TWIK-related two-pore domain potassium channel TREK-1 in carotid endothelium of normotensive and hypertensive mice.

AIMS: Potassium channels are essential elements of endothelial function. Recently, evidence emerged that the TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)-related K+ channel (TREK-1) of the two-pore domain potassium channel gene family (K2P) may be involved in the regulation of vascular tone. However, the functional and molecular characterization of vascular TREK-1 is incomplete. In this study, we therefore analysed the functional expression of TREK-1 in the endothelium. Moreover, we hypothesized that changes in channel expression may contribute to altered endothelial vasodilator response under conditions of elevated blood pressure. METHODS AND RESULTS: Gene expression and function of endothelial TREK-1 were analysed by single-cell RT-PCR, the patch-clamp technique and pressure myography in murine carotid arteries (CA). K+ outward currents displaying the characteristics of TREK-1 were observed following various TREK-1-activating stimuli such as membrane stretch, intracellular acidosis, polyunsaturated fatty acids, isoflurane (ISOFL), riluzole, and acetylcholine (ACh). In K(Ca)3.1(-/-) mice exhibiting elevated blood pressure, endothelial TREK-1 currents and TREK-1 mRNA expression were enhanced as compared with normotensive control mice. TREK-1-mediated vasodilator responses to alpha-linolenic acid, ISOFL, or ACh were increased. A similar up-regulation of endothelial TREK-1 was observed in spontaneously hypertensive rats. CONCLUSION: We have found that TREK-1 is an endothelial K+ channel capable of producing hyperpolarization and vasodilation. A correlation between hypertension and up-regulation of TREK-1 was observed in two different animal models of elevated blood pressure. Thus, TREK-1 may play a protective role in the cardiovascular system by providing a novel type of endothelial hyperpolarization-mediated vasodilator response.

Arterial response to shear stress critically depends on endothelial TRPV4 expression.

BACKGROUND: In blood vessels, the endothelium is a crucial signal transduction interface in control of vascular tone and blood pressure to ensure energy and oxygen supply according to the organs' needs. In response to vasoactive factors and to shear stress elicited by blood flow, the endothelium secretes vasodilating or vasocontracting autacoids, which adjust the contractile state of the smooth muscle. In endothelial sensing of shear stress, the osmo- and mechanosensitive Ca(2+)-permeable TRPV4 channel has been proposed to be candidate mechanosensor. Using TRPV4(-/-) mice, we now investigated whether the absence of endothelial TRPV4 alters shear-stress-induced arterial vasodilation. METHODOLOGY/PRINCIPAL FINDINGS: In TRPV4(-/-) mice, loss of the TRPV4 protein was confirmed by Western blot, immunohistochemistry and by in situ-patch-clamp techniques in carotid artery endothelial cells (CAEC). Endothelium-dependent vasodilation was determined by pressure myography in carotid arteries (CA) from TRPV4(-/-) mice and wild-type littermates (WT). In WT CAEC, TRPV4 currents could be elicited by TRPV4 activators 4alpha-phorbol-12,13-didecanoate (4alphaPDD), arachidonic acid (AA), and by hypotonic cell swelling (HTS). In striking contrast, in TRPV4(-/-) mice, 4alphaPDD did not produce currents and currents elicited by AA and HTS were significantly reduced. 4alphaPDD caused a robust and endothelium-dependent vasodilation in WT mice, again conspicuously absent in TRPV4(-/-) mice. Shear stress-induced vasodilation could readily be evoked in WT, but was completely eliminated in TRPV4(-/-) mice. In addition, flow/reperfusion-induced vasodilation was significantly reduced in TRPV4(-/-) vs. WT mice. Vasodilation in response to acetylcholine, vasoconstriction in response to phenylephrine, and passive mechanical compliance did not differ between genotypes, greatly underscoring the specificity of the above trpv4-dependent phenotype for physiologically relevant shear stress. CONCLUSIONS/SIGNIFICANCE: Genetically encoded loss-of-function of trpv4 results in a loss of shear stress-induced vasodilation, a response pattern critically dependent on endothelial TRPV4 expression. Thus, Ca(2+)-influx through endothelial TRPV4 channels is a molecular mechanism contributing significantly to endothelial mechanotransduction.

Impaired endothelium-derived hyperpolarizing factor-mediated dilations and increased blood pressure in mice deficient of the intermediate-conductance Ca2+-activated K+ channel.

