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A number of techniques developed to investigate protein structure and function depend on chemically modifying and/or labeling of proteins. However, in the case of homooligomeric proteins, the presence of multiple identical subunits obstructs the introduction of residue-specific labels to only one or several subunits, selectively. Here, in order to study the initial conformational changes of a homopentameric mechanosensitive ion channel during its gating, we developed a method for labeling a defined number of subunits of the channel with two different cysteine-specific compounds simultaneously. The first one is a light-sensitive channel activator that determines the degree of openness of the ion channel upon irradiation. The second one is a spin label, containing an unpaired electron, which allows following the resulting structural changes upon channel gating by electron paramagnetic resonance spectroscopy. With this method, we could open MscL into different sub-open states. As the number of light switches per channel increased, the intersubunit spin–spin interactions became less, indicating changes in intersubunit proximities and opening of the channel. The ability of controlled activation of MscL into different open states with a noninvasive trigger and following the resulting conformational changes by spectroscopy will pave the way for detailed spectroscopic studies in the area of mechanosensation. Electronic supplementary material The online version of this article (doi:10.1007/s00249-015-1063-4) contains supplementary material, which is available to authorized users.

We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherichia coli and have discovered pressure-activated ion channels. The channels have the following properties: activation by slight positive or negative pressure; voltage dependence; large conductance; selectivity for anions over cations; dependence of activity on the species of permeant ions. We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.

Mechanosensitive ion channels use mechanical energy to gate the dissipation of electrochemical gradients across cell membranes. This function is fundamental to physiological processes such as hearing and touch. In electrophysiological studies of ion channels in the plasma membrane of the yeast Saccharomyces cerevisiae, channels were observed that were activated by, and adapted to, stretching of the membrane. Adaptation of channel activity to mechanical stimuli was voltage-dependent. Because these mechanosensitive channels pass both cations and anions, they may play a role in turgor regulation in this walled organism.

Mechanical loading and inflammation interact to cause degenerative disc disease and low back pain (LBP). However, the underlying mechanosensing and mechanotransductive pathways are poorly understood. This results in untargeted pharmacological treatments that do not take the mechanical aspect of LBP into account. We investigated the role of the mechanosensitive ion channel TRPV4 in stretch-induced inflammation in human annulus fibrosus (AF) cells. The cells were cyclically stretched to 20% hyperphysiological strain. TRPV4 was either inhibited with the selective TRPV4 antagonist GSK2193874 or knocked out (KO) via CRISPR-Cas9 gene editing. The gene expression, inflammatory mediator release and MAPK pathway activation were analyzed. Hyperphysiological cyclic stretching significantly increased the IL6, IL8, and COX2 mRNA, PGE2 release, and activated p38 MAPK. The TRPV4 pharmacological inhibition significantly attenuated these effects. TRPV4 KO further prevented the stretch-induced upregulation of IL8 mRNA and reduced IL6 and IL8 release, thus supporting the inhibition data. We provide novel evidence that TRPV4 transduces hyperphysiological mechanical signals into inflammatory responses in human AF cells, possibly via p38. Additionally, we show for the first time the successful gene editing of human AF cells via CRISPR-Cas9. The pharmacological inhibition or CRISPR-based targeting of TRPV4 may constitute a potential therapeutic strategy to tackle discogenic LBP in patients with AF injury.

