Mechanosensitive ion channels and the peptide inhibitor GsMTx-4: History, properties, mechanisms and pharmacology

Bowman, Charles L.; Gottlieb, Philip A.; Suchyna, Thomas M.; Murphy, Yolanda K.; Sachs, Frederick

doi:10.1016/j.toxicon.2006.09.030

Cited by 164 publications

(128 citation statements)

References 134 publications

(149 reference statements)

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“…1 and 2). As we have shown for a variety of stimulation modalities and stem cell reprogramming, the probes appear to be applicable to all physiological variables (35).…”

Section: Discussionmentioning

confidence: 89%

Actin stress in cell reprogramming

Guo

Wang

Sachs

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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103

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Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin-cpstFRET-actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFPtagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals.actin | force probe | stem cell | reprogramming | cell mechanics C ells have only three sources of free energy-chemical, electrical, and mechanical potential (1)-and life is driven by the flow of energy between them. The flow of mechanical energy has not been well studied in living cells outside of muscle, yet all cells are subject to endogenous and exogenous mechanical forces. The lack of progress in measuring these forces (stress) has primarily been due to a lack of suitable probes, but that has now been remedied with the development of genetically coded FRETbased force sensors (2). The literature shows many macroscopic effects of mechanical stress on cellular processes including cell motility, embryogenesis, stem cell replication and differentiation (3, 4), bone and muscle homeostasis, gene expression, protein folding (5), and membrane potential (6). However, these studies lacked the ability to measure stress in specific structural proteins, and the analyses have tended to treat cytoskeletal stresses as uniform, which we now know is not true (2, 7). We, and other groups (8), have shown that the distribution of forces is nonuniform in both time and space and protein specificity (9).The force sensors consist of a FRET pair (typically CFP/ YFP) connected by a protein linker, a biological analog of a mechanic...

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“…1 and 2). As we have shown for a variety of stimulation modalities and stem cell reprogramming, the probes appear to be applicable to all physiological variables (35).…”

Section: Discussionmentioning

confidence: 89%

Actin stress in cell reprogramming

Guo

Wang

Sachs

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

103

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“…We next tested the susceptibility of hNSPC SAC currents to GsMTx-4, a peptide isolated from the venom of the Chilean rose tarantula spider, Grammostola rosea. GsMTx-4 is the only known drug to inhibit cationic SACs specifically without inhibiting other ion-channel families such as voltage-gated sodium, potassium, and calcium channels (27) or potassium-selective SACs (28). It acts as a gating modifier by partitioning into the cell membrane and affecting force transfer to the channel protein (29).…”

Section: Resultsmentioning

confidence: 99%

Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells

Pathak¹,

Nourse

Tran³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

503

509

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Neural stem cells are multipotent cells with the ability to differentiate into neurons, astrocytes, and oligodendrocytes. Lineage specification is strongly sensitive to the mechanical properties of the cellular environment. However, molecular pathways transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification remain unclear. We found that the mechanically gated ion channel Piezo1 is expressed by brainderived human neural stem/progenitor cells and is responsible for a mechanically induced ionic current. Piezo1 activity triggered by traction forces elicited influx of Ca 2+ , a known modulator of differentiation, in a substrate-stiffness-dependent manner. Inhibition of channel activity by the pharmacological inhibitor GsMTx-4 or by siRNA-mediated Piezo1 knockdown suppressed neurogenesis and enhanced astrogenesis. Piezo1 knockdown also reduced the nuclear localization of the mechanoreactive transcriptional coactivator Yes-associated protein. We propose that the mechanically gated ion channel Piezo1 is an important determinant of mechanosensitive lineage choice in neural stem cells and may play similar roles in other multipotent stem cells. (5) and that the mechanical properties of the culturing environment before transplantation can influence the outcome of in vivo stem cell transplants (6). Hence, a molecular and mechanistic understanding of how stem cells process mechanical cues and how this processing results in downstream signaling events and ultimately in fate decisions is needed for greater control over the fate of transplanted cells.Studies in mesenchymal and neural stem cells have revealed the involvement of focal adhesion zones and cytoskeletal proteins, such as integrins, nonmuscle myosin II (7), Rho GTPases (8-10), and vinculin (11), that participate in the generation of cellular traction forces. Recent work also has identified the nucleoskeletal protein lamin-A (12) and the transcriptional coactivators Yap (Yesassociated protein) and Taz (transcriptional coactivator with PDZbinding motif) (13) in mechanotransduction in mesenchymal stem cells. However, the mechanisms by which mechanical cues detected by cellular traction forces are transduced to downstream intracellular pathways of differentiation remain unclear.Ion channels are involved, directly or indirectly, in the transduction of all forms of physical stimuli-including sound, light, temperature, mechanical force, and even gravity-into intracellular signaling pathways. Hence, we wondered whether ion channels could be involved in transducing matrix mechanical cues to intracellular signaling pathways linked to lineage specification. In particular, we focused here on cationic stretch-activated channels (SACs) because they are known to detect mechanical forces with high sensitivity and broad dynamic range and because they are permeable to Ca 2+ , an important second messenger implicated in cell fate (14,15). We examined the role of SACs in neural stem cells, for which mechanical cues influence specification alo...

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“…We are well aware of the fact that the mechanosensitivity of TRPC1 is discussed controversially at present and that especially the activity of heterologously overexpressed TRPC1 in mammalian cells is problematic [14]. The spider venom GsMTx-4 is a blocker of mechanosensitive cation channels like TRPC6 [3,40] and was used in this study to determine the impact of such channels on the displacement of TRPC1 knockdown and hTRPC1-HA-overexpressing cells. GsMTx-4 is three times more effective in cells overexpressing TRPC1-HA than in cells with reduced expression.…”

Section: Discussionmentioning

confidence: 99%

“…To inhibit mechanosensitive cation channels, we employed a peptide (GsMTx-4, Peptides International, USA) isolated from venom of the tarantula, Grammostola spatulata [3,40,41]. The effect of GsMTx-4 was studied in paired experiments.…”

Section: Analysis Of Cell Migrationmentioning

confidence: 99%

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TRPC1 channels regulate directionality of migrating cells

Fabian

Fortmann

Dieterich

et al. 2008

Pflugers Arch - Eur J Physiol

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Cell migration depends on the generation of structural asymmetry and on different steps: protrusion and adhesion at the front and traction and detachment at the rear part of the cell. The activity of Ca 2+ channels coordinate these steps by arranging intracellular Ca 2+ signals along the axis of movement. Here, we investigated the role of the putative mechanosensitive canonical transient receptor potential channel 1 (TRPC1) in cell migration. We analyzed its function in transformed renal epithelial (Madin-Darby canine kidney-focus) cells with variation of TRPC1 expression. As shown by time lapse video microscopy, TRPC1 knockdown cells have partially lost their polarity and the ability to persistently migrate into a given direction. This failure is linked to the suppression of a local Ca 2+ gradient at the front of migrating TRPC1 knockdown cells, whereas TRPC1 overexpression leads to steeper Ca 2+ gradients. We propose that the Ca 2+ signaling events regulated by TRPC1 within the lamellipodium determine polarity and directed cell migration.

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Mechanosensitive ion channels and the peptide inhibitor GsMTx-4: History, properties, mechanisms and pharmacology

Cited by 164 publications

References 134 publications

Actin stress in cell reprogramming

Actin stress in cell reprogramming

Stretch-activated ion channel Piezo1 directs lineage choice in human neural stem cells

TRPC1 channels regulate directionality of migrating cells

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