Simultaneous cardiac and respiratory inhibition during seizure precedes death in the DBA/1 audiogenic mouse model of SUDEP.

This study was designed to evaluate cardiac and respiratory dysfunction in a mouse model of sudden unexpected death in epilepsy i.e., SUDEP. We simultaneously monitored respiration via plethysmography and the electrocardiogram via telemetry before, during, and after an audiogenic seizure. DBA/1 mice responded to an acoustic stimulus with one or two cycles of circling and jumping before entering a clonic/tonic seizure. This was followed by death unless the mice were resuscitated by mechanical ventilation using room air. During the initial clonic phase, respiration declined and cardiac rhythm is slowed. By the tonic phase, respiration had ceased, atrial P-waves were absent or dissociated from the QRS complex, and heart rate had decreased from 771±11 to 252±16 bpm. Heart rate further deteriorated terminating in asystole unless the mice were resuscitated at the end of the tonic phase which resulted in abrupt recovery of P-waves and a return to normal sinus rhythm, associated with gasping. Interestingly, P-waves were preserved in the mice treated with methylatropine during the pre-ictal period (to block parasympathetic stimulation) and heart rate remained unchanged through the end of the tonic phase (765±8 vs. 748±21 bpm), but as in control, methylatropine treated mice died from respiratory arrest. These results demonstrate that a clonic/tonic seizure in the DBA/1 mouse results in abrupt and simultaneous respiratory and cardiac depression. Although death clearly results from respiratory arrest, our results suggest that seizure activates two central nervous system pathways in this model-one inhibits respiratory drive, whereas the other inhibits cardiac function via vagal efferents. The abrupt and simultaneous recovery of both respiration and cardiac function with mechanical ventilation within an early post-ictal timeframe shows that the vagal discharge can be rapidly terminated. Understanding the central mechanism associated with the abrupt cardiorespiratory dysfunction and equally abrupt recovery may provide clues for therapeutic targets for SUDEP.

Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2).

Albuminuria and tubular atrophy are among the highest risks for CKD progression to ESRD. A parsimonious mechanism involves leakage of albumin-bound nonesterified fatty acids (NEFAs) across the damaged glomerular filtration barrier and subsequent reabsorption by the downstream proximal tubule, causing lipoapoptosis. We sought to identify the apical proximal tubule transporter that mediates NEFA uptake and cytotoxicity. We observed transporter-mediated uptake of fluorescently labeled NEFA in cultured proximal tubule cells and microperfused rat proximal tubules, with greater uptake from the apical surface than from the basolateral surface. Protein and mRNA expression analyses revealed that kidney proximal tubules express transmembrane fatty acid transporter-2 (FATP2), encoded by Slc27a2, but not the other candidate transporters CD36 and free fatty acid receptor 1. Kidney FATP2 localized exclusively to proximal tubule epithelial cells along the apical but not the basolateral membrane. Treatment of mice with lipidated albumin to induce proteinuria caused a decrease in the proportion of tubular epithelial cells and an increase in the proportion of interstitial space in kidneys from wild-type but not Slc27a2-/- mice. Ex vivo microperfusion and in vitro experiments with NEFA-bound albumin at concentrations that mimic apical proximal tubule exposure during glomerular injury revealed significantly reduced NEFA uptake and palmitate-induced apoptosis in microperfused Slc27a2-/- proximal tubules and Slc27a2-/- or FATP2 shRNA-treated proximal tubule cell lines compared with wild-type or scrambled oligonucleotide-treated cells, respectively. We conclude that FATP2 is a major apical proximal tubule NEFA transporter that regulates lipoapoptosis and may be an amenable target for the prevention of CKD progression.

TRPV4 mediates myofibroblast differentiation and pulmonary fibrosis in mice.

