Modulation of hERG potassium currents in HEK‐293 cells by protein kinase C. Evidence for direct phosphorylation of pore forming subunits

Cockerill, Sarah; Tobin, Andrew B.; Torrecilla, Ignacio; Willars, Gary B.; Standen, NB; Mitcheson, John S.

doi:10.1113/jphysiol.2006.123414

Cited by 48 publications

(74 citation statements)

References 41 publications

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“…hERG1 currents in NIH-3T3 cells were measured at room temperature using the whole-cell configuration of the patch-clamp technique as described previously (Cockerill et al, 2007). A561V and G628S hERG1 properties and the stability and efficacy of dofetilide and terfenadine in cell-culture conditions were investigated on hERG1 currents expressed in Xenopus oocytes by two-electrode voltage clamp (Perry et al, 2004).…”

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confidence: 99%

Long-Term Channel Block Is Required to Inhibit Cellular Transformation by Human Ether-à-Go-Go–Related Gene (hERG1) Potassium Channels

et al. 2014

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Both human ether-à-go-go-related gene (hERG1) and the closely related human ether-à-go-go (hEAG1) channel are aberrantly expressed in a large proportion of human cancers. In the present study, we demonstrate that transfection of hERG1 into mouse fibroblasts is sufficient to induce many features characteristic of malignant transformation. An important finding of this work is that this transformation could be reversed by chronic incubation (for 2-3 weeks) with the hERG channel blocker dofetilide (100 nM), whereas more acute applications (for 1-2 days) were ineffective. The hERG1 expression resulted in a profound loss of cell contact inhibition, multiple layers of overgrowing cells, and high saturation densities. Cells also changed from fibroblast-like to a more spindle-shaped morphology, which was associated with a smaller cell size, a dramatic increase in cell polarization, a reduction in the number of actin stress fibers, and less punctate labeling of focal adhesions. Analysis of single-cell migration and scratch-wound closure clearly demonstrated that hERG1-expressing cells migrated more rapidly than vector-transfected control cells. In contrast to previous studies on hEAG1, there were no increases in rates of proliferation, or loss of growth factor dependency; however, hERG1-expressing cells were capable of substrateindependent growth. Allogeneic transplantation of hERG1-expressing cells into nude mice resulted in an increased incidence of tumors. In contrast to hEAG1, the mechanism of cellular transformation is dependent on ion conduction. Trafficking-deficient and conduction-deficient hERG1 mutants also prevented cellular transformation. These results provide evidence that hERG1 expression is sufficient to induce cellular transformation by a mechanism distinct from hEAG1. The most important conclusion of this study is that selective hERG1 channel blockers have therapeutic potential in the treatment of hERG1-expressing cancers.

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confidence: 99%

Long-Term Channel Block Is Required to Inhibit Cellular Transformation by Human Ether-à-Go-Go–Related Gene (hERG1) Potassium Channels

et al. 2014

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“…A physiological interpretation of the observation falls beyond the scope of this study; it is worth noting, however, that general PKC sites have long been identified in several domains of Kv11.1, where some have been shown to be pathologically relevant. 14 Our results indicated a strongest effect for PKCε modulator peptides, followed closely by the PKCβI and PKCα, whereas PKCη and βII isoforms displayed effects of much lesser amplitudes. It was previously reported that PKCε also played a significant role in the regulation of many other voltage-gated ion channels (VGIC), as in the case of the L-type Ca 2+ channels, and the sodium channels Nav1.7 or Nav1.8.…”

Section: Discussionmentioning

confidence: 54%

“…A linear correlation could be drawn between V 1/2 and τ activation, as shown in Figure 4. The linear fit to the data indicates that a coupling between these parameters is preserved despite the differential effects caused by specific PKC isoform peptides 0.7 ± 0.1 (6) 420 ± 30 (6) 322 ± 34 (5) 1 ± 2 (6) -24 ± 2 (6) βIV5-3 0.7 ± 0.1 (14) 404 ± 28 (14) 339 ± 19 (15) 4 ± 1 (14) -23 ± 1 (14) αC2-4 0.6 ± 0.1 (14) 367 ± 26 (14) 422 ± 20 (12) 9 ± 1 (14) -22 ± 1 (14) ηV1-2 0.7 ± 0.1 (7) 293 ± 39 (7) 386 ± 25 (7) 11 ± 2 (6) -22 ± 1 (6) βIV5-3 0.6 ± 0.1 (7) 306 ± 23 (7) 454 ± 52 (5) 13 ± 2 (7) -22 ± 1 (7) δV1-1 0.6 ± 0.1 (6) 339 ± 50 (6) 598 ± 42 (4) 19 ± 1 (5) -24 ± 2 (4) εV1-7 0.7 ± 0.1 (7) 293 ± 39 (7) 539 ± 39 (6) 8 ± 1(7) -24 ± 1 (7) Values of Kv11.1 properties following treatments with the protein kinase C (PKC) modulators. The table is sorted to present the maximal effect on V 1/2 at the top.…”

Section: Effect Of Pkc Modulators On the Kinetics Of Kv111 Tail Currmentioning

confidence: 96%

“…12 The long QT syndrome, a channelopathy of primarily Kv11.1 and Kv7.1, 13 can lead to the development of "Torsades de Pointes" when the cardiac action potential fails to repolarize and immediately stops the heart in tetanus, eventually causing sudden death if left uncorrected. It has long been known that PKC modulates Kv11.1, 14 although to the best of our knowledge, no data are currently available as to the roles of specific PKC isoforms and their differential implications in regulating the channel biophysics.…”

Section: Research-article2014mentioning

confidence: 99%

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Roles of PKC Isoforms in PMA-Induced Modulation of the hERG Channel (Kv11.1)

Radresa

Guia²,

Baroudi

2014

SLAS Discovery

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Protein kinases C (PKC) modulate the activity of the Kv11.1 ion channel current (hERG). However, the differential effects of specific PKC subtypes on the biophysics of the channel are unknown. The pharmaceutical tools to selectively modulate PKC subtypes are not membrane permeable and must be added directly to the intracellular solution in electrophysiology studies. Here, the PatchXpress electrophysiology robot was used to voltage clamp up to 16 cells simultaneously yet asynchronously across individual Sealchip chambers. The precision afforded by repeats of automation procedures minimized the experimental errors typical of these assays. Eight well-known PKC selective peptidomimmetics and general synthetic modulators were used to modulate the protein-protein interactions between hERG and the major PKC subtypes. We identified a specific role for the PKCε inhibitory peptidomimmetics in decreasing PKC-induced hERG τ activation (80%) and half-maximum activation voltage (90%) at steady state; a specific PKCε activator exhibited the opposite effect. Disruption of PKCβ, PKCα, and PKCη interactions also showed significant effects albeit of lower magnitudes. The effect of PKCδ inhibitor was only marginal. A significant correlation was observed between the shifts in τ activation and half-maximum voltage at steady state (R 2 = 0.85). Peak current amplitudes and time constant of deactivation remained unaffected in all conditions.

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“…This effect is dependent on consumption of PIP 2 at the membrane and direct binding of PIP 2 to HERG but occurs independently of calcium signaling or PKC activity (Bian et al, 2004;Bian & McDonald, 2007)). PKC regulation of HERG remains an active area of investigation where conclusive results await (Thomas, 2003) (Cockerill et al, 2007). For KCNQ1/KCNE1 and I Ks , Varnum et al showed that PKC stimulation decreased in I Ks due to KCNE1 phosphorylation at serine-102 (Varnum et al, 1993).…”

Section: Regulationmentioning

confidence: 99%