[Show abstract][Hide abstract] ABSTRACT: Despite the substantial knowledge on the antidiabetic, antiobesity and antihypertensive actions of tungstate, information on its primary target/s is scarce. Tungstate activates both the ERK1/2 pathway and the vascular voltage- and Ca2+-dependent large-conductance BKαβ1 potassium channel, which modulates vascular smooth muscle cell (VSMC) proliferation and function, respectively. Here, we have assessed the possible involvement of BKαβ1 channels in the tungstate-induced ERK phosphorylation and its relevance for VSMC proliferation. Western blot analysis in HEK cell lines showed that expression of vascular BKαβ1 channels potentiates the tungstate-induced ERK1/2 phosphorylation in a Gi/o protein-dependent manner. Tungstate activated BKαβ1 channels upstream of G proteins as channel activation was not altered by the inhibition of G proteins with GDPβS or pertussis toxin. Moreover, analysis of Gi/o protein activation measuring the FRET among heterologously expressed Gi protein subunits suggested that tungstate-targeting of BKαβ1 channels promotes G protein activation. Single channel recordings on VSMCs from wild-type and β1-knockout mice indicated that the presence of the regulatory β1 subunit was essential for the tungstate-mediated activation of BK channels in VSMCs. Moreover, the specific BK channel blocker iberiotoxin lowered tungstate-induced ERK phosphorylation by 55% and partially reverted (by 51%) the tungstate-produced reduction of platelet-derived growth factor (PDGF)-induced proliferation in human VSMCs. Our observations indicate that tungstate-targeting of BKαβ1 channels promotes activation of PTX-sensitive Gi proteins to enhance the tungstate-induced phosphorylation of ERK, and inhibits PDGF-stimulated cell proliferation in human vascular smooth muscle.
PLoS ONE 02/2015; 10(2):e0118148. DOI:10.1371/journal.pone.0118148 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser(1048) of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser(1048) hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser(1048) increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons.
[Show abstract][Hide abstract] ABSTRACT: Benign paroxysmal torticollis of infancy (BPTI) is a rare paroxysmal disorder characterized by recurrent episodes of head tilt and accompanying general symptoms which remit spontaneously. The rare association with gain-of-function CACNA1A mutations, similar to hemiplegic migraine, has been reported. We report here two new BPTI patients from the same family carrying a heterozygous mutation in the CACNA1A gene leading to the change p.Glu533Lys. Functional analysis revealed that this mutation induces a loss of channel function due to impaired gating by voltage and much lower current density. Our data suggest that BPTI, a periodic syndrome commonly considered a migraine precursor, constitutes an age-specific manifestation of defective neuronal calcium channel activity.
European journal of paediatric neurology: EJPN: official journal of the European Paediatric Neurology Society 01/2014; 14(3). DOI:10.1016/j.ejpn.2013.12.011 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing.
[Show abstract][Hide abstract] ABSTRACT: Tungstate, a compound with antidiabetic, antiobesity, and antihypertensive properties, activates the large-conductance voltage- and Ca(2+)-dependent K(+) (BK) channel containing either β1 or β4 subunits. The BK activation by tungstate is Mg(2+)-dependent and promotes arterial vasodilation, but only in precontracted mouse arteries expressing β1. In this study, we further explored how the β1 subunit participates in tungstate activation of BK channels. Activation of heterologously expressed human BKαβ1 channels in inside-out patches is fully dependent on the Mg(2+) sensitivity of the BK α channel subunit even at high (10 μM) cytosolic Ca(2+) concentration. Alanine mutagenesis of β1 extracellular residues Y74 or S104, which destabilize the active voltage sensor, greatly decreased the tungstate-induced left-shift of the BKαβ1 G-V curves in either the absence or presence of physiologically relevant cytosolic Ca(2+) levels (10 μM). The weakened tungstate activation of the BKαβ1Y74A and BKαβ1S104A mutant channels was not related to decreased Mg(2+) sensitivity. These results, together with previously published reports, support the idea that the putative binding site for tungstate-mediated BK channel activation is located in the pore-forming α channel subunit, around the Mg(2+) binding site. The role of β1 in tungstate-induced channel activation seems to rely on its interaction with the BK α subunit to modulate channel activity. Loop residues that are essential for the regulation of voltage sensor activation and gating of the BK channel are also relevant for BK activation by tungstate.