The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca(2+)-activated K(+) channels (K(Ca)), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance K(Ca) (IK(Ca)/K(Ca)3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of K(Ca)3.1. K(Ca)3.1-deficient mice (K(Ca)3.1(-/-)) were generated by conventional gene-targeting strategies. Endothelial K(Ca) currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the K(Ca)3.1 gene abolished endothelial K(Ca)3.1 currents and significantly diminished overall current through K(Ca) channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in K(Ca)3.1(-/-) mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in K(Ca)3.1(-/-) mice. Moreover, the loss of K(Ca)3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial K(Ca)3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.

Mitogenic modulation of Ca2+ -activated K+ channels in proliferating A7r5 vascular smooth muscle cells.

Modulation of Ca(2+)-activated K(+) channels (K(Ca)) has been implicated in the control of proliferation in vascular smooth muscle cells (VSMC) and other cell types. In the present study, we investigated the underlying signal transduction mechanisms leading to mitogen-induced alterations in the expression pattern of intermediate-conductance K(Ca) in VSMC. Regulation of expression of IK(Ca)/rK(Ca)3.1 and BK(Ca)/rK(Ca)1.1 in A7r5 cells, a cell line derived from rat aortic VSMC, was investigated by patch-clamp technique, quantitative RT-PCR, immunoblotting procedures, and siRNA strategy.PDGF stimulation for 2 and 48 h induced an 11- and 3.5-fold increase in rK(Ca)3.1 transcript levels resulting in a four- and seven-fold increase in IK(Ca) currents after 4 and 48 h, respectively. Upregulation of rK(Ca)3.1 transcript levels and channel function required phosphorylation of extracellular signal-regulated kinases (ERK1/2) and Ca(2+) mobilization, but not activation of p38-MAP kinase, c-Jun NH(2)-terminal kinase, protein kinase C, calcium-calmodulin kinase II and Src kinases. In contrast to rK(Ca)3.1, mRNA expression and functions of BK(Ca)/rK(Ca)1.1 were decreased by half following mitogenic stimulation. Downregulation of rK(Ca)1.1 did not require ERK1/2 phosphorylation or Ca(2+) mobilization. In an in vitro-proliferation assay, knockdown of rK(Ca)3.1 expression by siRNA completely abolished functional IK(Ca) channels and mitogenesis. Mitogen-induced upregulation of rK(Ca)3.1 expression is mediated via activation of the Raf/MEK- and ERK-signaling cascade in a Ca(2+)-dependent manner. Upregulation of rK(Ca)3.1 promotes VSMC proliferation and may thus represent a pharmacological target in cardiovascular disease states characterized by abnormal cell proliferation.

Evidence for a functional role of endothelial transient receptor potential V4 in shear stress-induced vasodilatation.

OBJECTIVE: Ca2+-influx through transient receptor potential (TRP) channels was proposed to be important in endothelial function, although the precise role of specific TRP channels is unknown. Here, we investigated the role of the putatively mechanosensitive TRPV4 channel in the mechanisms of endothelium-dependent vasodilatation. METHODS AND RESULTS: Expression and function of TRPV4 was investigated in rat carotid artery endothelial cells (RCAECs) by using in situ patch-clamp techniques, single-cell RT-PCR, Ca2+ measurements, and pressure myography in carotid artery (CA) and Arteria gracilis. In RCAECs in situ, TRPV4 currents were activated by the selective TRPV4 opener 4alpha-phorbol-12,13-didecanoate (4alphaPDD), arachidonic acid, moderate warmth, and mechanically by hypotonic cell swelling. Single-cell RT-PCR in endothelial cells demonstrated mRNA expression of TRPV4. In FURA-2 Ca2+ measurements, 4alphaPDD increased [Ca2+]i by &140 nmol/L above basal levels. In pressure myograph experiments in CAs and A gracilis, 4alphaPDD caused robust endothelium-dependent and strictly endothelium-dependent vasodilatations by &80% (K(D) 0.3 microL), which were suppressed by the TRPV4 blocker ruthenium red (RuR). Shear stress-induced vasodilatation was similarly blocked by RuR and also by the phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3). 4alphaPDD produced endothelium-derived hyperpolarizing factor (EDHF)-type responses in A gracilis but not in rat carotid artery. Shear stress did not produce EDHF-type vasodilatation in either vessel type. CONCLUSIONS: Ca2+ entry through endothelial TRPV4 channels triggers NO- and EDHF-dependent vasodilatation. Moreover, TRPV4 appears to be mechanistically important in endothelial mechanosensing of shear stress.

