curves of many voltage-gated ion channels exhibit hysteresis but such curves are also a direct measure of free energy of channel gating and, hence, should be path-independent. Here, we identify conditions to measure steady-state charge-voltage curves and show that these are curves are not hysteretic. Charged residues in transmembrane segments of voltage-gated ion channels (VGICs) sense and respond to changes in the electric field.

Cryo-EM structure of the human Kv3.1 channel reveals gating control by the cytoplasmic T1 domain.

Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-electron microscopy structures of the human Kv3.

Mapping Electromechanical Coupling Pathways in Voltage-Gated Ion Channels: Challenges and the Way Forward.

Inter- and intra-molecular allosteric interactions underpin regulation of activity in a variety of biological macromolecules. In the voltage-gated ion channel superfamily, the conformational state of the voltage-sensing domain regulates the activity of the pore domain via such long-range allosteric interactions. Although the overall structure of these channels is conserved, allosteric interactions between voltage-sensor and pore varies quite dramatically between the members of this superfamily.

Helix breaking transition in the S4 of HCN channel is critical for hyperpolarization-dependent gating.

In contrast to most voltage-gated ion channels, hyperpolarization- and cAMP gated (HCN) ion channels open on hyperpolarization. Structure-function studies show that the voltage-sensor of HCN channels are unique but the mechanisms that determine gating polarity remain poorly understood. All-atom molecular dynamics simulations (~20 μs) of HCN1 channel under hyperpolarization reveals an initial downward movement of the S4 voltage-sensor but following the transfer of last gating charge, the S4 breaks into two sub-helices with the lower sub-helix becoming parallel to the membrane.

The contribution of voltage clamp fluorometry to the understanding of channel and transporter mechanisms.

Key advances in single particle cryo-EM methods in the past decade have ushered in a resolution revolution in modern biology. The structures of many ion channels and transporters that were previously recalcitrant to crystallography have now been solved. Yet, despite having atomistic models of many complexes, some in multiple conformations, it has been challenging to glean mechanistic insight from these structures.

Bipolar switching by HCN voltage sensor underlies hyperpolarization activation.

Despite sharing a common architecture with archetypal voltage-gated ion channels (VGICs), hyperpolarization- and cAMP-activated ion (HCN) channels open upon hyperpolarization rather than depolarization. The basic motions of the voltage sensor and pore gates are conserved, implying that these domains are inversely coupled in HCN channels. Using structure-guided protein engineering, we systematically assembled an array of mosaic channels that display the full complement of voltage-activation phenotypes observed in the VGIC superfamily.

Light-driven quinone reduction in heliobacterial membranes.

Photosynthetic reaction centers (RCs) evolved > 3 billion years ago and have diverged into Type II RCs reducing quinones and Type I RCs reducing soluble acceptors via iron-sulfur clusters. Photosystem I (PSI), the exemplar Type I RC, uses modified menaquinones as intermediate electron transfer cofactors, but it has been controversial if the Type I RC of heliobacteria (HbRC) uses its two bound menaquinones in the same way. The sequence of the quinone-binding site in PSI is not conserved in the HbRC, and the recently solved crystal structure of the HbRC does not reveal a quinone in the analogous site.

Structure and dynamics underlying elementary ligand binding events in human pacemaking channels.

Although molecular recognition is crucial for cellular signaling, mechanistic studies have relied primarily on ensemble measures that average over and thereby obscure underlying steps. Single-molecule observations that resolve these steps are lacking due to diffraction-limited resolution of single fluorophores at relevant concentrations. Here, we combined zero-mode waveguides with fluorescence resonance energy transfer (FRET) to directly observe binding at individual cyclic nucleotide-binding domains (CNBDs) from human pacemaker ion channels critical for heart and brain function.

Thermodynamics of the Electron Acceptors in Heliobacterium modesticaldum: An Exemplar of an Early Homodimeric Type I Photosynthetic Reaction Center.

The homodimeric type I reaction center in heliobacteria is arguably the simplest known pigment-protein complex capable of conducting (bacterio)chlorophyll-based conversion of light into chemical energy. Despite its structural simplicity, the thermodynamics of the electron transfer cofactors on the acceptor side have not been fully investigated. In this work, we measured the midpoint potential of the terminal [4Fe-4S](2+/1+) cluster (FX) in reaction centers from Heliobacterium modesticaldum.

Expression and characterization of cytochrome c553 from Heliobacterium modesticaldum.

Cytochrome c553 of Heliobacterium modesticaldum is the donor to P800 (+), the primary electron donor of the heliobacterial reaction center (HbRC). It is a membrane-anchored 14-kDa cytochrome that accomplishes electron transfer from the cytochrome bc complex to the HbRC. The petJ gene encoding cyt c 553 was cloned and expressed in Escherichia coli with a hexahistidine tag replacing the lipid attachment site to create a soluble donor that could be made in a preparative scale.

Purification of the photosynthetic reaction center from Heliobacterium modesticaldum.

We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC-MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al.

Double reduction of plastoquinone to plastoquinol in photosystem 1.

In Photosystem 1 (PS1), phylloquinone (PhQ) acts as a secondary electron acceptor from chlorophyll ec(3) and also as an electron donor to the iron-sulfur cluster F(X). PS1 possesses two virtually equivalent branches of electron transfer (ET) cofactors from P(700) to F(X), and the lifetime of the semiquinone intermediate displays biphasic kinetics, reflecting ET along the two different branches. PhQ in PS1 serves only as an intermediate in ET and is not normally fully reduced to the quinol form.

Influence of intermittent hypoxia on myocardial and hepatic P-glycoprotein expression in a rodent model.

Study Objective: Patients with obstructive sleep apnea who receive drug therapy for cardiovascular disease may experience resistant hypertension, arrhythmias, or more severe heart failure, and many of the drugs used to treat these conditions are substrates for P-glycoprotein (P-gp) transporters. Therefore, we sought to determine if intermittent hypoxia, which mimics obstructive sleep apnea, would upregulate myocardial and hepatic P-gp expression and Abcb1a and Abcb1b messenger RNA (mRNA) expression (genes that encode for P-gp) in an animal model.

Design: Prospective, randomized, blinded, parallel-design animal study.