Pflügers Archiv - European Journal of Physiology 10/2013; 466(7). DOI:10.1007/s00424-013-1379-9 · 4.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca2+-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca2+ signal. ATP-induced MUC5AC secretion depended strongly on Ca2+ influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca2+ entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca2+ and by inhibition of the Na+/Ca2+ exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na+ entry to promote Ca2+ uptake via an NCX to trigger MUC5AC secretion. DOI: http://dx.doi.org/10.7554/eLife.00658.001
[Show abstract][Hide abstract] ABSTRACT: Tungstate reduces blood pressure in experimental animal models of both hypertension and metabolic syndrome, although the underlying mechanisms are not fully understood. Given that the large-conductance voltage- and Ca(2+)-dependent K(+) (BK) channel is a key element in the control of arterial tone, our aim was to evaluate whether BK channel modulation by tungstate can contribute to its antihypertensive effect.
Patch-clamp studies of heterologously expressed human BK channels (α + β(1-4) subunits) revealed that cytosolic tungstate (1 mM) induced a significant left shift (∼20 mV) in the voltage-dependent activation curve only in BK channels containing αβ(1) or αβ(4) subunits, but reduced the amplitude of K(+) currents through all BK channels tested. The β(1)-dependent activation of BK channels by tungstate was enhanced at cytosolic Ca(2+) levels reached during myocyte contraction, and prevented either by removal of cytosolic Mg(2+) or by mutations rendering the channel insensitive to Mg(2+). A lower concentration of tungstate (0.1 mM) induced voltage-dependent activation of the vascular BKαβ(1) channel without reducing current amplitude, and consistently exerted a vasodilatory action on wild-type but not on β(1)-knockout mouse arteries pre-contracted with endothelin-1.
Tungstate activates BK channels in a β subunit- and Mg(2+)-dependent manner and induces vasodilatation only in mouse arteries that express the BK β(1) subunit.
Cardiovascular Research 04/2012; 95(1):29-38. DOI:10.1093/cvr/cvs139 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T-type calcium channels represent a key pathway for Ca2+ entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold
exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here,
we report the existence of a syntaxin-1A/Cav3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein
interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Cav3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated
by syntaxin-1A, but to a smaller extent. Overexpression of Cav3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels
from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca2+ channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold
Journal of Biological Chemistry 01/2012; 287(4):2810-2818. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transient receptor potential (TRP) superfamily of non-selective cationic channels are involved in several processes plausibly relevant to migraine pathophysiology, including multimodal sensory and pain perception, central and peripheral sensitization, and regulation of calcium homeostasis. With the aim of identifying single nucleotide polymorphisms (SNPs) in TRP genes that may confer increased genetic susceptibility to migraine, we carried out a case-control genetic association study with replication, including a total of 1,040 cases and 1,037 controls. We genotyped 149 SNPs covering 14 TRP genes with known brain expression. The two-stage study comprised samples of 555 and 485 Spanish, Caucasian patients, selected according to the ICHD-II criteria for the diagnosis of migraine without aura (MO) or migraine with aura (MA). In the discovery sample, 19 SNPs in ten TRP genes showed nominal association (P < 0.05) with MO, MA, or overall migraine. In the replication sample, nominal association was confirmed for TRPV3 rs7217270 in MA and TRPV1 rs222741 in the overall migraine group. Risk haplotypes were identified for seven of the genes showing nominal association in the discovery set, but none of them was replicated. The present findings suggest that members of the vanilloid TRPV subfamily of receptors contribute to the genetic susceptibility to migraine in the Spanish population.
American Journal of Medical Genetics Part B Neuropsychiatric Genetics 01/2012; 159B(1):94-103. DOI:10.1002/ajmg.b.32007 · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.
[Show abstract][Hide abstract] ABSTRACT: Familial hemiplegic migraine (FHM)-causing mutations in the gene encoding the P/Q Ca(2+) channel alpha(1A) subunit (CACNA1A) locate to the pore and voltage sensor regions and normally involve gain-of-channel function. We now report on a mutation identified in the first intracellular loop of CACNA1A (alpha(1A(A454T))) that does not cause FHM but is associated with the absence of sensorimotor symptoms in a migraine with aura pedigree. Alpha(1A(A454T)) channels showed weakened regulation of voltage-dependent steady-state inactivation by Ca(V)beta subunits. More interestingly, A454T mutation suppressed P/Q channel modulation by syntaxin 1A or SNAP-25 and decreased exocytosis. Our findings reveal the importance of I-II loop structural integrity in the functional interaction between P/Q channel and proteins of the vesicle-docking/fusion machinery, and that genetic variation in CACNA1A may be not only a cause but also a modifier of migraine phenotype.
