Mechanosensitive K2P Channels Research Articles

Membrane phospholipids control activity of the mechanosensitive K2P channel TREK1

Tandem pore (K2P) potassium ion channels play vital roles in human physiology, chiefly responsible for maintaining cellular resting membrane potential. Modulation of K2P channel activity controls cellular excitability, altering the degree of input signal required to reach action potential thresholds. For the mechanosensitive subfamily of K2Ps, including the TREK1, TREK2, and TRAAK channels, a wide range of diverse and dissimilar inputs alter channel activity, including pH, heat, mechanical stretch, lipids, post-translational modification, and an array of pharmacologically active agents including volatile anesthetics, antidepressants, and neuroprotective agents.

Trabecular Meshwork TREK-1 Channels Function as Polymodal Integrators of Pressure and pH.

PurposeThe concentration of protons in the aqueous humor (AH) of the vertebrate eye is maintained close to blood pH; however, pathologic conditions and surgery may shift it by orders of magnitude. We investigated whether and how changes in extra- and intracellular pH affect the physiology and function of trabecular meshwork (TM) cells that regulate AH outflow.MethodsElectrophysiology, in conjunction with pharmacology, gene knockdown, and optical recording, was used to track the pH dependence of transmembrane currents and mechanotransduction in primary and immortalized human TM cells.ResultsExtracellular acidification depolarized the resting membrane potential by inhibiting an outward K+-mediated current, whereas alkalinization hyperpolarized the cells and augmented the outward conductance. Intracellular acidification with sodium bicarbonate hyperpolarized TM cells, whereas removal of intracellular protons with ammonium chloride depolarized the membrane potential. The effects of extra- and intracellular acid and alkaline loading were abolished by quinine, a pan-selective inhibitor of two-pore domain potassium (K2P) channels, and suppressed by shRNA-mediated downregulation of the mechanosensitive K2P channel TREK-1. Extracellular acidosis suppressed, whereas alkalosis facilitated, the amplitude of the pressure-evoked TREK-1–mediated outward current.ConclusionsThese results demonstrate that TM mechanotransduction mediated by TREK-1 channels is profoundly sensitive to extra- and intracellular pH shifts. Intracellular acidification might modulate aqueous outflow and IOP by stimulating TREK-1 channels.

Studying Mechanosensitivity of Two-Pore Domain K+ Channels in Cellular and Reconstituted Proteoliposome Membranes.

Mechanical force sensation is fundamental to a wide breadth of biology from the classic senses of touch, pain, hearing, and balance to less conspicuous sensations of proprioception, blood pressure, and osmolarity and basic aspects of cell growth, differentiation, and development. These diverse and essential systems use force-gated (or mechanosensitive) ion channels that convert mechanical stimuli into cellular electrical signals. TRAAK, TREK1, and TREK2 are K+-selective ion channels of the two-pore domain K+ (K2P) family that are mechanosensitive: they are gated open by increasing membrane tension. TRAAK and TREK channels are thought to play roles in somatosensory and other mechanosensory processes in neuronal and non-neuronal tissues. Here, we present protocols for three assays to study mechanical activation of these channels in cell membranes: (1) cell swelling, (2) cell poking, and (3) patched membrane stretching. Patched membrane stretching is also applicable to the study of mechanosensitive K2P channel activity in a cell-free system and a procedure for proteoliposome reconstitution and patching is also presented. These approaches are alsoreadily applicable to the study of other mechanosensitive ion channels.

Stretch-activated two-pore-domain (K2P) potassium channels in the heart: Focus on atrial fibrillation and heart failure

Two-pore-domain potassium (K2P) channels modulate cellular excitability. The significance of stretch-activated cardiac K2P channels (K2P2.1, TREK-1, KCNK2; K2P4.1, TRAAK, KCNK4; K2P10.1, TREK-2, KCNK10) in heart disease has not been elucidated in detail. The aim of this work was to assess expression and remodeling of mechanosensitive K2P channels in atrial fibrillation (AF) and heart failure (HF) patients in comparison to murine models.Cardiac K2P channel levels were quantified in atrial (A) and ventricular (V) tissue obtained from patients undergoing open heart surgery. In addition, control mice and mouse models of AF (cAMP-response element modulator (CREM)-IbΔC-X transgenic animals) or HF (cardiac dysfunction induced by transverse aortic constriction, TAC) were employed. Human and murine KCNK2 displayed highest mRNA abundance among mechanosensitive members of the K2P channel family (V > A). Disease-associated K2P2.1 remodeling was studied in detail. In patients with impaired left ventricular function, atrial KCNK2 (K2P2.1) mRNA and protein expression was significantly reduced. In AF subjects, downregulation of atrial and ventricular KCNK2 (K2P2.1) mRNA and protein levels was observed. AF-associated suppression of atrial Kcnk2 (K2P2.1) mRNA and protein was recapitulated in CREM-transgenic mice. Ventricular Kcnk2 expression was not significantly altered in mouse models of disease.In conclusion, mechanosensitive K2P2.1 and K2P10.1 K+ channels are expressed throughout the heart. HF- and AF-associated downregulation of KCNK2 (K2P2.1) mRNA and protein levels suggest a mechanistic contribution to cardiac arrhythmogenesis.