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disorder with no effective medical treatments available. The generation of myofibroblasts, which are critical for fibrogenesis, requires both a mechanical signal and activated TGF-ß; however, it is not clear how fibroblasts sense and transmit the mechanical signal(s) that promote differentiation into myofibroblasts. As transient receptor potential vanilloid 4 (TRPV4) channels are activated in response to changes in plasma membrane stretch/matrix stiffness, we investigated whether TRPV4 contributes to generation of myofibroblasts and/or experimental lung fibrosis. We determined that TRPV4 activity is upregulated in lung fibroblasts derived from patients with IPF. Moreover, TRPV4-deficient mice were protected from fibrosis. Furthermore, genetic ablation or pharmacological inhibition of TRPV4 function abrogated myofibroblast differentiation, which was restored by TRPV4 reintroduction. TRPV4 channel activity was elevated when cells were plated on matrices of increasing stiffness or on fibrotic lung tissue, and matrix stiffness-dependent myofibroblast differentiation was reduced in response to TRVP4 inhibition. TRPV4 activity modulated TGF-ß1-dependent actions in a SMAD-independent manner, enhanced actomyosin remodeling, and increased nuclear translocation of the α-SMA transcription coactivator (MRTF-A). Together, these data indicate that TRPV4 activity mediates pulmonary fibrogenesis and suggest that manipulation of TRPV4 channel activity has potential as a therapeutic approach for fibrotic diseases.

Lipotoxic disruption of NHE1 interaction with PI(4,5)P2 expedites proximal tubule apoptosis.

Chronic kidney disease progression can be predicted based on the degree of tubular atrophy, which is the result of proximal tubule apoptosis. The Na+/H+ exchanger NHE1 regulates proximal tubule cell survival through interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], but pathophysiologic triggers for NHE1 inactivation are unknown. Because glomerular injury permits proximal tubule luminal exposure and reabsorption of fatty acid/albumin complexes, we hypothesized that accumulation of amphipathic, long-chain acyl-CoA (LC-CoA) metabolites stimulates lipoapoptosis by competing with the structurally similar PI(4,5)P2 for NHE1 binding. Kidneys from mouse models of progressive, albuminuric kidney disease exhibited increased fatty acids, LC-CoAs, and caspase-2-dependent proximal tubule lipoapoptosis. LC-CoAs and the cytosolic domain of NHE1 directly interacted, with an affinity comparable to that of the PI(4,5)P2-NHE1 interaction, and competing LC-CoAs disrupted binding of the NHE1 cytosolic tail to PI(4,5)P2. Inhibition of LC-CoA catabolism reduced NHE1 activity and enhanced apoptosis, whereas inhibition of proximal tubule LC-CoA generation preserved NHE1 activity and protected against apoptosis. Our data indicate that albuminuria/lipiduria enhances lipotoxin delivery to the proximal tubule and accumulation of LC-CoAs contributes to tubular atrophy by severing the NHE1-PI(4,5)P2 interaction, thereby lowering the apoptotic threshold. Furthermore, these data suggest that NHE1 functions as a metabolic sensor for lipotoxicity.

Protein S-glutathionylation enhances Ca2+-induced Ca2+ release via the IP3 receptor in cultured aortic endothelial cells.

In non-excitable cells, thiol-oxidizing agents have been shown to evoke oscillations in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by increasing the sensitivity of the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) to IP(3). Although thiol modification of the IP(3)R is implicated in this response, the molecular nature of the modification(s) responsible for changes in channel activity is still not well understood. Diamide is a chemical oxidant that selectively converts reduced glutathione (GSH) to its disulfide (GSSG) and promotes the formation of protein­glutathione (P-SSG) mixed disulfide, i.e. glutathionylation. In the present study, we examined the effect of diamide, and the model oxidant hydrogen peroxide (H(2)O(2)), on oscillations in [Ca(2+)](i) in fura-2-loaded bovine (BAECs) and human (HAECs) aortic endo-thelial cells using time-lapse fluorescence video microscopy. In the absence of extracellular Ca(2+), acute treatment with either diamide or H(2)O(2) increased the number of BAECs exhibiting asynchronous Ca(2+) oscillations, whereas HAECs were unexpectedly resistant. Diamide pretreatment increased the sensitivity of HAECs to histamine-stimulated Ca(2+) oscillations and BAECs to bradykinin-stimulated Ca(2+) oscillations. Moreover, in both HAECs and BAECs, diamide dramatically increased both the rate and magnitude of the thapsigargin-induced Ca(2+) transient suggesting that Ca(2+)-induced Ca(2+) release (CICR) via the IP(3)R is enhanced by glutathionylation. Similar to diamide, H(2)O(2) increased the sensitivity of HAECs to both histamine and thapsigargin. Lastly, biochemical studies showed that glutathionylation of native IP(3)R(1) is increased in cells challenged with H(2)O(2). Collectively our results reveal that thiol-oxidizing agents primarily increase the sensitivity of the IP(3)R to Ca(2+), i.e. enhanced CICR, and suggest that glutathionylation may represent a fundamental mechanism for regulating IP(3)R activity during physiological redox signalling and during pathologicalical oxidative stress.

