The Piezo2 ion channel is mechanically activated by low-threshold positive pressure.

Recent parallel studies clearly indicated that Merkel cells and the mechanosensitive piezo2 ion channel play critical roles in the light-touch somatosensation. Moreover, piezo2 was suggested to be a light-touch sensing ion channel without a role in pain sensing in mammals. However, biophysical characteristics of piezo2, such as single channel conductance and sensitivities to various mechanical stimuli, are unclear, hampering a precise understanding of its role in touch sensation. Here, we describe the biophysical properties of piezo2 in human Merkel cell carcinoma (MCC)-13 cells; piezo2 is a low-threshold, positive pressure-specific, curvature-sensitive, mechanically activated cation channel with a single channel conductance of ~28.6 pS. Application of step indentations under the whole-cell mode of the patch-clamp technique, and positive pressures ≥5 mmHg under the cell-attached mode, activated piezo2 currents in MCC-13 and human embryonic kidney 293 T cells where piezo2 was overexpressed. By contrast, application of a negative pressure failed to activate piezo2 in these cells, whereas both positive and negative pressure activated piezo1 in a similar manner. Our results are the first to demonstrate single channel recordings of piezo2. We anticipate that our findings will be a starting point for a more sophisticated understanding of piezo2 roles in light-touch sensation.

Generation of Integration-free Induced Neural Stem Cells from Mouse Fibroblasts.

The viral vector-mediated overexpression of the defined transcription factors, Brn4/Pou3f4, Sox2, Klf4, and c-Myc (BSKM), could induce the direct conversion of somatic fibroblasts into induced neural stem cells (iNSCs). However, viral vectors may be randomly integrated into the host genome thereby increasing the risk for undesired genotoxicity, mutagenesis, and tumor formation. Here we describe the generation of integration-free iNSCs from mouse fibroblasts by non-viral episomal vectors containing BSKM. The episomal vector-derived iNSCs (e-iNSCs) closely resemble control NSCs, and iNSCs generated by retrovirus (r-iNSCs) in morphology, gene expression profile, epigenetic status, and self-renewal capacity. The e-iNSCs are functionally mature, as they could differentiate into all the neuronal cell types both in vitro and in vivo Our study provides a novel concept for generating functional iNSCs using a non-viral, non-integrating, plasmid-based system that could facilitate their biomedical applicability.

Impaired Inactivation of L-Type Ca2+ Current as a Potential Mechanism for Variable Arrhythmogenic Liability of HERG K+ Channel Blocking Drugs.

The proarrhythmic effects of new drugs have been assessed by measuring rapidly activating delayed-rectifier K+ current (IKr) antagonist potency. However, recent data suggest that even drugs thought to be highly specific IKr blockers can be arrhythmogenic via a separate, time-dependent pathway such as late Na+ current augmentation. Here, we report a mechanism for a quinolone antibiotic, sparfloxacin-induced action potential duration (APD) prolongation that involves increase in late L-type Ca2+ current (ICaL) caused by a decrease in Ca2+-dependent inactivation (CDI). Acute exposure to sparfloxacin, an IKr blocker with prolongation of QT interval and torsades de pointes (TdP) produced a significant APD prolongation in rat ventricular myocytes, which lack IKr due to E4031 pretreatment. Sparfloxacin reduced peak ICaL but increased late ICaL by slowing its inactivation. In contrast, ketoconazole, an IKr blocker without prolongation of QT interval and TdP produced reduction of both peak and late ICaL, suggesting the role of increased late ICaL in arrhythmogenic effect. Further analysis showed that sparfloxacin reduced CDI. Consistently, replacement of extracellular Ca2+ with Ba2+ abolished the sparfloxacin effects on ICaL. In addition, sparfloxacin modulated ICaL in a use-dependent manner. Cardiomyocytes from adult mouse, which is lack of native IKr, demonstrated similar increase in late ICaL and afterdepolarizations. The present findings show that sparfloxacin can prolong APD by augmenting late ICaL. Thus, drugs that cause delayed ICaL inactivation and IKr blockage may have more adverse effects than those that selectively block IKr. This mechanism may explain the reason for discrepancies between clinically reported proarrhythmic effects and IKr antagonist potencies.

Hydrogen peroxide induces vasorelaxation by enhancing 4-aminopyridine-sensitive Kv currents through S-glutathionylation.

Hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor. Since opposing vasoactive effects have been reported for H2O2 depending on the vascular bed and experimental conditions, this study was performed to assess whether H2O2 acts as a vasodilator in the rat mesenteric artery and, if so, to determine the underlying mechanisms. H2O2 elicited concentration-dependent relaxation in mesenteric arteries precontracted with norepinephrine. The vasodilatory effect of H2O2 was reversed by treatment with dithiothreitol. H2O2-elicited vasodilation was significantly reduced by blocking 4-aminopyridine (4-AP)-sensitive Kv channels, but it was resistant to blockers of big-conductance Ca(2+)-activated K(+) channels and inward rectifier K(+) channels. A patch-clamp study in mesenteric arterial smooth muscle cells (MASMCs) showed that H2O2 increased Kv currents in a concentration-dependent manner. H2O2 speeded up Kv channel activation and shifted steady state activation to hyperpolarizing potentials. Similar channel activation was seen with oxidized glutathione (GSSG). The H2O2-mediated channel activation was prevented by glutathione reductase. Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester showed incorporation of glutathione (GSH) in the Kv channel proteins in the presence of H2O2. Interestingly, conditions of increased oxidative stress within MASMCs impaired the capacity of H2O2 to stimulate Kv channels. Not only was the H2O2 stimulatory effect much weaker, but the inhibitory effect of H2O2 was unmasked. These data suggest that H2O2 activates 4-AP-sensitive Kv channels, possibly through S-glutathionylation, which elicits smooth muscle relaxation in rat mesenteric arteries. Furthermore, our results support the idea that the basal redox status of MASMCs determines the response of Kv currents to H2O2.

Distinct activity of BK channel ß1-subunit in cerebral and pulmonary artery smooth muscle cells.

This study was designed to test a hypothesis that the functional activity of big-conductance, Ca(2+)-activated K(+) (BK) channels is different in cerebral and pulmonary artery smooth muscle cells (CASMCs and PASMCs). Using patch-clamp recordings, we found that the activity of whole cell and single BK channels were significantly higher in CASMCs than in PASMCs. The voltage and Ca(2+) sensitivity of BK channels were greater in CASMCs than in PASMCs. Targeted gene knockout of ß(1)-subunits significantly reduced BK currents in CASMCs but had no effect in PASMCs. Western blotting experiments revealed that BK channel α-subunit protein expression level was comparable in CASMCs and PASMCs; however, ß(1)-subunit protein expression level was higher in CASMCs than in PASMCs. Inhibition of BK channels by the specific blocker iberiotoxin enhanced norepinephrine-induced increase in intracellular calcium concentration in CASMCs but not in PASMCs. Systemic artery blood pressure was elevated in ß(1)(-/-) mice. In contrast, pulmonary artery blood pressure was normal in ß(1)(-/-) mice. These findings provide the first evidence that the activity of BK channels is higher in cerebral than in PASMCs. This heterogeneity is primarily determined by the differential ß(1)-subunit function and contributes to diverse cellular responses in these two distinct types of cells.

Serotonin depolarizes the membrane potential in rat mesenteric artery myocytes by decreasing voltage-gated K+ currents.

We hypothesized that voltage-gated K+ (Kv) currents regulate the resting membrane potential (Em), and that serotonin (5-HT) causes Em depolarization by reducing Kv currents in rat mesenteric artery smooth muscle cells (MASMCs). The resting Em was about -40 mV in the nystatin-perforated patch configuration, and the inhibition of Kv currents by 4-aminopyridine caused marked Em depolarization. The inhibition of Ca2+-activated K+ (KCa) currents had no effect on Em. 5-HT (1 microM) depolarized Em by approximately 11 mV and reduced the Kv currents to approximately 63% of the control at -20 mV. Similar 5-HT effects were observed with the conventional whole-cell configuration with a weak Ca2+ buffer in the pipette solution, but not with a strong Ca2+ buffer. In the presence of tetraethylammonium (1mM), 5-HT caused Em depolarization similar to the control condition. These results indicate that the resting Em is largely under the regulation of Kv currents in rat MASMCs, and that 5-HT depolarizes Em by reducing Kv currents in a [Ca2+]i-dependent manner.