Constitutive expression of yeast phospholipid biosynthetic genes by variants of Ino2 activator defective for interaction with Opi1 repressor.

Regulated expression of structural genes involved in yeast phospholipid biosynthesis is mediated by inositol/choline-responsive element (ICRE) upstream motifs, bound by the heterodimeric activator complex Ino2 + Ino4. Gene repression occurs in the presence of sufficient inositol and choline, requiring an intact Opi1 repressor which binds to Ino2. For a better understanding of interactions among regulators, we mapped an 18 aa repressor interaction domain (RID, aa 118-135) within Ino2 necessary and sufficient for binding by Opi1. By alanine scanning mutagenesis of the entire RID we were able to identify nine residues critical for Opi1-dependent repression of Ino2 function. Consequently, the corresponding dominant Ino2 variants conferred constitutive expression of an ICRE-dependent reporter gene and were no longer inhibited even by overproduction of Opi1. Interestingly, Ino2 RID partially overlaps with transcriptional activation domain TAD2. As certain mutations exclusively affect repression while others affect both repression and activation, both functions of Ino2 can be functionally uncoupled. Correspondingly, we mapped the RID-binding activator interaction domain (AID, aa 321-380) at the C-terminus of Opi1 and introduced missense mutations at selected positions. An Opi1 variant simultaneously mutated at three highly conserved positions showed complete loss of repressor function, confirming RID-AID interaction as the crucial step of regulated expression of ICRE-dependent genes.

Negative regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by a Candida albicans orthologue of OPI1.

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are coordinately regulated by a UAS element, designated ICRE (inositol/choline-responsive element). Opi1 is a negative regulator responsible for repression of ICRE-dependent genes in the presence of an excess of inositol and choline. Gene regulation by phospholipid precursors has been also reported for the pathogenic yeast Candida albicans. Screening of a data base containing raw sequences of the C. albicans genome project allowed us to identify an open reading frame exhibiting weak similarity to Opi1. Expression of the putative CaOPI1 in an opi1 mutant of S. cerevisiae could restore repression of an ICRE-dependent reporter gene. Similar to OPI1, overexpression of CaOPI1 strongly inhibited derepression of ICRE-driven genes leading to inositol-requiring transformants. Previous work has shown that Opi1 mediates gene repression by interaction with the pleiotropic repressor Sin3. The genome of C. albicans also encodes a protein similar to Sin3 (CaSin3). By two-hybrid analyses and in vitro studies for protein-protein interaction we were able to show that CaOpi1 binds to ScSin3. ScOpi1 could also interact with CaSin3, while CaOpi1 failed to bind to CaSin3. Despite of some conservation of regulatory mechanisms between both yeasts, these results suggest that repression of phospholipid biosynthetic genes in C. albicans is mediated by a mechanism which does not involve recruitment of CaSin3 by CaOpi1.

TFIIB and subunits of the SAGA complex are involved in transcriptional activation of phospholipid biosynthetic genes by the regulatory protein Ino2 in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, genes involved in phospholipid biosynthesis are activated by ICRE (inositol/choline-responsive element) up-stream motifs and the corresponding heterodimeric binding factor, Ino2 + Ino4. Both Ino2 and Ino4 contain basic helix-loop-helix (bHLH) domains required for ICRE binding, whereas transcriptional activation is mediated exclusively by Ino2. In this work, we describe a molecular analysis of functional minimal domains responsible for specific DNA recognition and transcriptional activation (TAD1 and TAD2). We also define the importance of individual amino acids within the more important activation domain TAD1. Random mutagenesis at five amino acid positions showed the importance of acidic as well as hydrophobic residues within this minimal TAD. We also investigated the contribution of known general transcription factors and co-activators for Ino2-dependent gene activation. Although an ada5 single mutant and a gal11 paf1 double mutant were severely affected, a partial reduction in activation was found for gcn5 and srb2. Ino2 interacts physically with the basal transcription factor Sua7 (TFIIB of yeast). Interestingly, interaction is mediated by the HLH dimerization domain of Ino2 and by two non-overlapping domains within Sua7. Thus, Sua7 may compete with Ino4 for binding to the Ino2 activator, creating the possibility of positive and negative influence of Sua7 on ICRE-dependent gene expression.