Phosphoinositide binding differentially regulates NHE1 Na+/H+ exchanger-dependent proximal tubule cell survival.

Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2).

Effect of protein S-glutathionylation on Ca2+ homeostasis in cultured aortic endothelial cells.

Diamide is a membrane-permeable, thiol-oxidizing agent that rapidly and reversibly oxidizes glutathione to GSSG and promotes formation of protein-glutathione mixed disulfides. In the present study, the acute effect of diamide on free cytosolic Ca2+ concentration ([Ca2+]i) was examined in fura-2-loaded bovine aortic endothelial cells. At low concentrations (50, 100 µM), diamide reversibly increased spontaneous, asynchronous Ca2+ oscillations, whereas, at higher concentrations (250, 500 µM), diamide caused an immediate synchronized Ca2+ oscillation in essentially all cells of the monolayer, followed by a time-dependent rise in basal [Ca2+]i. The effects of diamide on [Ca2+]i dynamics were independent of extracellular Ca2+. Inhibition of phospholipase C by U-73122 prevented the observed changes in [Ca2+]i. Additionally, the diamide-induced oscillations, but not the rise in basal [Ca2+]i, were blocked by inhibition of the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) by 2-aminoethyl diphenyl borate. However, diamide failed to alter the plasmalemmal distribution of a green fluorescent protein-tagged phosphatidylinositol-4,5-bisphosphate binding protein, demonstrating that diamide does not activate phospholipase C. Inhibition of glutathione reductase by N,N'-bis(2-chloroethyl)-N-nitrosourea or depletion of glutathione by l-buthionine-sulfoximine enhanced the effects of diamide, which, under these conditions, could only be reversed by addition of dithiothreitol to the wash buffer. Biochemical assays showed that both the IP3R and the plasmalemmal Ca2+-ATPase pump could be reversibly glutathionylated in response to diamide. These results demonstrate that diamide promotes Ca2+ release from IP3-sensitive internal Ca2+ stores and elevates basal [Ca2+]i in the absence of extracellular Ca2+, effects that may be related to a diamide-induced glutathionylation of the IP3R and the plasmalemmal Ca2+-ATPase Ca2+ pump, respectively.

Role of TRPC3 channels in ATP-induced Ca2+ signaling in principal cells of the inner medullary collecting duct.

The transient receptor potential channel TRPC3 is exclusively expressed in the apical membrane of principal cells of the collecting duct (CD) both in vivo and in the mouse CD cell line IMCD-3. Previous studies revealed that ATP-induced apical-to-basolateral transepithelial Ca(2+) flux across IMCD-3 monolayers is increased by overexpression of TRPC3 and attenuated by a dominant negative TRPC3 construct, suggesting that Ca(2+) entry across the apical membrane occurs via TRPC3 channels. To test this hypothesis, we selectively measured the Ca(2+) permeability of the apical membrane of fura-2-loaded IMCD-3 cells using the Mn(2+) quench technique. Mn(2+) influx across the apical membrane was increased 12- to 16-fold by apical ATP and was blocked by the pyrazole derivative BTP2, a known inhibitor of TRPC3 channels, with an IC(50) value <100 nM. In contrast, Mn(2+) influx was only increased approximately 2-fold by basolateral ATP. Mn(2+) influx was also activated by apical, but not basolateral, 1-stearoyl-2-acetyl-sn-glycerol (SAG), a known activator of TRPC3 channels. Apical ATP- and SAG-induced Mn(2+) influx was increased by overexpression of TRPC3 and completely blocked by expression of the dominant negative TRPC3 construct. Mn(2+) influx was also stimulated approximately 2-fold by thapsigargin applied to either the apical or basolateral side. Thapsigargin-induced flux was blocked by BTP2 but was unaffected by overexpression of TRPC3 or by dominant negative TRPC3. Apical ATP, but not basolateral ATP, increased transepithelial (45)Ca(2+) flux. These results demonstrate that the apical membrane of IMCD-3 cells has two distinct Ca(2+) influx pathways: 1) a store-operated channel activated by thapsigargin and basolateral ATP and 2) TRPC3 channels activated by apical ATP. Only activation of TRPC3 leads to net transepithelial apical-to-basolateral Ca(2+) flux. Furthermore, these results demonstrate that native TRPC3 is not a store-operated channel in IMCD-3 cells.

Role of cAMP/PKA signaling cascade in vasopressin-induced trafficking of TRPC3 channels in principal cells of the collecting duct.

Transient receptor potential channels TRPC3 and TRPC6 are expressed in principal cells of the collecting duct (CD) along with the water channel aquaporin-2 (AQP2) both in vivo and in the cultured mouse CD cell line IMCD-3. The channels are primarily localized to intracellular vesicles, but upon stimulation with the antidiuretic hormone arginine vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined using immunohistochemical techniques and by biotinylation of surface membrane proteins. Both in vivo in rat kidney and in IMCD-3 cells, translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino-Cys(1), d-Arg(8)]-vasopressin (dDAVP), a specific V2-receptor agonist, and blocked by [adamantaneacetyl(1), O-Et-d-Tyr(2), Val(4), aminobutyryl(6), Arg(8,9)]-vasopressin (AEAVP), a specific V2-receptor antagonist. In IMCD-3 cells, translocation of TRPC3 and AQP2 was activated by forskolin, a direct activator of AC, or by dibutyryl-cAMP, a membrane-permeable cAMP analog. AVP-, dDAVP-, and forskolin-induced translocation in IMCD-3 cells was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by dibutyryl-cAMP was unaffected by AEAVP but could be blocked by H89. AVP- and forskolin-induced translocation of TRPC3 in IMCD-3 cells was also blocked by two additional inhibitors of PKA, specifically Rp-cAMPS and the myristoylated inhibitor of PKA (m-PKI). Quantification of TRPC3 membrane insertion in IMCD-3 cells under each assay condition using a surface membrane biotinylation assay, confirmed the translocation results observed by immunofluorescence. Importantly, AVP-induced translocation of TRPC3 as estimated by biotinylation was blocked on average 95.2 +/- 1.0% by H89, Rp-cAMPS, or m-PKI. Taken together, these results demonstrate that AVP stimulation of V2 receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade.

Maitotoxin converts the plasmalemmal Ca(2+) pump into a Ca(2+)-permeable nonselective cation channel.

Maitotoxin (MTX) activates Ca(2+)-permeable nonselective cation channels and causes a dramatic increase in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in every cell examined to date, but the molecular identity of the channels involved remains unknown. A clue came from studies of a structurally related marine toxin called palytoxin (PTX). PTX binds to the plasmalemmal Na(+)-K(+)-ATPase (NKA) and converts the Na(+) pump into a nonselective cation channel. Given the high permeability of the MTX channel for Ca(2+), we considered the possibility that MTX may bind to the plasmalemmal Ca(2+)-ATPase (PMCA) pump, and like PTX, convert the pump into a channel. To test this hypothesis, the PMCA was overexpressed in Spodoptera frugiperda (Sf9) insect cells and in human embryonic kidneys (HEK) 293 cells. In both cell types, enhanced expression of the PMCA was associated with a significant increase in MTX-induced whole cell membrane currents. The effect of MTX on whole cell currents in both wild-type and PMCA overexpressing HEK cells was sensitive to pump ligands including Ca(2+) and ATP. MTX-induced currents were significantly reduced by knockdown of PMCA1 in HEK cells using small interfering RNA or in mouse embryonic fibroblasts from genetically modified mice with the PMCA1(+/-) PMCA4(-/-) genotype. Finally, PMCA catalytic activity (i.e., Ca(2+)-ATPase) in isolated membranes, or in purified PMCA preparations, was inhibited by MTX. Together, these results suggest that MTX binds to and converts the PMCA pump into a Ca(2+)-permeable nonselective cation channel.

