A potassium channel agonist protects hearing function and promotes outer hair cell survival in a mouse model for age-related hearing loss.

Age-related hearing loss (ARHL) is the most common sensory impairment mainly caused by degeneration of sensory hair cells in the cochlea with no causal medical treatment available. Auditory function and sensory hair cell survival critically depend on the Kv7.4 (KCNQ4) channel, a voltage-gated potassium channel expressed in outer hair cells (OHCs), with its impaired function or reduced activity previously associated with ARHL.

Three-Dimensional Control of Ion Channel Function through Optogenetics and Co-Culture.

The lack of miniaturized and cost-effective methods to control cellular excitability with dosable and temporally precise electrical perturbations represents a long-lasting and unsolved bottleneck for ion channel drug discovery pipelines. Here we developed a high-throughput-compatible fluorescent-based cellular assay that combines optogenetics and co-culture approaches to obtain spatial, temporal, and quantitative control of ion channel activity. The modularity and increased flexibility of control of this light-tandem assay, combined with contained costs and compatibility with conventional drug-screening platforms, make this system suitable for temporally precise screening of ion channel function in controlled conformations and can also be used to recapitulate other complexly regulated biological processes.

Mitochondrial Ca(2+) mobilization is a key element in olfactory signaling.

In olfactory sensory neurons (OSNs), cytosolic Ca(2+) controls the gain and sensitivity of olfactory signaling. Important components of the molecular machinery that orchestrates OSN Ca(2+) dynamics have been described, but key details are still missing. Here, we demonstrate a critical physiological role of mitochondrial Ca(2+) mobilization in mouse OSNs.

Purinergic signalling mobilizes mitochondrial Ca²⁺ in mouse Sertoli cells.

Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca(2+) signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria.

From c-Photina mouse embryonic stem cells to high-throughput screening of differentiated neural cells via an intermediate step enriched in neural precursor cells.

The use of engineered mouse embryonic stem (mES) cells in high-throughput screening (HTS) can offer new opportunities for studying complex targets in their native environment, increasing the probability of discovering more meaningful hits. The authors have generated and developed a mouse embryonic stem cell line called c-Photina mES stably expressing a Ca(2+)-activated photoprotein as a reporter gene. This reporter cell line retains the ability to differentiate into any cell lineage and can be used for miniaturized screening processes in 384-well microplates.

A photoprotein in mouse embryonic stem cells measures Ca2+ mobilization in cells and in animals.

Exogenous expression of pharmacological targets in transformed cell lines has been the traditional platform for high throughput screening of small molecules. However, exogenous expression in these cells is limited by aberrant dosage, or its toxicity, the potential lack of interaction partners, and alterations to physiology due to transformation itself. Instead, primary cells or cells differentiated from precursors are more physiological, but less amenable to exogenous expression of reporter systems.

Engineering of a monomeric fluorescent protein AsGFP499 and its applications in a dual translocation and transcription assay.

The tetrameric green fluorescent protein AsGFP(499) from the sea anemone Anemonia sulcata was converted into a dimeric and monomeric protein by site-directed mutagenesis. The protein was engineered without prior knowledge of its crystal structure based on a sequence alignment of multiple proteins belonging to the GFP-family. Crucial residues for oligomerisation of AsGFP(499) were predicted and selected for mutation.

Development of a Ca(2+)-activated photoprotein, Photina, and its application to high-throughput screening.

The present work describes the engineering and characterization of a new Ca(2+)-activated photoprotein (Photina) and its use in mammalian cell lines for implementation of flash luminescence cell-based assays for high-throughput screening (HTS). When used to measure the activation of 2 G protein-coupled receptors (GPCRs), targeting Photina to the mitochondria increased the signal strength as compared to the normal cytoplasmic expression of Photina. The mitochondrial-targeted Photina also produced a higher signal-to-noise ratio than conventional calcium dyes and a consistently stronger signal than aequorin when tested under equivalent conditions.

An innovative cell-based assay for the detection of modulators of soluble guanylate cyclase.

Guanylate cyclase (GC) catalyzes the biosynthesis of cyclic guanosine 3',5'- monophosphate (cGMP) from GTP. GC exists in two isoenzyme forms: soluble and membrane-bound. The soluble GC (sGC) is a heterodimer composed of an alpha and a beta subunit, and it contains heme as a prosthetic group.