Mechanosensitive Potassium Channel Research Articles

Ultrasound-Induced Membrane Hyperpolarization in Motor Axons and Muscle Fibers of the Crayfish Neuromuscular Junction.

Focused ultrasound (FUS) can modulate neuronal activity by depolarization or hyperpolarization. Although FUS-evoked depolarization has been studied extensively, the mechanisms underlying FUS-evoked hyperpolarization (FUSH) have received little attention. In the study described here, we developed a procedure using FUS to selectively hyperpolarize motor axons in crayfish. As a previous study had reported that these axons express mechano- and thermosensitive two-pore domain potassium (K2P) channels, we tested the hypothesis that K2P channels underlie FUSH. Intracellular recordings from a motor axon and a muscle fiber were obtained simultaneously from the crayfish opener neuromuscular preparation. FUSH was examined while K2P channel activities were modulated by varying temperature or by K2P channel blockers. FUSH in the axons did not exhibit a coherent temperature dependence, consistent with predicted K2P channel behavior, although changes in the resting membrane potential of the same axons indicated well-behaved K2P channel temperature dependence. The same conclusion was supported by pharmacological data; namely, FUSH was not suppressed by K2P channel blockers. Comparison between the FUS-evoked responses recorded in motor axons and muscle fibers revealed that the latter exhibited very little FUSH, indicating that the FUSH was specific to the axons. It is not likely that K2P channels are the underlying mechanism for FUSH in motor axons. Alternative mechanisms such as sonophore and axon-specific potassium channels were considered. Although the sonophore hypothesis could account for electrophysiological features of axonal recordings, it is not consistent with the lack of FUSH in muscle fibers. An axon-specific and mechanosensitive potassium channel is also a possible explanation.

Mechanosensitive Potassium Channels in the Afferent Limb of the Cardio-Renal Baroreflex

Mechanosensitive Potassium Channels in the Afferent Limb of the Cardio-Renal Baroreflex

Modulation of Mechanosensitive Potassium Channels by a Membrane-targeted Nongenetic Photoswitch.

Mechanosensitive ion channels are present in the plasma membranes of all cells. They play a fundamental role in converting mechanical stimuli into biochemical signals and are involved in several physiological processes such as touch sensation, hearing, and blood pressure regulation. This protein family includes TWIK-related arachidonic acid-stimulated K+ channel (TRAAK), which is specifically implicated in the maintenance of the resting membrane potential and in the regulation of a variety of important neurobiological functions. Dysregulation of these channels has been linked to various diseases, including blindness, epilepsy, cardiac arrhythmia, and chronic pain. For these reasons, mechanosensitive channels are targets for the treatment of several diseases. Here, we propose a new approach to investigate TRAAK ion channel modulation that is based on nongenetic photostimulation. We employed an amphiphilic azobenzene, named Ziapin2. In the dark, Ziapin2 preferentially dwells in the plasma membrane, causing a thinning of the membrane. Upon light irradiation, an isomerization occurs, breaking the dimers and inducing membrane relaxation. To study the effect of Ziapin2 on the mechanosensitive channels, we expressed human TRAAK (hTRAAK) channels in HEK293T cells. We observed that Ziapin2 insertion in the membrane is able per se to recruit hTRAAK, permitting the exit of K+ ions outside the cells with a consequent hyperpolarization of the cell membrane. During light stimulation, membrane relaxation induces hTRAAK closure, generating a consistent and compensatory depolarization. These results add information to the Ziapin2 mechanism and suggest that membrane deformation can be a tool for the nonselective modulation of mechanosensitive channels.

Freestanding lipid bilayer tensiometer for the study of mechanosensitive ion channels

Mechanical forces modify the cell membrane potential by opening mechanosensitive ion channels. We report the design and construction of a lipid bilayer tensiometer to study channels that respond to lateral membrane tension, [Formula: see text] , in the range 0.2 to 1.4 [Formula: see text] (0.8 to 5.7 [Formula: see text] ). The instrument consists of a black-lipid-membrane bilayer, a custom-built microscope, and a high-resolution manometer. Values of [Formula: see text] are obtained from the determination of the bilayer curvature as a function of applied pressure by means of the Young-Laplace equation. We demonstrate that [Formula: see text] can be determined by calculating the bilayer radius of curvature from fluorescence microscopy imaging or from measurements of the bilayer's electrical capacitance, both yielding similar results. Using electrical capacitance, we show that the mechanosensitive potassium channel TRAAK responds to [Formula: see text] , not curvature. TRAAK channel open probability increases as [Formula: see text] is increased from 0.2 to 1.4 [Formula: see text] but open probability never reaches 0.5. Thus, TRAAK opens over a wide range of [Formula: see text] , but with a tension sensitivity about one-fifth that of the bacterial mechanosensitive channel MscL.

Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate

Atrial fibrillation is the most common sustained cardiac arrhythmia in humans. Current atrial fibrillation antiarrhythmic drugs have limited efficacy and carry the risk of ventricular proarrhythmia. GsMTx4, a mechanosensitive channel–selective inhibitor, has been shown to suppress arrhythmias through the inhibition of stretch-activated channels (SACs) in the heart. The cost of synthesizing this peptide is a major obstacle to clinical use. Here, we studied two types of short peptides derived from GsMTx4 for their effects on a stretch-activated big potassium channel (SAKcaC) from the heart. Type I, a 17-residue peptide (referred to as Pept 01), showed comparable efficacy, whereas type II (i.e., Pept 02), a 10-residue peptide, exerted even more potent inhibitory efficacy on SAKcaC compared with GsMTx4. We identified through mutagenesis important sequences required for peptide functions. In addition, molecular dynamics simulations revealed common structural features with a hydrophobic head followed by a positively charged protrusion that may be involved in peptide channel–lipid interactions. Furthermore, we suggest that these short peptides may inhibit SAKcaC through a specific modification to the mechanogate, as the inhibitory effects for both types of peptides were mostly abolished when tested with a mechano-insensitive channel variant (STREX-del) and a nonmechanosensitive big potassium (mouse Slo1) channel. These findings may offer an opportunity for the development of a new class of drugs in the treatment of cardiac arrhythmia generated by excitatory SACs in the heart.

Supramolecular Mechanosensitive Potassium Channel Formed by Fluorinated Amphiphilic Cyclophane.

Inspired by mechanosensitive potassium channels found in nature, we developed a fluorinated amphiphilic cyclophane composed of fluorinated rigid aromatic units connected via flexible hydrophilic octa(ethylene glycol) chains. Microscopic and emission spectroscopic studies revealed that the cyclophane could be incorporated into the hydrophobic layer of the lipid bilayer membranes and self-assembled to form a supramolecular transmembrane ion channel. Current recording measurements using cyclophane-containing planer lipid bilayer membranes successfully demonstrated an efficient transmembrane ion transport. We also demonstrated that the ion transport property was sensitive to the mechanical forces applied to the membranes. In addition, ion transport assays using pH-sensitive fluorescence dye revealed that the supramolecular channel possesses potassium ion selectivity. We also performed all-atom hybrid quantum-mechanical/molecular mechanical simulations to assess the channel structures at atomic resolution and the mechanism of selective potassium ion transport. This research demonstrated the first example of a synthetic mechanosensitive potassium channel, which would open a new door to sensing and manipulating biologically important processes and purification of key materials in industries.