A defining pathophysiological hallmark of Alzheimer's disease (AD) is the amyloid plaque; an extracellular deposit of aggregated fibrillar Aβ1-42 peptides. Amyloid plaques are hard, brittle structures scattered throughout the hippocampus and cerebral cortex and are thought to cause hyperphosphorylation of tau, neurofibrillary tangles, and progressive neurodegeneration. Reactive astrocytes and microglia envelop the exterior of amyloid plaques and infiltrate their inner core. Glia are highly mechanosensitive cells and can almost certainly sense the mismatch between the normally soft mechanical environment of the brain and very stiff amyloid plaques via mechanosensing ion channels. Piezo1, a non-selective cation channel, can translate extracellular mechanical forces to intracellular molecular signaling cascades through a process known as mechanotransduction. Here, we utilized an aging transgenic rat model of AD (TgF344-AD) to study expression of mechanosensing Piezo1 ion channels in amyloid plaque-reactive astrocytes. We found that Piezo1 is upregulated with age in the hippocampus and cortex of 18-month old wild-type rats. However, more striking increases in Piezo1 were measured in the hippocampus of TgF344-AD rats compared to age-matched wild-type controls. Interestingly, repeated urinary tract infections with Escherichia coli bacteria, a common comorbidity in elderly people with dementia, caused further elevations in Piezo1 channel expression in the hippocampus and cortex of TgF344-AD rats. Taken together, we report that aging and peripheral infection augment amyloid plaque-induced upregulation of mechanoresponsive ion channels, such as Piezo1, in astrocytes. Further research is required to investigate the role of astrocytic Piezo1 in the Alzheimer's brain, whether modulating channel opening will protect or exacerbate the disease state, and most importantly, if Piezo1 could prove to be a novel drug target for age-related dementia.

Investigations on the effects of applied magnetic fields on mechanosensitive (MS) ion channel activity in Escherichia coli reveal an enhancement of subconducting activity with field exposure. In nine of 10 experimental runs, more subconducting activity was observed during the application of a 1.35 millitesla (mT) DC magnetic field when compared to control periods before field application (p=.1). This is an indication that these weak fields may interfere with the function of MS channel subunits in this bacterium and may have implications for the interaction of applied magnetic fields with human MS ion channels.

KCNK2 is a 2 pore domain potassium channel involved in maintaining cellular membrane resting potentials. Although KCNK2 is regarded as a mechanosensitive ion channel, it can also be gated chemically. Previous research indicates that KCNK2 expression is particularly enriched in neuronal and cardiac tissues. In this respect, KCNK2 plays an important role in neuroprotection and has also been linked to cardiac arrhythmias. KCNK2 has subsequently become an attractive pharmacologic target for developing preventative/curative strategies for neuro/cardio pathophysiological conditions. Zebrafish represent an important in vivo model for rapidly analysing pharmacological compounds. We therefore sought to identify and characterise zebrafish kcnk2 to allow this model system to be incorporated into therapeutic research. Our data indicates that zebrafish possess two kcnk2 orthologs, kcnk2a and kcnk2b. Electrophysiological analysis of both zebrafish Kcnk2 orthologs shows that, like their human counterparts, they are activated by different physiological stimuli such as mechanical stretch, polyunsaturated fatty acids and intracellular acidification. Furthermore, both zebrafish Kcnk2 channels are inhibited by the human KCNK2 inhibitory peptide spadin. Taken together, our results demonstrate that both Kcnk2a and Kcnk2b share similar biophysiological and pharmacological properties to human KCNK2 and indicate that the zebrafish will be a useful model for developing KCNK2 targeting strategies.

Summary The mechanosensitive two-pore domain (K2P) K + channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the "down" to "up" conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer.

Sodium absorption in epithelial cells is rate-limited by the epithelial sodium channel (ENaC) activity in lungs, kidney and the distal colon. Pathophysiological conditions, such as cystic fibrosis and Liddle syndrome, result from water-electrolyte imbalance partly due to malfunction of ENaC's molecular regulation. The molecular mechanism(s) of pathologically implicated mutations in ENaC subunits are largely unknown due to absence of structural models for ENaC's oligomers. Here, we propose a dynamics-driven mechanism of the gain of function mutation αW493R implicated in atypical cystic fibrosis. We utilize a combination of discrete molecular dynamics simulations (DMD) of the extracellular region of ENaC's heterotrimer αβγ and functional data from whole-cell electrophysiology experiments. Structure-function studies suggest that tetramers and trimers are the major characterized functional oligomeric states of ENaC. Using the crystal structure of the acid sensing ion channel, ENaC's structural homolog in the ENaC/degenerin mechanosensitive ion channel family, we have constructed homology models of ENaC subunits alpha, beta and gamma, in addition to the heterotrimers αβγ, αγβ and the heterotetramer αβαγ. Electrophysiology data show constitutive activity of αW493Rβγ in oocytes indicated by higher open probability and elevated basal activity compared to WT. Our DMD simulation provides an allosteric framework, which agrees with experimental data. W493R rewires the electrostatically mediated inter-residue interaction network in close proximity to W493, resulting in widening of the pore geometry in the outer mouth of the pore in the extracellular region. Rewiring effect of inter-residue interactions in 493R mutant pocket allosterically propagates across the channel resulting in a more stabilized global conformational ensemble of the channel. These findings predict a novel mechanism of ENaC's constitutive activity, in which changes in local dynamics can affect the relative population of the channel's active states and its open probability.