Proceedings of the National Academy of Sciences 01/2010; 107(4):1672-7. DOI:10.1073/pnas.0908359107 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Disorders of water balance are among the most common and morbid of the electrolyte disturbances, and are reflected clinically as abnormalities in the serum sodium concentration. The transient receptor potential vanilloid 4 (TRPV4) channel is postulated to comprise an element of the central tonicity-sensing mechanism in the mammalian hypothalamus, and is activated by hypotonic stress in vitro. A nonsynonymous polymorphism in the TRPV4 gene gives rise to a Pro-to-Ser substitution at residue 19. We show that this polymorphism is significantly associated with serum sodium concentration and with hyponatremia (serum sodium concentration < or =135 mEq/L) in 2 non-Hispanic Caucasian male populations; in addition, mean serum sodium concentration is lower among subjects with the TRPV4(P19S) allele relative to the wild-type allele. Subjects with the minor allele were 2.4-6.4 times as likely to exhibit hyponatremia as subjects without the minor allele (after inclusion of key covariates). Consistent with these observations, a human TRPV4 channel mutated to incorporate the TRPV4(P19S) polymorphism showed diminished response to hypotonic stress (relative to the wild-type channel) and to the osmotransducing lipid epoxyeicosatrienoic acid in heterologous expression studies. These data suggest that this polymorphism affects TRPV4 function in vivo and likely influences systemic water balance on a population-wide basis.
Proceedings of the National Academy of Sciences 09/2009; 106(33):14034-9. DOI:10.1073/pnas.0904084106 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Voltage-dependent potassium channels (Kv) play a crucial role in the activation and proliferation of leukocytes. Kv channels are either homo- or hetero-oligomers. This composition modulates their surface expression and serves as a mechanism for regulating channel activity. Kv channel interaction with accessory subunits provides mechanisms for channels to respond to stimuli beyond changes in membrane potential. Here, we demonstrate that KCNE4 (potassium voltage-gated channel subfamily E member 4), but not KCNE2, functions as an inhibitory Kv1.3 partner in leukocytes. Kv1.3 trafficking, targeting and activity are altered by the presence of KCNE4. KCNE4 decreases current density, slows activation, accelerates inactivation, increases cumulative inactivation, retains Kv1.3 in the ER and impairs channel targeting to lipid raft microdomains. KCNE4 associates with Kv1.3 in the ER and decreases the number of Kv1.3 channels at the cell surface, which diminishes cell excitability. Kv1.3 and KCNE4 are differentially regulated upon activation or immunosuppression in macrophages. Thus, lipopolysaccharide-induced activation increases Kv1.3 and KCNE4 mRNA, whereas dexamethasone triggers a decrease in Kv1.3 with no changes in KCNE4. The channelosome composition determines the activity and affects surface expression and membrane localization. Therefore, KCNE4 association might play a crucial role in controlling immunological responses. Our results indicate that KCNE ancillary subunits could be new targets for immunomodulation.
[Show abstract][Hide abstract] ABSTRACT: Migraine is a common neurological disorder with a complex inheritance pattern. Mutations in genes encoding proteins that are involved in ion transport across the neuronal membrane have been linked to rare monogenic variants of migraine. These or other related genes and proteins are also candidates to be involved in the inherited predisposition to the more common forms of migraine without aura (MO) or migraine with aura (MA). One of these proteins, syntaxin 1A, encoded by the STX1A gene, is a key molecule in ion channel regulation and synaptic exocytosis. We assessed the contribution of STX1A to migraine by analyzing three SNPs that cover the entire gene (rs6951030-rs941298-rs4363087), in a case-control association study in 210 migraine patients (102 MO, 86 MA, 22 hemiplegic migraine) and 210 sex-matched unrelated controls. The single-marker analysis revealed significant differences in both allele frequencies (P=0.0087, OR=1.48) and genotype distributions (P=0.0133) of the rs941298 SNP between migraineurs and controls, with an overrepresentation of T-allele carriers in the migraine sample (OR=1.78). We subsequently performed a haplotype-based analysis and observed evidence of an overrepresentation of the A-T-G (rs6951030-rs941298-rs4363087) allelic combination in migraine patients and an increased frequency of carriers of this risk haplotype (P=0.008, OR=1.71). These differences remained significant when patients were subdivided into MO and MA. When the control series was enlarged for rs941298, we confirmed the association only with the whole migraine group.
[Show abstract][Hide abstract] ABSTRACT: We report a patient with typical features of episodic ataxia type 2 (EA2) but with onset in the sixth decade and associated interictal hand dystonia. He was found to bear the novel heterozygous missense mutation p.Gly638Asp (c.1913G>A) in the CACNA1A gene. Functional analysis of the mutation on P/Q channels expressed in HEK 293 cells revealed a reduction of Ca(2+) current densities, a left-shift in the apparent reversal potential, the slowing of inactivation kinetics and the increase in the rate of current recovery from inactivation. These results are consistent with a decrease in Ca(2+) permeability through mutant P/Q channels. To our knowledge, this is just the second patient with late onset EA2 linked to a CACNA1A mutation and the first to carry a loss-of-function missense mutation.
Journal of the neurological sciences 03/2009; 280(1-2):10-4. DOI:10.1016/j.jns.2009.01.005 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in the gene encoding the pore-forming alpha(1A) subunit of P/Q Ca(2+) channels (CACNA1A) are linked to familial hemiplegic migraine. CACNA1A Y1245C is the first missense mutation described in a subject affected with childhood periodic syndromes that evolved into hemiplegic migraine. Y1245C is also the first amino acid change described in any S1 segment of CACNA1A in a hemiplegic migraine background. We found that Y1245C induced a 9-mV left shift in the current-voltage activation curve, accelerated activation kinetics, and slowed deactivation kinetics within a wide range of voltage depolarizations. Y1245C also left-shifted the voltage-dependent steady-state inactivation with a significant increase in steepness, suggesting a direct effect on the P/Q channel voltage sensor. Moreover, Y1245C reduced Gbetagamma subunits-dependent channel inhibition probably by favoring Gbetagamma dissociation from the channel; an effect also observed using action-potential-like waveforms of different durations. The formation of a new disulfide bridge between cysteines may contribute to the Y1245C effects on activation and Gbetagamma inhibition of the channel, as they were significantly reversed by the sulphydryl-reducing agent dithiothreitol. Together, our data suggest that Y1245C alters the structure of the alpha(1A) voltage sensor producing an overall gain of channel function that may explain the observed clinical phenotypes.
Pflügers Archiv - European Journal of Physiology 03/2009; 458(3):489-502. DOI:10.1007/s00424-009-0637-3 · 4.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The large conductance Ca2+ -dependent potassium channel plays a critical role in the control of vascular tone, coupling local increases in intracellular Ca2+ to membrane hyperpolarization and vascular relaxation. It also impacts blood pressure by modulating the renin-angiotensin-aldosterone system. Previous studies have shown that a polymorphism in the beta1 regulatory subunit of the Ca2+ -dependent potassium channel modulates the risk of diastolic hypertension in humans.
We have studied polymorphisms in the pore-forming alpha subunit gene (KCNMA1) and their association to hypertension and myocardial infarction.
Sequencing of the KCNMA1 gene revealed two genetic variants (polymorphisms C864T and IVS17) in population-based epidemiological studies (4786 participants). We detected a significant increase in the frequency of the IVS17+37T>C polymorphism with severe systolic hypertension (48.3% for normotensive vs. 69% for severe systolic hypertension, P=0.03) and with severe general hypertension (48.7 vs. 65.8%, P=0.04), although the adjusted odd ratios did not reach statistical significance. Four C864T/IVS17 haplotypes were identified. Haplotype 4 (encompassing the C allele of the IVS17 polymorphism and the T allele of the C864T polymorphism) was related with increased severity of systolic and general hypertension as well as increased risk of myocardial infarction.
Our study provides genetic evidence that highlights the relevance of the Ca2+ -dependent potassium channel in the control of human blood pressure and its impact on cardiovascular disease.