Differential regulation of P2X7 receptor activation by extracellular nicotinamide adenine dinucleotide and ecto-ADP-ribosyltransferases in murine macrophages and T cells.

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X(7) purinergic receptor (P2X(7)R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X(7)R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X(7)R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X(7)R function in murine macrophages. Coexpression of the cloned murine P2X(7)R with ART2.1 or ART2.2 in HEK293 cells verified that P2X(7)R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X(7)R. Rather, NAD potentiated ATP-dependent P2X(7)R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X(7)R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X(7)R channel opening in T cells, this P2X(7)R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X(7)R signaling in murine macrophages and also suggest that the cellular context in which P2X(7)R signaling occurs differs between myeloid and lymphoid leukocytes.

Vasopressin-induced membrane trafficking of TRPC3 and AQP2 channels in cells of the rat renal collecting duct.

The canonical transient receptor potential channels TRPC3 and TRPC6 are abundantly expressed along with the water channel aquaporin-2 (AQP2) in principal cells of the cortical and medullary collecting duct. Although TRPC3 is selectively localized to the apical membrane and TRPC6 is found in both the apical and basolateral domains, immunofluorescence is often observed in the cytoplasm, suggesting that TRPC3 and TRPC6 may exist in intracellular vesicles and may shuttle to and from the membrane in response to receptor stimulation. To test this hypothesis, the effect of arginine-vasopressin (AVP) on the subcellular distribution of TRPC3, TRPC6, and AQP2 was examined in the rat kidney and in cultured cell lines from the cortical (M1) and inner medullary (IMCD-3) collecting duct. Immunofluorescence analysis revealed that TRPC3, but not TRPC6, colocalized with AQP2 in intracellular vesicles. AVP caused the insertion and accumulation of TRPC3 and AQP2 in the apical membrane but had no effect on the subcellular distribution of TRPC6. TRPC3, but not TRPC6, coimmunoprecipitated with AQP2 from both medulla and M1 and IMCD-3 cell lysates. Apical-to-basolateral transepithelial 45Ca2+ flux in polarized IMCD-3 cell monolayers was stimulated by diacylglycerol analogs or by the purinergic receptor agonist ATP but not by thapsigargin. Stimulated 45Ca2+ flux was increased by overexpression of TRPC3 and attenuated by a dominant-negative TRPC3 construct. Furthermore, 45Ca2+ flux was greatly reduced by the pyrazole-derivative BTP2, a known inhibitor of TRPC3 channels. These results demonstrate that 1) TRPC3 and TRPC6 exist in different vesicle populations, 2) TRPC3 physically associates with APQ2 and shuttles to the apical membrane in response to AVP, and 3) TRPC3 is responsible for transepithelial Ca2+ flux in principal cells of the renal collecting duct.

TRPC3 channels colocalize with Na+/Ca2+ exchanger and Na+ pump in axial component of transverse-axial tubular system of rat ventricle.

