Genetically encoded voltage indicator proteins revealed differential effects of hyperosmotic stress on yeast plasma membrane potential imposed by different stress conditions.

Cells can be affected by several causes of osmotic stress, by which they are forced to adapt. An essential aspect of adaptation is ion regulation, and many insights into such complex processes can be obtained through measurement of the plasma membrane potential (PMP) of cells during stress. We recently established genetically encoded voltage indicator proteins that could be utilized to report the yeast PMP change in real time. In this work, we employed this method to monitor the early change in the PMP of yeast Saccharomyces cerevisiae with intact cell wall, immediately following hyperosmotic up-shock due to various stress agents. The results pointed to differential effects of NaCl, sorbitol and polyethylene glycol (PEG) 6000. Yeast PMP was more responsive toward PEG 6000 than NaCl and sorbitol at comparable osmotic pressure, and PEG 6000 stimulated the largest response magnitude, followed by sorbitol and NaCl, respectively. After prolonged treatment, PEG 6000 also instigated distinct cell morphology from NaCl and sorbitol. Accordingly, this study presents new evidence supporting multiple pathways underlying yeast adaptation to varying hyperosmotic conditions, enabled through the optical physiology approach. Our findings promote better understanding of the yeast cellular response to hyperosmotic stress, with tenable relevance to the physiologically related plant cells.

Real-time detection of changes in yeast plasma membrane potential using genetically encoded voltage indicator proteins.

In yeast, adaptation to varying conditions often requires proper regulation of the plasma membrane potential. To determine yeast membrane potential change, optical methods involving potentiometric dyes have been supplemental to the direct electrode-based method. However, the hydrophobic nature of the dyes and their slow distribution across the membrane still limits their utilization. Genetically encoded voltage indicator (GEVI) proteins employed in neuroscience offer a tantalizing alternative for monitoring yeast membrane potential change. In this work, several widely used GEVI proteins were assessed in Saccharomyces cerevisiae for their expression and function as a voltage reporter. Among them, only ArcLight and Accelerated Sensor of Action Potential (ASAP) proteins could be expressed and transported to the plasma membrane. While the voltage-sensing capability was demonstrated for both ArcLight and ASAP, ArcLight fluorescence was sensitive to the intracellular pH change concurrently with the voltage change. Therefore, we established that ASAP is the more suitable GEVI protein for reporting yeast membrane potential change. This voltage-sensing reporter for yeast based on ASAP offers a new effective strategy for real-time optical detection of yeast membrane potential change, which potentially facilitates many areas of yeast research including optimizing growth conditions for industrial use and investigating yeast ion transport system.

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.

The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors.

Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain.

Heterologous expression and nonsense suppression provide insights into agonist behavior at α6ß2 nicotinic acetylcholine receptors.

The α6-containing subtypes of the nicotinic acetylcholine receptor (nAChR) are localized to presynaptic terminals of the dopaminergic pathways of the central nervous system. Selective ligands for these nAChRs are potentially useful in both Parkinson's disease and addiction. For these and other goals, it is important to distinguish the binding behavior of agonists at the α6-ß2 binding site versus other subtypes. To study this problem, we apply nonsense suppression-based non-canonical amino acid mutagenesis. We report a combination of four mutations in α6ß2 that yield high-level heterologous expression in Xenopus oocytes. By varying mRNA injection ratios, two populations were observed with unique characteristics, likely due to differing stoichiometries. Responses to nine known nAChR agonists were analyzed at the receptor, and their corresponding EC50 values and efficacies are reported. The system is compatible with nonsense suppression, allowing structure-function studies between Trp149 - a conserved residue on loop B found to make a cation-π interaction at several nAChR subtypes - and several agonists. These studies reveal that acetylcholine forms a strong cation-π interaction with the conserved tryptophan, while nicotine and TC299423 do not, suggesting a unique pharmacology for the α6ß2 nAChR.

Subtype-specific mechanisms for functional interaction between α6ß4* nicotinic acetylcholine receptors and P2X receptors.