Journal of Hypertension 11/2008; 26(11):2147-53. DOI:10.1097/HJH.0b013e32831103d8 · 4.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium-dependent potassium channels are implicated in electrolyte transport, cell volume regulation and mechanical responses in epithelia, although the pathways for calcium entry and their coupling to the activation of potassium channels are not fully understood. We now show molecular evidence for the presence of TRPV4, a calcium permeable channel sensitive to osmotic and mechanical stress, and its functional coupling to the large conductance calcium-dependent potassium channel (BK(Ca)) in a human bronchial epithelial cell line (HBE). Reverse transcriptase polymerase chain reaction, intracellular calcium imaging and whole-cell patch-clamp experiments using HBE cells demonstrated the presence of TRPV4 messenger and Ca(2+) entry, and outwardly rectifying cationic currents elicited by the TRPV4 specific activator 4alpha-phorbol 12,13-didecanoate (4alphaPDD). Cell-attached and whole-cell patch-clamp of HBE cells exposed to 4alphaPDD, and hypotonic and high-viscosity solutions (related to mechanical stress) revealed the activation of BK(Ca) channels subsequent to extracellular Ca(2+) influx via TRPV4, an effect lost upon antisense-mediated knock-down of TRPV4. Further analysis of BK(Ca) modulation after TRPV4 activation showed that the Ca(2+) signal can be generated away from the BK(Ca) location at the plasma membrane, and it is not mediated by intracellular Ca(2+) release via ryanodine receptors. Finally, we have shown that, unlike the reported disengagement of TRPV4 and BK(Ca) in response to hypotonic solutions, cystic fibrosis bronchial epithelial cells (CFBE) preserve the functional coupling of TRPV4 and BK(Ca) in response to high-viscous solutions.
Pflügers Archiv - European Journal of Physiology 06/2008; 457(1):149-59. DOI:10.1007/s00424-008-0516-3 · 4.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mechanical and osmotic sensitivity of the transient receptor potential vanilloid 4 (TRPV4) channel depends on phospholipase A2 (PLA2) activation and the subsequent production of the arachidonic acid metabolites, epoxyeicosatrienoic acid (EET). We show that both high viscous loading and hypotonicity stimuli in native ciliated epithelial cells use PLA2-EET as the primary pathway to activate TRPV4. Under conditions of low PLA2 activation, both also use extracellular ATP-mediated activation of phospholipase C (PLC)-inositol trisphosphate (IP3) signaling to support TRPV4 gating. IP3, without being an agonist itself, sensitizes TRPV4 to EET in epithelial ciliated cells and cells heterologously expressing TRPV4, an effect inhibited by the IP3 receptor antagonist xestospongin C. Coimmunoprecipitation assays indicated a physical interaction between TRPV4 and IP3 receptor 3. Collectively, our study suggests a functional coupling between plasma membrane TRPV4 channels and intracellular store Ca2+ channels required to initiate and maintain the oscillatory Ca2+ signal triggered by high viscosity and hypotonic stimuli that do not reach a threshold level of PLA2 activation.
The Journal of General Physiology 06/2008; 131(5):i2. DOI:10.1085/JGP1315OIA2 · 4.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mechanical and osmotic sensitivity of the transient receptor potential vanilloid 4 (TRPV4) channel depends on phospholipase
A2 (PLA2) activation and the subsequent production of the arachidonic acid metabolites, epoxyeicosatrienoic acid (EET). We show that
both high viscous loading and hypotonicity stimuli in native ciliated epithelial cells use PLA2–EET as the primary pathway to activate TRPV4. Under conditions of low PLA2 activation, both also use extracellular ATP-mediated activation of phospholipase C (PLC)–inositol trisphosphate (IP3) signaling to support TRPV4 gating. IP3, without being an agonist itself, sensitizes TRPV4 to EET in epithelial ciliated cells and cells heterologously expressing
TRPV4, an effect inhibited by the IP3 receptor antagonist xestospongin C. Coimmunoprecipitation assays indicated a physical interaction between TRPV4 and IP3 receptor 3. Collectively, our study suggests a functional coupling between plasma membrane TRPV4 channels and intracellular
store Ca2+ channels required to initiate and maintain the oscillatory Ca2+ signal triggered by high viscosity and hypotonic stimuli that do not reach a threshold level of PLA2 activation.
The Journal of Cell Biology 04/2008; 181(1):143-155. DOI:10.1083/jcb.200712058 · 9.83 Impact Factor