Kinetic and thermodynamic modeling of a voltage-gated sodium channel.

Like all biological and chemical reactions, ion channel kinetics are highly sensitive to changes in temperature. Therefore, it is prudent to investigate channel dynamics at physiological temperatures. However, most ion channel investigations are performed at room temperature due to practical considerations, such as recording stability and technical limitations.

Optical probing of local membrane potential with fluorescent polystyrene beads.

The study of electrical activity in single cells and local circuits of excitable cells, such as neurons, requires an easy-to-use, high-throughput methodology that allows for the measurement of membrane potential. Investigating the electrical properties in specific subcompartments of neurons, or in a specific type of neurons, introduces additional complexity. An optical voltage-imaging technique that allows high spatial and temporal resolution could be an ideal solution.

Point-localized, site-specific membrane potential optical recording by single fluorescent nanodiscs.

Nanodisc technology was implemented as a platform for voltage nanosensors. A fluorescence (Förster) resonance energy transfer (FRET)- based voltage-sensing scheme employing fluorescent nanodiscs and the hydrophobic ion dipicrylamine was developed and utilized to optically record membrane potentials on the single-nanodisc level. Ensemble and single-nanosensor recordings were demonstrated for HEK293 cells and primary cortical neuron cells.

Interfacing the Cell with "Biomimetic Membrane Proteins".

Integral membrane proteins mediate a myriad of cellular processes and are the target of many therapeutic drugs. Enhancement and extension of the functional scope of membrane proteins can be realized by membrane incorporation of engineered nanoparticles designed for specific diagnostic and therapeutic applications. In contrast to hydrophobic insertion of small amphiphilic molecules, delivery and membrane incorporation of particles on the nanometric scale poses a crucial barrier for technological development.

Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin.

The broad-spectrum anthelmintic drug ivermectin (IVM) activates and stabilizes an open-channel conformation of invertebrate chloride-selective glutamate receptors (GluClRs), thereby causing a continuous inflow of chloride ions and sustained membrane hyperpolarization. These effects suppress nervous impulses and vital physiological processes in parasitic nematodes. The GluClRs are pentamers.

Trapping of ivermectin by a pentameric ligand-gated ion channel upon open-to-closed isomerization.

Ivermectin (IVM) is a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. By activating invertebrate pentameric glutamate-gated chloride channels (GluCl receptors; GluClRs), IVM induces sustained chloride influx and long-lasting membrane hyperpolarization that inhibit neural excitation in nematodes. Although IVM activates the C.

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel.

The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds.

Molecular dissection of Cl--selective Cys-loop receptor points to components that are dispensable or essential for channel activity.

Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that bind neurotransmitters to open an intrinsic transmembrane ion channel pore. The recent crystal structure of a prokaryotic pLGIC from the cyanobacterium Gloeobacter violaceus (GLIC) revealed that it naturally lacks an N-terminal extracellular α helix and an intracellular domain that are typical of eukaryotic pLGICs. GLIC does not respond to neurotransmitters acting at eukaryotic pLGICs but is activated by protons.

Probing pore constriction in a ligand-gated ion channel by trapping a metal ion in the pore upon agonist dissociation.

Eukaryotic pentameric ligand-gated ion channels (pLGICs) are receptors activated by neurotransmitters to rapidly transport ions across cell membranes, down their electrochemical gradients. Recent crystal structures of two prokaryotic pLGICs were interpreted to imply that the extracellular side of the transmembrane pore constricts to close the channel (Hilf, R. J.

Selective interaction of syntaxin 1A with KCNQ2: possible implications for specific modulation of presynaptic activity.

KCNQ2/KCNQ3 channels are the molecular correlates of the neuronal M-channels, which play a major role in the control of neuronal excitability. Notably, they differ from homomeric KCNQ2 channels in their distribution pattern within neurons, with unique expression of KCNQ2 in axons and nerve terminals. Here, combined reciprocal coimmunoprecipitation and two-electrode voltage clamp analyses in Xenopus oocytes revealed a strong association of syntaxin 1A, a major component of the exocytotic SNARE complex, with KCNQ2 homomeric channels resulting in a approximately 2-fold reduction in macroscopic conductance and approximately 2-fold slower activation kinetics.

A tale of switched functions: from cyclooxygenase inhibition to M-channel modulation in new diphenylamine derivatives.

Cyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.

Pre- and postsynaptic activation of M-channels by a novel opener dampens neuronal firing and transmitter release.

The M-type K(+) current (M-current), encoded by Kv7.2/3 (KCNQ2/3) K(+) channels, plays a critical role in regulating neuronal excitability because it counteracts subthreshold depolarizations. Here we have characterized the functions of pre- and postsynaptic M-channels using a novel Kv7.