Optical recordings of organellar membrane potentials and the components of membrane conductance in lysosomes.

The eukaryotic cell is highly compartmentalized with organelles. Owing to their function in transporting metabolites, metabolic intermediates and byproducts of metabolic activity, organelles are important players in the orchestration of cellular function. Recent advances in optical methods for interrogating the different aspects of organellar activity promise to revolutionize our ability to dissect cellular processes with unprecedented detail.

Cold blooded vertebrates help unveil a heat-dependent trigger.

In a recent study, Hori and colleagues demonstrated that two specific residues located in the first ankyrin repeat of TRPV1 channels modulate the threshold for temperature activation. This study highlights the importance of considering natural diversity and comparative biology when approaching biophysical questions.

Intracellular Helix-Loop-Helix Domain Modulates Inactivation Kinetics of Mammalian TRPV5 and TRPV6 Channels.

TRPV5 and TRPV6 are calcium-selective ion channels expressed at the apical membrane of epithelial cells. Important for systemic calcium (Ca) homeostasis, these channels are considered gatekeepers of this cation transcellular transport. Intracellular Ca exerts a negative control over the activity of these channels by promoting inactivation.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies.

A serotonergic axon-cilium synapse drives nuclear signaling to alter chromatin accessibility.

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons.

Sequence and structural conservation reveal fingerprint residues in TRP channels.

Transient receptor potential (TRP) proteins are a large family of cation-selective channels, surpassed in variety only by voltage-gated potassium channels. Detailed molecular mechanisms governing how membrane voltage, ligand binding, or temperature can induce conformational changes promoting the open state in TRP channels are still a matter of debate. Aiming to unveil distinctive structural features common to the transmembrane domains within the TRP family, we performed phylogenetic reconstruction, sequence statistics, and structural analysis over a large set of TRP channel genes.

Studying ion channel conformation dynamics by encoding coumarin as unnatural amino acid.

Monitoring the conformational changes of proteins is critical to understand their function. Ion channels are membrane-bound minute machines controlling the passage of ions across biological membranes. The precise labeling of ion channels with fluorescent probes allows studying their dynamics and facilitates their characterization by high-resolution optical techniques.

Imaging the electrical activity of organelles in living cells.

Eukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoS, an optical method to monitor changes in the membrane potential of subcellular membranes.

A rationally designed orthogonal synthetase for genetically encoded fluorescent amino acids.

The incorporation of non-canonical amino acids into proteins has emerged as a promising strategy to manipulate and study protein structure-function relationships with superior precision and . To date, fluorescent non-canonical amino acids (f-ncAA) have been successfully incorporated in proteins expressed in bacterial systems, oocytes, and HEK-293T cells. Here, we describe the rational generation of a novel orthogonal aminoacyl-tRNA synthetase based on the tyrosine synthetase that is capable of encoding the f-ncAA tyr-coumarin in HEK-293T cells.

Gating and calcium-sensing mechanisms of TRPA1 channels revealed.

Novel structures of the human TRPA1 channel were determined in the presence of the agonist iodoacetamide and the antagonist A-967079, to reveal the open and closed states of the channel, respectively. The structures further revealed the location of Ca modulatory site that is likely conserved among several TRP subgroups.

Evolutionary analyses reveal independent origins of gene repertoires and structural motifs associated to fast inactivation in calcium-selective TRPV channels.

Essential for calcium homeostasis, TRPV5 and TRPV6 are calcium-selective channels belonging to the transient receptor potential (TRP) gene family. In this study, we investigated the evolutionary history of these channels to add an evolutionary context to the already available physiological information. Phylogenetic analyses revealed that paralogs found in mammals, sauropsids, amphibians, and chondrichthyes, are the product of independent duplication events in the ancestor of each group.

Conformational dynamics in TRPV1 channels reported by an encoded coumarin amino acid.

TRPV1 channels support the detection of noxious and nociceptive input. Currently available functional and structural data suggest that TRPV1 channels have two gates within their permeation pathway: one formed by a 'bundle-crossing' at the intracellular entrance and a second constriction at the selectivity filter. To describe conformational changes associated with channel gating, the fluorescent non-canonical amino acid coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter.