Identification of Druggable Binding Sites and Small Molecules as Modulators of TMC1.

Our ability to hear and maintain balance relies on the proper functioning of inner ear sensory hair cells, which translate mechanical stimuli into electrical signals via mechano-electrical transducer (MET) channels, composed of TMC1/2 proteins. However, the therapeutic use of ototoxic drugs, such as aminoglycosides and cisplatin, which can enter hair cells through MET channels, often leads to profound auditory and vestibular dysfunction. Despite extensive research on otoprotective compounds targeting MET channels, our understanding of how small molecule modulators interact with these channels remains limited, hampering the discovery of novel compounds. Here, we propose a structure-based screening approach, integrating 3D-pharmacophore modeling, molecular simulations, and experimental validation. Our pipeline successfully identified several novel compounds and FDA-approved drugs that reduced dye uptake in cultured cochlear explants, indicating MET modulation activity. Molecular docking and free-energy estimations for binding allowed us to identify three potential drug binding sites within the channel pore, phospholipids, and key amino acids involved in modulator interactions. We also identified shared ligand-binding features between TMC and structurally related TMEM16 protein families, providing novel insights into their distinct inhibition, while potentially guiding the rational design of MET-channel-specific modulators. Our pipeline offers a broad application to discover small molecule modulators for a wide spectrum of mechanosensitive ion channels.

Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices.

The transmembrane (TM) channel-like 1 (TMC1) and TMC2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of 12 mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining TM4 and TM6 helices modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.

Function and Dysfunction of TMC Channels in Inner Ear Hair Cells.

The TMC1 channel was identified as a protein essential for hearing in mouse and human, and recognized as one of a family of eight such proteins in mammals. The TMC family is part of a superfamily of seven branches, which includes the TMEM16s. Vertebrate hair cells express both TMC1 and TMC2. They are located at the tips of stereocilia and are required for hair cell mechanotransduction. TMC1 assembles as a dimer and its similarity to the TMEM16s has enabled a predicted tertiary structure with an ion conduction pore in each subunit of the dimer. Cysteine mutagenesis of the pore supports the role of TMC1 and TMC2 as the core channel proteins of a larger mechanotransduction complex that includes PCDH15 and LHFPL5, and perhaps TMIE, CIB2 and others.

TMC1 Forms the Pore of Mechanosensory Transduction Channels in Vertebrate Inner Ear Hair Cells.

The proteins that form the permeation pathway of mechanosensory transduction channels in inner-ear hair cells have not been definitively identified. Genetic, anatomical, and physiological evidence support a role for transmembrane channel-like protein (TMC) 1 in hair cell sensory transduction, yet the molecular function of TMC proteins remains unclear. Here, we provide biochemical evidence suggesting TMC1 assembles as a dimer, along with structural and sequence analyses suggesting similarity to dimeric TMEM16 channels. To identify the pore region of TMC1, we used cysteine mutagenesis and expressed mutant TMC1 in hair cells of Tmc1/2-null mice. Cysteine-modification reagents rapidly and irreversibly altered permeation properties of mechanosensory transduction. We propose that TMC1 is structurally similar to TMEM16 channels and includes ten transmembrane domains with four domains, S4-S7, that line the channel pore. The data provide compelling evidence that TMC1 is a pore-forming component of sensory transduction channels in auditory and vestibular hair cells.

A facile approach for the in vitro assembly of multimeric membrane transport proteins.

Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter.

The Outer Pore and Selectivity Filter of TRPA1.

TRPA1 (transient-receptor-potential-related ion channel with ankyrin domains) is a direct receptor or indirect effector for a wide variety of nociceptive signals, and thus is a compelling target for development of analgesic pharmaceuticals such as channel blockers. Recently, the structure of TRPA1 was reported, providing insights into channel assembly and pore architecture. Here we report whole-cell and single-channel current recordings of wild-type human TRPA1 as well as TRPA1 bearing point mutations of key charged residues in the outer pore. These measurements demonstrate that the glutamate at position 920 plays an important role in collecting cations into the mouth of the pore, by changing the effective surface potential by ~16 mV, while acidic residues further out have little effect on permeation. Electrophysiology experiments also confirm that the aspartate residue at position 915 represents a constriction site of the TRPA1 pore and is critical in controlling ion permeation.

Plug-N-Play: Mechanotransduction Goes Modular.

Mechanosensitive ion channels initiate sensory signals by converting mechanical information into electrochemical signals. In this issue of Neuron (Zhao et al., 2016), a data-rich structure-function study on mammalian mechanosensitive Piezo channels reveals a modular protein architecture that includes a central pore module surrounded by a force-sensing module.

Transport domain unlocking sets the uptake rate of an aspartate transporter.

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.

Structural brain imaging in children and adolescents following prenatal cocaine exposure: preliminary longitudinal findings.

The brain morphometry of 21 children, who were followed from birth and underwent structural brain magnetic resonance imaging at 8-10 years, was studied. This cohort included 11 children with prenatal cocaine exposure (CE) and 10 noncocaine-exposed children (NCE). We compared the CE versus NCE groups using FreeSurfer to automatically segment and quantify the volume of individual brain structures. In addition, we created a pediatric atlas specifically for this population and demonstrate the enhanced accuracy of this approach. We found an overall trend towards smaller brain volumes among CE children. The volume differences were significant for cortical gray matter, the thalamus and the putamen. Here, reductions in thalamic and putaminal volumes showed a robust inverse correlation with exposure levels, thus highlighting effects on dopamine-rich brain regions that form key components of brain circuitry known to play important roles in behavior and attention. Interestingly, head circumferences (HCs) at birth as well as at the time of imaging showed a tendency for smaller size among CE children. HCs at the time of imaging correlated well with the cortical volumes for all subjects. In contrast, HCs at birth were predictive of the cortical volume only for the CE group. A subgroup of these subjects (6 CE, 4 NCE) was also scanned at 13-15 years of age. In subjects who were scanned twice, we found that the trend for smaller structures continued into teenage years. We found that the differences in structural volumes between the CE and NCE groups are largely diminished when the HCs are controlled for or matched by study design. Participants in this study were drawn from a unique longitudinal cohort and, while the small sample size precludes strong conclusions regarding the longitudinal findings reported, the results point to reductions in HCs and in specific brain structures that persist through teenage years in children who were exposed to cocaine in utero.

Transport dynamics in a glutamate transporter homologue.

Glutamate transporters are integral membrane proteins that catalyse neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons. Their mechanism of action involves transitions between extracellular (outward)-facing and intracellular (inward)-facing conformations, whereby substrate binding sites become accessible to either side of the membrane. This process has been proposed to entail transmembrane movements of three discrete transport domains within a trimeric scaffold. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods. We propose that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent transmembrane movements and may be rate determining in the transport cycle.