Polycystin-1 and -2 dosage regulates pressure sensing.

Autosomal-dominant polycystic kidney disease, the most frequent monogenic cause of kidney failure, is induced by mutations in the PKD1 or PKD2 genes, encoding polycystins TRPP1 and TRPP2, respectively. Polycystins are proposed to form a flow-sensitive ion channel complex in the primary cilium of both epithelial and endothelial cells. However, how polycystins contribute to cellular mechanosensitivity remains obscure. Here, we show that TRPP2 inhibits stretch-activated ion channels (SACs). This specific effect is reversed by coexpression with TRPP1, indicating that the TRPP1/TRPP2 ratio regulates pressure sensing. Moreover, deletion of TRPP1 in smooth muscle cells reduces SAC activity and the arterial myogenic tone. Inversely, depletion of TRPP2 in TRPP1-deficient arteries rescues both SAC opening and the myogenic response. Finally, we show that TRPP2 interacts with filamin A and demonstrate that this actin crosslinking protein is critical for SAC regulation. This work uncovers a role for polycystins in regulating pressure sensing.

Sequencing of the ANKYRIN 3 gene (ANK3) encoding ankyrin G in bipolar disorder reveals a non-conservative amino acid change in a short isoform of ankyrin G.

Significant association between polymorphisms at the ANK3 gene with bipolar disorder has previously been reported and confirmed in several samples. Here we report on association between ANK3 and bipolar disorder in a new sample of 593 patients and 642 controls (UCL2) as well as the results of sequencing of the exons and flanking regions of ANK3 from bipolar patients. Single nucleotide polymorphisms (SNPs) associated with bipolar disorder in our original GWA study (UCL1) were genotyped and tested for association in the new sample. Novel SNPs found by sequencing were genotyped in both samples to test for association with bipolar disorder. None of the SNPs previously associated with bipolar disorder were associated in the UCL2 sample. One of the four SNPs associated in the UCL1 sample, rs1938526, was still significantly associated with bipolar disorder when the UCL1 and UCL2 samples were combined (P = 0.0095). The results demonstrate the impact of heterogeneity on replication of allelic associations even within well-defined ancestral populations. DNA sequencing revealed a novel low frequency (0.007) ANK3 SNP (ss469104599) which causes a non-conservative amino acid change at position 794 in the shorter isoforms of the ankyrin G protein. Protein-function analysis software predicted the amino acid change to be "probably damaging" and it could therefore be detrimental to the function of this isoform. Given that there was only a modest increase in the allele frequency of ss469104599 in cases compared to controls further association studies are needed in additional samples to establish a possible etiological role for this amino acid change.

Pkd1-inactivation in vascular smooth muscle cells and adaptation to hypertension.

Autosomal dominant polycystic kidney disease (ADPKD) is a multisystem disorder characterized by renal, hepatic and pancreatic cyst formation and cardiovascular complications. The condition is caused by mutations in the PKD1 or PKD2 gene. In mice with reduced expression of Pkd1, dissecting aneurysms with prominent media thickening have been seen. To study the effect of selective disruption of Pkd1 in vascular smooth muscle cells (SMCs), we have generated mice in which a floxed part of the Pkd1 gene was deleted by Cre under the control of the SM22 promotor (SM22-Pkd1(del/del) mice). Cre activity was confirmed by X-gal staining using lacZ expressing Cre reporter mice (R26R), and quantitative PCR indicated that in the aorta Pkd1 gene expression was strongly reduced, whereas Pkd2 levels remained unaltered. Histopathological analysis revealed cyst formation in pancreas, liver and kidneys as the result of extravascular Cre activity in pancreatic ducts, bile ducts and in the glomerular Bowman's capsule. Remarkably, we did not find any spontaneous gross structural blood vessel abnormalities in mice with somatic Pkd1 gene disruption in SMCs or simultaneous disruption of Pkd1 in SMCs and endothelial cells (ECs). Extensive isometric myographic analysis of the aorta did not reveal differences in response to KCl, acetylcholine, phenylephrin or serotonin, except for a significant increase in contractility induced by phenylephrin on arteries from 40 weeks old Pkd1(del/+) germ-line mice. However, SM22-Pkd1(del/del) mice showed significantly reduced decrease in heart rate on angiotensin II-induced hypertension. The present findings further demonstrate in vivo, that adaptation to hypertension is altered in SM22-Pkd1(del/del) mice.

TRPC1 transcript variants, inefficient nonsense-mediated decay and low up-frameshift-1 in vascular smooth muscle cells.