Molecular and cellular insights into mechanosensation and volume regulation in the nervous system

The transduction of physical forces into cellular electrical signals is fundamental to a wide breadth of biology including the classic senses of touch, pain, hearing, and balance; less conspicuous sensations of proprioception, blood pressure, and osmolarity; and basic aspects of neural growth, signaling, and death. In this talk, I will discuss recent work aimed at understanding the basis of mechanosensation and volume regulation in the nervous system. First, I will present a model for the mechanism of membrane tension sensing and physiological roles of a mechanosensitive potassium channel based on a combination of structural, electrophysiological, and localization data. Second, I will present a model for ionic strength sensing and channel gating in a family of volume regulated anion channels based on structural and functional studies.Support or Funding InformationNew York Stem Cell Foundation ‐ Robertson Neuroscience Investigator Award, McKnight Scholar Award, Klingenstein‐Simons Fellowship Award, NIGMS New Innovator Award, Rose Hill Innovator Award

Functional expression of mechanosensitive two-pore domain potassium channel in human bladder carcinoma cells

Bladder cancer is a common cancer in smoking men and may correlate with mechanosensitive potassium channels because the urinary bladder is a stretch sensing organ. Two-pore K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to play a critical role in both cell apoptosis and tumorigenesis. Of the channels, TREK1 can be activated by many physiological stimuli, including polyunsaturated fatty acids, and intracellular pH, hypoxia, and neurotransmitters. Here we attempted to determine whether TREK1 is functionally expressed in bladder cancer 253J cells. K2P channels, including TREK1, TREK2, TASK1, TASK3, and TWIK1, were quantified in cultured human bladder cancer 253J cells using real time quantitative RT-PCR (qRT-PCR) analysis. Among them, TREK1-like channel was recorded at a single channel level using the patch-clamp technique. The TREKl-like channel, with single-channel conductance of ~90 pS at ?80 mV, was recorded in symmetrical 150 mM KCl using an excised inside-out patch configuration. The current-voltage relationships were linear and were insensitive to tetraethylammonium. The channel was activated by membrane stretch, free fatty acids, and intracellular acidosis. These results with electrophysiological properties resemble to those of K2P channel, for instance, TREK1. Therefore, we conclude that TREK1 channel is functionally present in bladder cancer 253J cells.

Effects of acute mechanical stretch on the expression of mechanosensitive potassium channel TREK-1 in rat left ventricle

To explore the role of mechanosensitive potassium channel TREK-1, Western blot analysis was used to investigate the expression changes of TREK-1 in left ventricle in acute mechanically stretched heart. Forty Wistar rats were randomly divided into 8 groups (n=5 in each group), subject to single Langendorff perfusion for 0, 30, 60, 120 min and acute mechanical stretch for 0, 30, 60, 120 min respectively. With Langendorff apparatus, an acute mechanically stretched heart model was established. There was no significant difference in the expression of TREK-1 among single Langendorff perfusion groups (P>0.05). As compared to non-stretched Langendorff-perfused heart, only the expression of TREK-1 in acute mechanically stretched heart (120 min) was greatly increased (P<0.05). This result suggested that some course of mechanical stretch could up-regulate the expression of TREK-1 in left ventricle. TREK-1 might play an important role in mechanoelectric feedback, so it could reduce the occurrence of arrhythmia that was induced by extra mechanical stretch.

Differential expression of the mechanosensitive potassium channel TREK‐1 in epicardial and endocardial myocytes in rat ventricle

Mechanoelectric feedback (MEF) is the process by which mechanical forces on the myocardium induce electrical responses. It is thought that MEF is important in controlling the beat to beat force of contraction in the ventricle, in response to fluctuations in load, and it may also play a role in controlling the dispersion of repolarization. The transduction mechanism for MEF is via stretch sensitive ion channels in the surface membrane of myocytes. Two types of stretch sensitive channels have been described; a non-selective cation channel, and a potassium selective channel. TREK-1 is a member of the recently cloned tandem pore potassium channels that has been shown to be mechanosensitive and to be expressed in rat heart. Here we report that the gene expression level of TREK-1, quantified using real-time RT-PCR against glyceraldehyde phosphate dehydrogenase (GAPDH) as a comparator gene, was found to be 0.34 +/- 0.14 in endocardial cells compared to 0.02 +/- 0.02 in epicardial cells (P < 0.05). To confirm that this is reflected in a different current density, whole cell TREK-1 currents, activated by chloroform, were recorded with patch clamp techniques in epicardial and endocardial cells. TREK-1 current density in epicardial and endocardial cells was 0.21 +/- 0.06 pA/pF and 0.8 +/- 0.27 pA/pF, respectively (P</= 0.05). We discuss the implications of this differential expression of TREK-1 for controlling action potential repolarization when the myocardium is stretched. We hypothesize that the gene expression of TREK-1 is controlled by the different amounts of stretch experienced by muscle cells across the ventricular wall.