Phosphoinositides are fundamental membrane lipids, e.g. phosphatidylinositol 4,5-bisphosphate (PIP2) is the long known precursor for second messengers such as inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). They are also involved in the regulation of ion channels, serve as membrane anchor for the cytoskeleton and required for hair cell function. The novel mechanically gated ion channel Piezo1 is a key player in erythrocyte volume regulation and Piezo2 is responsible for the rapidly adapting mechanically activated currents in sensory neurons. Mechanosensitive ion channels are proposed to be regulated by the cytoskeleton and membrane lipids in general. Piezo1 channel activity is inhibited by the disruption of the actin cytoskeleton, and its sensitivity to mechanical stimuli is increased by hypotonic swelling, however, the lipid regulation of these channels remains unexplored.Piezo channels are activated by mechanical stimuli when expressed in heterologous systems unlike most mechanosensitive ion channel candidates. Taking advantage of this we used electrophysiological techniques on HEK293 cells expressing Piezo channels to investigate the effects of Phospholipase C (PLC) activating pathways and specifically phosphoinositide depletion. PLC activation, which converts PIP2 into IP3 and DAG, inhibited mechanically activated currents in Piezo expressing cells. Phosphoinositide depletion using rapidly inducible lipid phosphatases also inhibited Piezo channel activity, but without generating IP3 or DAG. Our data provides evidence that phosphoinositides are involved in the regulation of Piezo channels, thus our study contributes to the understanding of lipid regulation of mechanically gated channels.

GsMTx4 is the only known peptide inhibitor of mechanosensitive channels. It is a gating modifier and presumably acts by modifying the arrangement of lipid molecules in the channel-lipid interface. Like other ICK peptide toxins GsMTx4 has a hydrophobic face which presumably facilitates partitioning of the molecule to the water/membrane interface of the bilayer. Prior studies also show that charged lysine residues may further influence the positioning of the peptide in the bilayer through interactions with the headgroups of anionic lipids. Coarse-grained simulations of GsMTx4 in the lipid bilayer suggest the existence of two binding modes: a shallow-binding mode and a deep-binding mode. The deep-binding mode in particular involves interactions between positively charged residues on the peptide and carbonyl atoms of the headgroups of inner-leaflet lipids. To provide insight into these interactions for GsMTx4 function, we investigated if reversing the charge by mutating lysine residues to glutamate affected the activity of GsMTx4. Each lysine residue in GsMTx4 was individually changed to glutamate to generate six mutants: K8E, K15E, K20E, K22E, K25E and K28E. When tested on outside-out patches from HEK cells transfected with mPiezo1 cDNA, all KtoE mutants showed reduced ability to inhibit the channel. In addition to reduced activity, K15E peptide altered the inactivation rate of the mPiezo1 currents. Even at saturating concentrations, K28E mutation reduced the activity of the peptide to 23% of that of the wild-type peptide. This study shows that charge reversing mutations to lysine residues diminish the efficacy of GsMTx4, and this phenomenon may involve disruption of peptide partitioning into the membrane and/or peptide-induced bending of the membrane bilayer.