Bilayer-Mediated Structural Transitions in the TREK-2 Mechanosensitive K2P Channel

Mammalian mechanosensitive ion channels are molecular sensors that transduce mechanical stimulus into altered electrical activity of cells. They are involved in numerous physiological processes such as touch, hearing, and cell growth. Recently, crystal structures for several mechanosensitive Two-Pore Domain Potassium channels (K2P) in different conformations have been published. However, functional annotation of these structures remains challenging, especially as these proteins were crystallized in the presence of detergents. Using crystal structures and functional studies of state-dependent inhibitor, we have recently shown that “Down” state of the TREK-2 channel most likely represents a non-stretched closed channel, whereas a more expanded “Up” conformation likely represents that of a stretch-activated channel {1}. To test this hypothesis, we now combine computational and experimental studies to demonstrate that TREK-2 channels can sense forces directly from their surrounding lipids. Molecular dynamics simulation of TREK-2 “Down” channels embedded in phospholipid bilayers reveal that these channels undergo structural transitions when the surrounding bilayer is stretched. The stretched conformations are remarkably similar to several of the “Up” structures published for TREK-2 and TRAAK channels. We further investigated the dynamics of stretch dependent conformational change in the TREK-2 channel and find that coordinated movement of TM4 with respect to TM2 and TM3, which involves interaction between the channel and phospholipids, is also required for successful transition between conformations. These finding demonstrate that stretching the surrounding bilayer leads to compensatory changes in TREK-2 structures and that this underlies mechanosensitive K2P channels’ ability to directly sense forces from the membrane.1. Dong YY et al. K2P channel gating mechanism revealed by structures of TREK-2 and a complex with Prozac Science 2015 Mar 13;347(6227):1256-9.

How ion channels sense mechanical force: insights from mechanosensitive K2P channels TRAAK, TREK1, and TREK2.

The ability to sense and respond to mechanical forces is essential for life and cells have evolved a variety of systems to convert physical forces into cellular signals. Within this repertoire are the mechanosensitive ion channels, proteins that play critical roles in mechanosensation by transducing forces into ionic currents across cellular membranes. Understanding how these channels work, particularly in animals, remains a major focus of study. Here, I review the current understanding of force gating for a family of metazoan mechanosensitive ion channels, the two-pore domain K(+) channels (K2Ps) TRAAK, TREK1, and TREK2. Structural and functional insights have led to a physical model for mechanical activation of these channels. This model of force sensation by K2Ps is compared to force sensation by bacterial mechanosensitive ion channels MscL and MscS to highlight principles shared among these evolutionarily unrelated channels, as well as differences of potential functional relevance. Recent advances address fundamental questions and stimulate new ideas about these unique mechanosensors.

A High-Throughput Functional Screen Identifies Small Molecule Regulators of Temperature- and Mechano-Sensitive K2P Channels

K2P (KCNK) potassium channels generate “leak” potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K2P currents poses for electrophysiology-based high-throughput screening strategies. Here, we present a yeast-based high-throughput screening assay that avoids this problem. Using a simple growth-based functional readout, we screened a library of 106,281 small molecules and identified two new inhibitors and three new activators of the mammalian K2P channel K2P2.1 (KCNK2, TREK-1). By combining biophysical, structure–activity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K2P channels. Biophysical studies show that ML67-33 reversibly increases channel currents by activating the extracellular selectivity filter-based C-type gate that forms the core gating apparatus on which a variety of diverse modulatory inputs converge. The new K2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K2P activity and facilitate the discovery and development of other K2P small molecule modulators.

276 MECHANOSENSITIVITY OF THE HUMAN DETRUSOR IN RESPONSE TO STRETCH IS REGULATED BY STRETCH-ACTIVATED TWO-PORE DOMAIN POTASSIUM CHANNELS