Expression and localization of TREK-1 K+ channels in human odontoblasts.

During tooth development, odontoblasts are the cells that form dentin and possibly mediate early stages of sensory processing in teeth. It is suggested that ion channels assist in these events. Indeed, mechanosensitive potassium currents, transducing mechanical stimuli into electrical cell signals, have been previously recorded in the human odontoblast cell membrane. Here, we show by RT-PCR that the mechanosensitive potassium channel TREK-1 (a member of the two-pore-domain potassium channel family) is overexpressed in these cultured cells compared with pulp cells in vitro. In situ hybridization showed that transcripts are detected in the odontoblast layer in vivo. The use of antibodies shows that TREK-1 is strongly expressed in the membrane of coronal odontoblasts and absent in the root. This distribution is related to the spatial distribution of nerve endings identified by labeling of the low-affinity nerve growth factor (NGF) receptor (p75(NTR)). These results demonstrate the expression of TREK-1 in human odontoblasts in vitro and in vivo.

Mechanosensitive potassium channels in rat colon sensory neurons.

Single-channel recording techniques were used to characterize mechanosensitive channels in identified (1.1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine methanesulfonate labeled) colon sensory neurons dissociated from adult S1 dorsal root ganglia. Channels were found in 30% (7/23) of patches in a cell-attached configuration and in 43% (48/111) of excised inside-out patches. Channels were highly selective for K(+), had a slope conductance of 54 pS in symmetrical solutions, and were blocked by tetraethylammonium, amiloride, and benzamil. Channels were also seen under Ca(2+)-free conditions. Gadolinium (Gd(3+)), a known blocker of mechanosensitive ion channels, did not block channel activity. Tetrodotoxin and 4-aminopyridine were also ineffective. The cytoskeletal disrupters colchicine and cytochalasin D reduced the percentage of patches containing mechanosensitive channels. These results indicate that rat colon sensory neurons contain K(+)-selective mechanosensitive channels that may modulate the membrane excitability induced by colonic distension.

Mechanosensitive potassium channels in locust muscle membrane

Patch clamp recordings have been made from adult locust (Schistocerca gregaria) muscle membrane to study the mechanosensitivity of potassium channels (BK and IK) in cell-attached patches by transiently applying measured pressures to the contents of the patch pipettes. The aim of the investigations was to demonstrate a novel gating behaviour by pressure of the BK channel in contrast to the familiar behaviour of the IK channel. The open probability (p 0) of the IK channel increased rapidly in response to a pressure step and monotonically during a pressure ramp. This gating was readily repeatable and rapidly reversible. The relationship between ln[p 0/(1–p 0)] and transmembrane pressure was linear. In comparison, p 0 for the BK channel was also increased by pressure, but its gating was delayed, cumulative, and hysteretic.

A mechanosensitive K + channel with fast-gating kinetics on human axons blocked by gadolinium ions

Current recordings from single mechanosensitive potassium channels were obtained from cell-attached and excised patches of human axons. The channel showed an inwardly rectifying I–V relationship with a slope conductance of 52 pS at negative potentials (symmetrical high potassium solution). The fast-gating kinetics of the channel were best described by one time constant for the open state and two time constants for the closed state. The open probability of this channel increased as negative pressure was applied to the patch pipette. The pressure dependence of the open probability of the mechanosensitive K + channel followed a sigmoid relationship. Gadolinium (0.5–1 mM) acted as a potent blocker when applied to the intra- and extracellular side of the patch membrane.