P2X receptors and nicotinic acetylcholine receptors (nAChRs) display functional and physical interactions in many cell types and heterologous expression systems, but interactions between α6ß4-containing (α6ß4*) nAChRs and P2X2 receptors and/or P2X3 receptors have not been fully characterized. We measured several types of crosstalk in oocytes coexpressing α6ß4 nAChRs and P2X2, P2X3, or P2X2/3 receptors. A novel form of crosstalk occurs between α6ß4 nAChRs and P2X2 receptors. P2X2 receptors were forced into a prolonged desensitized state upon activation by ATP through a mechanism that does not depend on the intracellular C terminus of the P2X2 receptors. Coexpression of α6ß4 nAChRs with P2X3 receptors shifts the ATP dose-response relation to the right, even in the absence of acetylcholine (ACh). Moreover, currents become nonadditive when ACh and ATP are coapplied, as previously reported for other Cys-loop receptors interacting with P2X receptors, and this crosstalk is dependent on the presence of the P2X3 C-terminal domain. P2X2 receptors also functionally interact with α6ß4ß3 but through a different mechanism from α6ß4. The interaction with P2X3 receptors is less pronounced for the α6ß4ß3 nAChR than the α6ß4 nAChR. We also measured a functional interaction between the α6ß4 nAChRs and the heteromeric P2X2/3 receptor. Experiments with the nAChR channel blocker mecamylamine on P2X2-α6ß4 oocytes point to the loss of P2X2 channel activity during the crosstalk, whereas the ion channel pores of the P2X receptors were fully functional and unaltered by the receptor interaction for P2X2-α6ß4ß3, P2X2/3-α6ß4, and P2X2/3-α6ß4ß3. These results may be relevant to dorsal root ganglion cells and to other neurons that coexpress these receptor subunits.

Key binding interactions for memantine in the NMDA receptor.

Memantine (Namenda) is prescribed as a treatment for moderate to severe Alzheimer's Disease. Memantine functions by blocking the NMDA receptor, but the key binding interactions between drug and receptor are not fully elucidated. To determine key binding interactions of memantine, we made side-by-side comparisons of IC(50) for memantine and amantadine, a structurally related drug, in the GluN1/GluN2B NMDA receptor. We identified hydrophobic binding pockets for the two methyl groups on memantine formed by the residues A645 and A644 on the third transmembrane helices of GluN1 and GluN2B, respectively. Moreover, we found that while adding two methyl groups to amantadine to produce memantine greatly improves affinity, adding a third methyl group to produce the symmetrical trimethylamantadine diminished affinity. Our results provide a better understanding of chemical-scale interactions between memantine and the NMDA channel, which will potentially benefit the development of new drugs for neurodegenerative diseases involving NMDA receptors.

A gating charge transfer center in voltage sensors.

Voltage sensors regulate the conformations of voltage-dependent ion channels and enzymes. Their nearly switchlike response as a function of membrane voltage comes from the movement of positively charged amino acids, arginine or lysine, across the membrane field. We used mutations with natural and unnatural amino acids, electrophysiological recordings, and x-ray crystallography to identify a charge transfer center in voltage sensors that facilitates this movement. This center consists of a rigid cyclic "cap" and two negatively charged amino acids to interact with a positive charge. Specific mutations induce a preference for lysine relative to arginine. By placing lysine at specific locations, the voltage sensor can be stabilized in different conformations, which enables a dissection of voltage sensor movements and their relation to ion channel opening.

Chemical scale studies of the Phe-Pro conserved motif in the cys loop of Cys loop receptors.

The functions of two conserved residues, Phe(135) and Pro(136), located at the apex of the Cys loop of the nicotinic acetylcholine receptor are investigated. Both residues were substituted with natural and unnatural amino acids, focusing on the role of aromaticity at Phe(135), backbone conformation at Pro(136), side chain polarity and volume, and the specific interaction between the aromatic side chain and the proline. NMR spectroscopy studies of model peptides containing proline and unnatural proline analogues following a Phe show a consistent increase in the population of the cis conformer relative to peptides lacking the Phe. In the receptor, a strong interaction between the Phe and Pro residues is evident, as is a strong preference for aromaticity and hydrophobicity at the Phe site. A similar influence of hydrophobicity is observed at the proline site. In addition, across a simple homologous series of proline analogues, the results reveal a correlation between receptor function and cis bias at the proline backbone. This could suggest a significant role for the cis proline conformer at this site in receptor function.

Reagentless functionalization of gold nanoparticles via a 3 + 2 Huisgen cycloaddition.

A mild method for functionalization of gold nanoparticles is reported. The reactions of azide functionalized nanoparticles with propynoic acid derivatives provide triazole functionalized nanoparticles under very mild reaction conditions. Characterization of the nanoparticle-bound triazoles using (1)H and (13)C NMR spectroscopy indicates that both the 1,4 and 1,5 triazole regioisomers are formed on the nanoparticle surface.