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable, nonselective cation channels activated after stimulation of G protein-coupled membrane receptors linked to phospholipase C (PLC). Although the PLC/inositol phosphate signaling pathway is known to exist in heart, expression and subcellular distribution of TRPC channel proteins in ventricular myocardium have not been evaluated. Of the six members of the TRPC channel family examined here, only TRPC3 was found by Western blot analysis of membrane proteins from rodent or canine ventricle. Likewise, only TRPC3 was observed in immunofluorescence analysis of thin sections from rat ventricle. TRPC3 was also the only family member observed in neonatal rat ventricular myocytes in culture. In longitudinal sections of rat ventricle, TRPC3 was predominantly localized to the intercalated disk region of the myocyte. However, transverse sections through heart muscle or single isolated adult myocytes revealed TRPC3-specific labeling in a vast network of intracellular membranes, where it colocalized with the Na(+)-K(+)-ATPase (NKA) pump and the Na(+)/Ca(2+) exchanger (NCX) but not with the ryanodine receptor or the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Reciprocal immunoprecipitation assays from rat or canine ventricle showed that TRPC3 associates with NKA and NCX but not with the plasmalemmal Ca(2+)-ATPase pump. Immunoprecipitations from Sf9 insect cells heterologously expressing TRPC3, NKA, and NCX in various combinations revealed that NKA and NCX interact and that TRPC3 and NCX interact, but that TRPC3 does not directly associate with NKA. Together, these results suggest that TRPC3 is localized in the ventricular myocyte to the axial component of the transverse-axial tubular system, where it exists in a signaling complex that includes NCX and NKA.

Palytoxin-induced cell death cascade in bovine aortic endothelial cells.

The plasmalemmal Na(+)-K(+)-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca(2+). However, the ability of PTX to directly increase cytosolic free Ca(2+) concentration ([Ca(2+)](i)) via Na(+) pump channels and to initiate Ca(2+) overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca(2+)](i) and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3-100 nM) produced a graded increase in [Ca(2+)](i) that was dependent on extracellular Ca(2+). The increase in [Ca(2+)](i) initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC(50) < 1 microM. The elevation in [Ca(2+)](i) could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca(2+)](i) occurs via the Na(+) pump. Subsequent to the rise in [Ca(2+)](i), PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca(2+) or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca(2+) overload.

Human TRPC6 expressed in HEK 293 cells forms non-selective cation channels with limited Ca2+ permeability.

TRPC6 is thought to be a Ca(2+)-permeable cation channel activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C (PLC). TRPC6 current is also activated by exogenous application of 1-oleoyl-acetyl-sn-glycerol (OAG) or by inhibiting 1,2-diacylglycerol (DAG) lipase activity using RHC80267. In the present study, both OAG and RHC80267 increased whole-cell TRPC6 current in cells from a human embryonic kidney cell line (HEK 293) stably expressing TRPC6, but neither compound increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) when the cells were bathed in high-K(+) buffer to hold the membrane potential near 0 mV. These results suggested that TRPC6 channels have limited Ca(2+) permeability relative to monovalent cation permeability and/or that Ca(2+) influx via TRPC6 is greatly attenuated by depolarization. To evaluate Ca(2+) permeability, TRPC6 currents were examined in extracellular buffer in which Ca(2+) was varied from 0.02 to 20 mm. The results were consistent with a pore-permeation model in which Ca(2+) acts primarily as a blocking ion and contributes only a small percentage ( approximately 4%) to whole-cell currents in the presence of extracellular Na(+). Measurement of single-cell fura-2 fluorescence during perforated-patch recording of TRPC6 currents showed that OAG increased [Ca(2+)](i) 50-100 nm when the membrane potential was clamped at between -50 and -80 mV, but had little or no effect if the membrane potential was left uncontrolled. These results suggest that in cells exhibiting a high input resistance, the primary effect of activating TRPC6 will be membrane depolarization. However, in cells able to maintain a hyperpolarized potential (e.g. cells with a large inwardly rectifying or Ca(2+)-activated K(+) current), activation of TRPC6 will lead to a sustained increase in [Ca(2+)](i). Thus, the contribution of TRPC6 current to both the kinetics and magnitude of the Ca(2+) response will be cell specific and dependent upon the complement of other channel types.

Identification and localization of TRPC channels in the rat kidney.

It is well established that transient receptor potential (TRP) channels are activated following stimulation of G protein-coupled membrane receptors linked to PLC, but their differential expression in various cells of the renal nephron has not been described. In the present study, immunoprecipitations from rat kidney lysates followed by Western blot analysis using TRPC-specific, affinity-purified antibodies revealed the presence of TRPC1, -C3, and -C6. TRPC4, -C5, and -C7 were nondetectable. TRPC1 immunofluorescence was detected in glomeruli and specific tubular cells of the cortex and outer medulla. TRPC1 colocalized with aquaporin-1, a marker for proximal tubule and thin descending limb, but not with aquaporin-2, a marker for connecting tubule and collecting duct cells. TRPC3 and -C6 immunolabeling was predominantly confined to glomeruli and specific tubular cells of the cortex and both the outer and inner medulla. TRPC3 and -C6 colocalized with aquaporin-2, but not with the Na(+)/Ca(2+) exchanger or peanut lectin. Thus TRPC3 and -C6 proteins are expressed in principle cells of the collecting duct. In polarized cultures of M1 and IMCD-3 collecting duct cells, TRPC3 was localized exclusively to the apical domain, whereas TRPC6 was found in both the basolateral and apical membranes. TRPC3 and TRPC6 were also detected in primary podocyte cultures, whereas TRPC1 was exclusively expressed in mesangial cell cultures. Specific immunopositive labeling for TRPC4, -C5, or -C7 was not observed in kidney sections or cell lines. These results suggest that TRPC1, -C3, and -C6 may play a functional role in PLC-dependent signaling in specific regions of the nephron.

Proteomic analysis of TRPC5- and TRPC6-binding partners reveals interaction with the plasmalemmal Na(+)/K(+)-ATPase.

Mammalian transient receptor potential canonical (TRPC) genes encode a family of nonselective cation channels that are activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. In Drosophila photoreceptor cells, TRP channels are found in large, multimolecular signaling complexes in association with the PDZ-containing scaffolding protein, INAD. A similar mammalian TRPC "signalplex" has been proposed, but has yet to be defined. In the present study, affinity-purified polyclonal antibodies against TRPC5 and TRPC6 were used to immunoprecipitate signalplex components from rat brain lysates. Immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and sequenced by mass spectrometry. Proteins identified in the immunoprecipitates included cytoskeletal proteins spectrin, myosin, actin, drebrin, tubulin, and neurabin; endocytic vesicle-associated proteins clathrin, dynamin and AP-2; and the plasmalemmal Na(+)/K(+)-ATPase (NKA) pump. Several of these interactions were confirmed by reciprocal immunoprecipitation followed by Western blot analysis. In lysates from rat kidney, TRPC6, but not TRPC3, was found to coimmunoprecipitate with the NKA pump. Likewise, TRPC6, stably expressed in human embryonic kidney (HEK) cells, coimmunoprecipitated with endogenous NKA and colocalized with the pump to the plasmalemma when examined by immunofluorescence microscopy. Cell surface biotinylation experiments in intact HEK cells, confirmed that both the Na(+) pump and TRPC6 were present in the surface membrane and appeared to interact. Lastly, TRPC6 coimmunoprecipitated with the NKA pump when the proteins were coexpressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. These observations suggest that TRPC6 and the Na(+) pump are part of a functional complex that may be involved in ion transport and homeostasis in both the brain and kidney.

TRPC channels as signal transducers.

The study of the TRPC cation channels as signal transducers in sensory neurons is in its infancy. Mechanoreceptors that monitor arterial pressure are prime candidates for the involvement of TRPC channels as either primary mechanical transducers or as modulators of the transduction process. Their activity patterns can be regulated by growth factors such as BDNF and by a variety of ligands that activate Gq-coupled receptors, mechanisms that have been shown in heterologous expression systems to activate TRPC channels. We investigated the distribution of TRPC1 and TRPC3-7 in nodose sensory neurons and in their peripheral axons that terminate as mechanosensitive receptors in the aortic arch of the rat. Using immunocytochemical techniques we identified these six TRPC proteins in the soma of the nodose neurons but only TRPC1 and TRPC3-5 were found to distribute to the peripheral axons and the mechanosensory terminals. TRPC1 and TRPC3 extended into the low threshold complex sensory endings with very strong labeling. In contrast, TRPC4 and TRPC5 were found primarily in major branches of the receptor but immunoreactivity was weak in the region where mechanotransduction is presumed to occur. Terminals arising from unmyelinated fibers also expressed TRPC1 and TRPC3-5 but not all fibers expressed all of the channels suggesting that specific TRPC protein may be aligned with previously described subclasses of the unmyelinated C-fibers.

Maitotoxin induces biphasic interleukin-1beta secretion and membrane blebbing in murine macrophages.

Maitotoxin (MTX) is a potent shellfish toxin widely used as an in vitro tool for increasing intracellular Ca2+ and studying Ca2+ -dependent processes. MTX also induces membrane blebbing and nonselective pores similar to those elicited by the P2X7 receptor (P2X7R), an ATP-gated cation channel expressed in inflammatory leukocytes. We therefore tested whether MTX treatment of lipopolysaccharide-primed murine macrophages would mimic the ability of activated P2X7R to induce secretion of the proinflammatory cytokine interleukin-1beta (IL-1beta). MTX at < or = 0.6 nM predominantly induced processing and nonlytic release of mature IL-1beta (mIL-1beta), whereas >0.6 nM of MTX induced cytolytic release of unprocessed proIL-1beta. MTX-dependent release of mIL-1beta (but not cytolysis) was inhibited by the elimination of the trans-plasma membrane K+ gradient. MTX-induced cytokine release and cytolysis were both abrogated in the absence of extracellular Ca2+. On the other hand, extracellular glycine (5 mM) blocked MTX-induced cytolytic release of proIL-1beta without affecting regulated secretion of mIL-1beta. Because MTX has profound effects on plasma membrane permeability, we used time-lapse videography to examine the morphologic response of individual macrophages to MTX. MTX treatment led to biphasic propidium dye uptake and dilated blebbing coincident with cytolysis. Glycine completely blocked the second, lytic phase of dye uptake and prevented MTX-induced bleb dilation. These results indicate that the inflammatory macrophage can assemble the necessary signaling components to initiate both regulated and lytic release of IL-1beta in response to MTX. This suggests that the hyperactivation of proinflammatory cytokine secretion may be a significant component of the in vivo response to MTX during shellfish seafood poisoning.

Association of immunophilins with mammalian TRPC channels.

Drosophila photoreceptor channels TRP and TRPL are held in a large signalplex by the scaffolding protein, INAD. Immunophilin FKBP59, another member of the signalplex, binds to both INAD and TRPL. Mutation P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709), eliminates TRPL interaction with FKBP59. The first leucylprolyl (LP) dipeptide in this region is conserved in mammalian TRPC channel proteins. However, the second LP is changed to isoleucylprolyl (IP) in TRPC1, -C4, and -C5, and valylprolyl (VP) in TRPC3, -C6, and -C7. The purpose of the present study was to determine if mammalian FKBP12 or FKBP52 interact with TRPC channel proteins. Using TRPC-specific antibodies, immunoprecipitations from Sf9 cells individually co-expressing each of the TRPC proteins along with the immunophilins showed that TRPC3, -C6, and -C7 interact with FKBP12, whereas TRPC1, -C4, and -C5 interact with FKBP52. The binding of FKBP12 and FKBP52 was specific and could be displaced by the immunosuppressant drug FK506, at concentrations of 0.5 and 10 microm, respectively. To evaluate TRPC-immunophilin interactions in vivo, immunoprecipitations were performed using membrane lysates of rat cerebral cortex. FKBP12 co-immunoprecipitated with TRPC3, -C6, and -C7 from rat brain, whereas FKBP52 was found to associate with TRPC1, -C4, and -C5. The association of immunophilins with the TRPC channels in rat brain lysates could be displaced by FK506. Receptor-mediated activation of TRPC6, stably expressed in HEK cells, was significantly inhibited by FK506, which also disrupted interaction between TRPC6 and the endogenous immunophilin found in HEK cells. Pro to Gln mutations in the first LP dipeptide in the putative FKBP binding domain eliminated FKBP12 and FKBP52 interaction with TRPC3 and -C6, and TRPC1 and -C4, respectively. However, mutual swap of VP and IP in TRPC3 and TRPC5 did not alter the association or the selectivity of the channels for their respective immunophilin binding partner. These results suggest that immunophilins are TRPC channel accessory proteins that play an important role in the mechanism of channel activation following receptor stimulation.