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology (I)1 Apr 2013276 MECHANOSENSITIVITY OF THE HUMAN DETRUSOR IN RESPONSE TO STRETCH IS REGULATED BY STRETCH-ACTIVATED TWO-PORE DOMAIN POTASSIUM CHANNELS Qi Lei, Xiao-Qing Pan, Stanley Malkowicz, Thomas Guzzo, and Anna Malykhina Qi LeiQi Lei Glenolden, PA More articles by this author , Xiao-Qing PanXiao-Qing Pan Glenolden, PA More articles by this author , Stanley MalkowiczStanley Malkowicz Philadelphia, PA More articles by this author , Thomas GuzzoThomas Guzzo Philadelphia, PA More articles by this author , and Anna MalykhinaAnna Malykhina Glenolden, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1660AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mechanosensitivity of the detrusor is defined as the ability of smooth muscle cells to generate mechanical activity independent of external stimuli. The mechanisms of bladder mechanosensitivity due to detrusor wall stretch are poorly understood. Animal data suggest that stretch-activated two-pore domain (K2P) K+ channels play a critical role in bladder response to filling. The objective of this study was to characterize the function of stretch-activated K+ channels in the human bladder and determine their physiological role in bladder mechanosensitivity. METHODS Full thickness bladder specimens were obtained from patients without symptoms of detrusor overactivity who underwent cystectomy for early stages of bladder cancer (T1-T2,N0-N1). Expression of mechanosensitive K2P channels was determined by immunohistochemistry (IHC) and Western blotting. Functional characterization and biophysical properties of the predominantly expressed member of K2P family, TREK-1 channel, were performed using path-clamp technique. RESULTS Western blot analysis evaluated protein expression of mechanosensitive channels TREK-1, TREK-2, and TRAAK in the human bladder, and established that TREK-1 is the predominantly expressed member of K2P family. IHC staining of the full thickness bladder wall identified TREK-1 expression in the urothelium, on single cells located in the lamina propria, and on bladder smooth muscle cells. Electrophysiological recordings from single smooth muscle cells established that TREK-1 channels are directly activated by mechanical stretch of the cell membrane. We next tested the effects of TREK-1 channel opener arachidonic acid (10 μM) in a whole cell patch clamp mode. Arachidonic acid caused a 5-fold increase in the current amplitude. Additionally, we performed IHC staining of cultured bladder smooth muscle cells with TREK-1 antibodies before and after treatment with Lantrunculin A, an agent which disrupts actin polymerization. Treatment with Lantrunculin A caused an increase in TREK-1 staining in bladder smooth muscle cells suggesting that these channels are linked to the cell cytoskeleton. CONCLUSIONS The results of our study provide direct evidence that the response of the human detrusor to mechanical stretch during bladder filling is regulated by TREK-1 channels. Impaired mechanosensation and mechanotransduction associated with changes in stretch-activated K2P channels may underlie overactive bladder disorders in humans. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e112-e113 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qi Lei Glenolden, PA More articles by this author Xiao-Qing Pan Glenolden, PA More articles by this author Stanley Malkowicz Philadelphia, PA More articles by this author Thomas Guzzo Philadelphia, PA More articles by this author Anna Malykhina Glenolden, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The TREK2 Channel Is Involved in the Proliferation of 253J Cell, a Human Bladder Carcinoma Cell

Bladder cancer is the seventh most common cancer in men that smoke, and the incidence of disease increases with age. The mechanism of occurrence has not yet been established. Potassium channels have been linked with cell proliferation. Some two-pore domain K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to be overexpressed in cancer cells. Here we focused on the relationship between cell growth and the mechanosensitive K2P channel, TREK2, in the human bladder cancer cell line, 253J. We confirmed that TREK2 was expressed in bladder cancer cell lines by Western blot and quantitative real-time PCR. Using the patch-clamp technique, the mechanosensitive TREK2 channel was recorded in the presence of symmetrical 150 mM KCl solutions. In 253J cells, the TREK2 channel was activated by polyunsaturated fatty acids, intracellular acidosis at -60 mV and mechanical stretch at -40 mV or 40 mV. Furthermore, small interfering RNA (siRNA)-mediated TREK2 knockdown resulted in a slight depolarization from -19.9 mV±0.8 (n=116) to -8.5 mV±1.4 (n=74) and decreased proliferation of 253J cells, compared to negative control siRNA. 253J cells treated with TREK2 siRNA showed a significant increase in the expression of cell cycle boundary proteins p21 and p53 and also a remarkable decrease in protein expression of cyclins D1 and D3. Taken together, the TREK2 channel is present in bladder cancer cell lines and may, at least in part, contribute to cell cycle-dependent growth.

Colitis decreases mechanosensitive K2P channel expression and function in mouse colon sensory neurons

TREK-1, TREK-2 and TRAAK are mechanosensitive two-pore domain K(+) (K(2P)) channels thought to be involved in the attenuation of mechanotransduction. Because colon inflammation is associated with colon mechanohypersensitivity, we hypothesized that the role of these channels in colon sensory (dorsal root ganglion, DRG) neurons would be reduced by colon inflammation. Accordingly, we studied the functional expression of mechanosensitive K(2P) channels in colon sensory neurons in both thoracolumbar (TL) and lumbosacral (LS) DRG that represent the splanchnic and pelvic nerve innervations of the colon, respectively. In colon DRG neurons identified by retrograde tracer previously injected into the colon wall, 62% of TL neurons and 83% of LS neurons expressed at least one of three K(2P) channel mRNAs; the proportion of neurons expressing the TREK-1 gene was greater in LS than in TL DRG. In electrophysiological studies, single-channel activities of TREK-1a, TREK-1b, TREK-2, and TRAAK-like channels were detected in cultured colon DRG neuronal membranes. After trinitrobenzene sulfonic acid-induced colon inflammation, we observed significant decreases in the amount of TREK-1 mRNA, in the response of TREK-2-like channels to membrane stretch, and in the whole cell outward current during osmotic stretch in LS colon DRG neurons. These findings document that the majority of DRG neurons innervating the mouse colon express mechanosensitive K(2P) channels and suggest that a decrease in their expression and activities contributes to the increased colon mechanosensitivity that develops in inflammatory bowel conditions.