BACKGROUND: Transient Receptor Potential Canonical 1 (TRPC1) is a widely-expressed mammalian cationic channel with functional effects that include stimulation of cardiovascular remodelling. The initial aim of this study was to investigate variation in TRPC1-encoding gene transcripts. RESULTS: Extensive TRPC1 transcript alternative splicing was observed, with exons 2, 3 and 5-9 frequently omitted, leading to variants containing premature termination codons. Consistent with the predicted sensitivity of such variants to nonsense-mediated decay (NMD) the variants were increased by cycloheximide. However it was notable that control of the variants by NMD was prominent in human embryonic kidney 293 cells but not human vascular smooth muscle cells. The cellular difference was attributed in part to a critical protein in NMD, up-frameshift-1 (UPF1), which was found to have low abundance in the vascular cells. Rescue of UPF1 by expression of exogenous UPF1 was found to suppress vascular smooth muscle cell proliferation. CONCLUSIONS: The data suggest: (i) extensive NMD-sensitive transcripts of TRPC1; (ii) inefficient clearance of aberrant transcripts and enhanced proliferation of vascular smooth muscle cells in part because of low UPF1 expression.

Molecular basis of the mammalian pressure-sensitive ion channels: focus on vascular mechanotransduction.

Mechano-gated ion channels are implicated in a variety of neurosensory functions ranging from touch sensitivity to hearing. In the heart, rhythm disturbance subsequent to mechanical effects is also associated with the activation of stretch-sensitive ion channels. Arterial autoregulation in response to hemodynamic stimuli, a vital process required for protection against hypertension-induced injury, is similarly dependent on the activity of force-sensitive ion channels. Seminal work in prokaryotes and invertebrates, including the nematode Caenorhabditis elegans and the fruit fly drosophila, greatly helped to identify the molecular basis of volume regulation, hearing and touch sensitivity. In mammals, more recent findings have indicated that members of several structural family of ion channels, namely the transient receptor potential (TRP) channels, the amiloride-sensitive ENaC/ASIC channels and the potassium channels K2P and Kir are involved in cellular mechanotransduction. In the present review, we will focus on the molecular and functional properties of these channel subunits and will emphasize on their role in the pressure-dependent arterial myogenic constriction and the flow-mediated vasodilation.

The mechano-gated K(2P) channel TREK-1.

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K+ channels. More than 80 genes encoding the main K+ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K+ channels. Mammalian two-pore domain K+ channels (K(2P)) make up one class of K+ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K+ channels. Each K(2P) channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including "background" or "leak" K+ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K(2P) channel TREK-1 and will emphasize on the polymodal function of this "unconventional" K+ channel.

A sphingosine-1-phosphate-activated calcium channel controlling vascular smooth muscle cell motility.

In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation.

TRPC channel lipid specificity and mechanisms of lipid regulation.

TRPC channels are a subset of the transient receptor potential (TRP) proteins widely expressed in mammalian cells. They are thought to be primarily involved in determining calcium or sodium entry and have broad-ranging functions that include regulation of cell proliferation, motility and contraction. The channels do not respond to a single stimulator but rather are activated or modulated by a multiplicity of factors, potentially existing as integrators at the plasma membrane. This review considers the sensitivity of TRPCs to lipid factors, with focus on sensitivities to diacylglycerols, lysophospholipids, arachidonic acid and its metabolites, sphingosine-1-phosphate (S1P), cholesterol and derivatives, and other lipid factors such as gangliosides. Promiscuous and selective lipid-sensing are apparent. In many cases the lipids stimulate channel function or increase insertion of channels in the membrane. Both direct and indirect (receptor-dependent) lipid effects are evident. Although information is limited, the lipid profiles are consistent with TRPCs having close working relationships with phospholipase C and A2 enzymes. We need much more information about lipid-sensing by TRPCs if we are to fully appreciate its significance, but the available data suggest that lipid-sensing is a key, but not exclusive, aspect of TRPC biology.

TRP channels and mechanosensory transduction: insights into the arterial myogenic response.

Mechano-gated ion channels are implicated in a variety of key physiological functions ranging from touch sensitivity to arterial pressure regulation. Seminal work in prokaryotes and invertebrates provided strong evidence for the role of specific ion channels in volume regulation, touch sensitivity, or hearing, specifically the mechanosensitive channel subunits of large and small conductances (MscL and MscS), the mechanosensory channel subunits (MEC) and the transient receptor potential channel subunits (TRP). In mammals, recent studies further indicate that members of the TRP channel family may also be considered as possible candidate mechanosensors responding to either tension, flow, or changes in cell volume. However, contradictory results have challenged whether these TRP channels, including TRPC1 and TRPC6, are directly activated by mechanical stimulation. In the present review, we will focus on the mechanosensory function of TRP channels, discuss whether a direct or indirect mechanism is at play, and focus on the proposed role for these channels in the arterial myogenic response to changes in intraluminal pressure.

Sensing of lysophospholipids by TRPC5 calcium channel.

TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. We have revealed that one of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine (LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurred downstream and independently of G-protein signaling. The effect was not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activated TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation showed dependence on side-chain length and the chemical head-group. The data revealed a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor.