Search

With an increase in urine flow there is a significant increase in shear stress against the renal epithelium including the inner medullary collecting duct, resulting in an increase in nitric oxide (NO) production. The mechanisms of the shear stress-mediated increases in NO are undetermined. Previous studies found that shear stress increases epithelial sodium channel (ENaC) open probability and endothelin (ET)-1 production in an ENaC-dependent mechanism in the collecting duct (CD). Given that ET-1 stimulates NO production in the CD, we hypothesized that shear stress-induced NO production is downstream of shear stress-induced ENaC activation and ET-1 production in a negative feedback loop. We determined that nitric oxide synthase 1 (NOS1) and NOS3 contribute to shear stress-mediated NO production in the CD, that is attenuated by low doses of the ENaC inhibitors amiloride and benzamil. Moreover, ETBreceptor blockade significantly blunted the shear stress-mediated NO production. We further elucidated whether mice lacking NOS1 in the collecting duct (CDNOS1KO) have an impaired renal ET-1 system in the CD. Although urinary ET-1 production and inner medullary ET receptor expression were similar between flox control and CDNOS1KO mice, acute ET-1 treatment significantly reduced ENaC open probability in CDs from flox mice but not CDNOS1KO mice compared with basal. Basal ENaC activity in CDs was similar between the genotypes. We conclude that during acute shear stress across the CD, ENaC acts in a negative feedback loop to stimulate NO production in an ETB/NOS1-dependent manner resulting in a decrease in ENaC open probability and promoting natriuresis.

cAMP is a key mediator of connecting tubule and collecting duct (CD) Na+and water reabsorption. Studies performed in vitro have suggested that CD adenylyl cyclase (AC)3 partly mediates the actions of vasopressin; however, the physiological role of CD AC3 has not been determined. To assess this, mice were developed with CD-specific disruption of AC3 [CD AC3 knockout (KO)]. Inner medullary CDs from these mice exhibited 100% target gene recombination and had reduced ANG II- but not vasopressin-induced cAMP accumulation. However, there were no differences in urine volume, urinary urea excretion, or urine osmolality between KO and control mice during normal water intake or varying degrees of water restriction in the presence or absence of chronic vasopressin administration. There were no differences between CD AC3 KO and control mice in arterial pressure or urinary Na+or K+excretion during a normal or high-salt diet, whereas plasma renin and vasopressin concentrations were similar between the two genotypes. Patch-clamp analysis of split-open cortical CDs revealed no difference in epithelial Na+channel activity in the presence or absence of vasopressin. Compensatory changes in AC6 were not responsible for the lack of a renal phenotype in CD AC3 KO mice since combined CD AC3/AC6 KO mice had similar arterial pressure and renal Na+and water handling compared with CD AC6 KO mice. In summary, these data do not support a significant role for CD AC3 in the regulation of renal Na+and water excretion in general or vasopressin regulation of CD function in particular.

The epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under tonic inhibition by a local purinergic signaling system responding to changes in dietary sodium intake. Normal BK(Ca) channel function is required for flow-sensitive ATP secretion in the ASDN. We tested here whether ATP secreted through connexin channels in a coupled manner with K(+) efflux through BK(Ca) channels is required for inhibitory purinergic regulation of ENaC in response to increases in sodium intake. Inhibition of connexin channels relieves purinergic inhibition of ENaC. Deletion of the BK-β4 regulatory subunit, which is required for normal BK(Ca) channel function and flow-sensitive ATP secretion in the ASDN, suppresses increases in urinary ATP in response to increases in sodium intake. As a consequence, ENaC activity, particularly in the presence of high sodium intake, is inappropriately elevated in BK-β4 null mice. ENaC in BK-β4 null mice, however, responds normally to exogenous ATP, indicating that increases in activity do not result from end-organ resistance but rather from lowered urinary ATP. Consistent with this, disruption of purinergic regulation increases ENaC activity in wild type but not BK-β4 null mice. Consequently, sodium excretion is impaired in BK-β4 null mice. These results demonstrate that the ATP secreted in the ASDN in a BK(Ca) channel-dependent manner is physiologically available for purinergic inhibition of ENaC in response to changes in sodium homeostasis. Impaired sodium excretion resulting form loss of normal purinergic regulation of ENaC in BK-β4 null mice likely contributes to their elevated blood pressure.

The epithelial Na + channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under negative-feedback regulation by the renin–angiotensin–aldosterone system in protection of sodium balance and blood pressure. We test here whether aldosterone is necessary and sufficient for ENaC expression and activity in the ASDN. Surprisingly, ENaC expression and activity are robust in adrenalectomized (Adx) mice. Exogenous mineralocorticoid increases ENaC activity equally well in control and Adx mice. Plasma [AVP] is significantly elevated in Adx vs. control mice. Vasopressin (AVP) stimulates ENaC. Inhibition of the V 2 AVP receptor represses ENaC activity in Adx mice. The absence of aldosterone combined with elevated AVP release compromises normal feedback regulation of ENaC in Adx mice in response to changes in sodium intake. These results demonstrate that aldosterone is sufficient but not necessary for ENaC activity in the ASDN. Aldosterone-independent stimulation by AVP shifts the role of ENaC in the ASDN from protecting Na + balance to promoting water reabsorption. This stimulation of ENaC likely contributes to the hyponatremia of adrenal insufficiency.

Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na+ excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na+ regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na+ intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na+ regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na+ feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na+ retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.

The epithelial Na+ channel (ENaC) is modulated by membrane lipid composition. However, the effect of an in vivo change of membrane composition is unknown. We examined the effect of a 70-day enhanced cholesterol diet (ECD) on ENaC and renal Na+ handling. Rats were fed a standard chow or one supplemented with 1% cholesterol and 0.5% cholic acid (ECD). ECD animals exhibited marked anti-diuresis and anti-natriuresis (40 and 47%), which peaked at 1–3 weeks. Secondary compensation returned urine output and urinary Na+ excretion to control levels by week 10. During these initial changes, there were no accompanying effects on systolic blood pressure, serum creatinine, or urinary creatinine excretion, indicating that the these effects of ECD preceded those which modify renal filtration and blood pressure. The effects of ECD on ENaC were evaluated by measuring the relative protein content of α, β, and γ subunits. α and γ blots were further examined for subunit cleavage (a process that activates ENaC). No significant changes were observed in α and β levels throughout the study. However, levels of cleaved γ were elevated, suggesting that ENaC was activated. The changes of γ persisted at week 10 and were accompanied by additional subunit fragments, indicating potential changes of γ-cleaving proteases. Enhanced protease activity, and specifically that which could act on the second identified cleavage site in γ, was verified in a newly developed urinary protease assay. These results predict enhanced ENaC activity, an effect that was confirmed in patch clamp experiments of principal cells of split open collecting ducts, where ENaC open probability was increased by 40% in the ECD group. These data demonstrate a complex series of events and a new regulatory paradigm that is initiated by ECD prior to the onset of elevated blood pressure. These events lead to changes of renal Na+ handling, which occur in part by effects on extracellular γ-ENaC cleavage.

Apical release of ATP and UTP can activate P2Y2 receptors in the aldosterone-sensitive distal nephron (ASDN) and inhibit the open probability (Po) of the epithelial sodium channel (ENaC). Little is known, however, about the regulation and physiological relevance of this system. Patch-clamp studies in freshly isolated ASDN provide evidence that increased dietary Na+ intake in wild-type mice lowers ENaC Po, consistent with a contribution to Na+ homeostasis, and is associated with increased urinary concentrations of UTP and the ATP hydrolytic product, ADP. Genetic deletion of P2Y2 receptors in mice (P2Y2−/−; littermates to wild-type mice) or inhibition of apical P2Y-receptor activation in wild-type mice prevents dietary Na+-induced lowering of ENaC Po. Although they lack suppression of ENaC Po by dietary NaCl, P2Y2−/− mice do not exhibit NaCl-sensitive blood pressure, perhaps as a consequence of compensatory down-regulation of aldosterone levels. Consistent with this hypothesis, clamping mineralocorticoid activity at high levels unmasks greater ENaC activity and NaCl sensitivity of blood pressure in P2Y2−/− mice. The studies indicate a key role of the apical ATP/UTP-P2Y2-receptor system in the inhibition of ENaC Po in the ASDN in response to an increase in Na+ intake, thereby contributing to NaCl homeostasis and blood pressure regulation.—Pochynyuk, O., Rieg, T., Bugaj, V., Schroth, J., Fridman, A., Boss, G. R., Insel, P. A., Stockand, J. D., Vallon, V. Dietary Na+ inhibits the open probability of the epithelial sodium channel in the kidney by enhancing apical P2Y2-receptor tone.

Thiazolidinediones are agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) that can induce fluid retention and weight gain through unclear mechanisms. To test a proposed role for the epithelial sodium channel ENaC in thiazolidinedione-induced fluid retention, we used mice with conditionally inactivated alphaENaC in the collecting duct (Scnn1a(loxloxCre) mice). In control mice, rosiglitazone did not alter plasma aldosterone levels or protein expression of ENaC subunits in the kidney, but did increase body weight, plasma volume, and the fluid content of abdominal fat pads, and decreased hematocrit. Scnn1a(loxloxCre) mice provided functional evidence for blunted Na+ uptake in the collecting duct, but still exhibited rosiglitazone-induced fluid retention. Moreover, treatment with rosiglitazone or pioglitazone did not significantly alter the open probability or number of ENaC channels per patch in isolated, split-open cortical collecting ducts of wild-type mice. Finally, patch-clamp studies in primary mouse inner medullary collecting duct cells did not detect ENaC activity but did detect a nonselective cation channel upregulated by pioglitazone. These data argue against a primary and critical role of ENaC in thiazolidinedione-induced fluid retention.

Growing evidence implicates a key role for extracellular nucleotides in cellular regulation, including of ion channels and renal function, but the mechanisms for such actions are inadequately defined. We investigated purinergic regulation of the epithelial Na+ channel (ENaC) in mammalian collecting duct. We find that ATP decreases ENaC activity in both mouse and rat collecting duct principal cells. ATP and other nucleotides, including UTP, decrease ENaC activity via apical P2Y2 receptors. ENaC in collecting ducts isolated from mice lacking this receptor have blunted responses to ATP. P2Y2 couples to ENaC via PLC; direct activation of PLC mimics ATP action. Tonic regulation of ENaC in the collecting duct occurs via locally released ATP; scavenging endogenous ATP and inhibiting P2 receptors, in the absence of other stimuli, rapidly increases ENaC activity. Moreover, ENaC has greater resting activity in collecting ducts from P2Y2-/- mice. Loss of collecting duct P2Y2 receptors in the knock-out mouse is the primary defect leading to increased ENaC activity based on the ability of direct PLC stimulation to decrease ENaC activity in collecting ducts from P2Y2-/- mice in a manner similar to ATP in collecting ducts from wild-type mice. These findings demonstrate that locally released ATP acts in an autocrine/paracrine manner to tonically regulate ENaC in mammalian collecting duct. Loss of this intrinsic regulation leads to ENaC hyperactivity and contributes to hypertension that occurs in P2Y2 receptor-/- mice. P2Y2 receptor activation by nucleotides thus provides physiologically important regulation of ENaC and electrolyte handling in mammalian kidney.

Activity of the epithelial sodium channel (ENaC) is limiting for Na+reabsorption at the distal nephron. Phosphoinositides, such as phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] modulate the activity of this channel. Activation of purinergic receptors triggers multiple events, including activation of PKC and PLC, with the latter depleting plasma membrane PI(4,5)P2. Here, we investigate regulation of ENaC in renal principal cells by purinergic receptors via PLC and PI(4,5)P2. Purinergic signaling rapidly decreases ENaC open probability and apical membrane PI(4,5)P2levels with similar time courses. Moreover, inhibiting purinergic signaling with suramin rescues ENaC activity. The PLC inhibitor U73122, but not U73343, its inactive analog, recapitulates the action of suramin. In contrast, modulating PKC signaling failed to affect purinergic regulation of ENaC. Unexpectedly, inhibiting either purinergic receptors or PLC in resting cells dramatically increased ENaC activity above basal levels, indicating tonic activation of purinergic signaling in these polarized renal epithelial cells. Increased ENaC activity was associated with elevation of apical membrane PI(4,5)P2levels. Subsequent treatment with ATP in the presence of inhibited purinergic signaling failed to decrease ENaC activity and apical membrane PI(4,5)P2levels. Dwell-time analysis reveals that depletion of PI(4,5)P2forces ENaC toward a closed state. In contrast, increasing PI(4,5)P2levels above basal values locks the channel in an open state interrupted by brief closings. Thus our results suggest that purinergic control of apical membrane PI(4,5)P2levels is a major regulator of ENaC activity in renal epithelial cells.

Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the aldosterone-sensitive distal nephron. Hormones, including aldosterone and insulin, increase ENaC activity, in part by stimulating phosphatidylinositide 3-OH kinase (PI3-K) signaling. Recent studies in heterologous expression systems reveal a close spatiotemporal coupling between PI3-K signaling and ENaC activity with the phospholipid product of this kinase, PI(3,4,5)P(3), in some cases, directly binding the channel and increasing open probability (P(o)). This study tested whether this tight coupling plays a physiologic role in modulating ENaC activity in native tissue and polarized epithelial cells. IGF-I was found to increase Na(+) reabsorption across mpkCCD(c14) principal cell monolayers in a PI3-K-sensitive manner. Inhibition of PI3-K signaling, moreover, rapidly decreased Na(+) reabsorption and ENaC activity in mpkCCD(c14) cells that were treated with corticosteroids and IGF-I. These decreases paralleled changes in apical membrane PI(3,4,5)P(3) levels, demonstrating tight spatiotemporal coupling between ENaC activity and PI3-K/PI(3,4,5)P(3) signaling within this membrane. For further probing of the mechanism underpinning this coupling, cortical collecting ducts (CCD) were isolated from rat and split open to expose the apical membrane for patch-clamp analysis. Inhibition of PI3-K signaling with wortmannin and LY294002 but not its inactive analogue rapidly and markedly decreased the P(o) of ENaC. Moreover, IGF-I acutely increased P(o) of ENaC in CCD principal cells in a PI3-K-sensitive manner. Together, these observations stress the importance of tight spatiotemporal coupling between PI3-K signaling and ENaC within the apical membrane of principal cells to the physiologic control of this ion channel.

Activation of phospholipase C (PLC)-linked signaling cascades in nonexcitable cells stimulates Ca2+ release from inositol-1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ entry via plasma membrane Ca2+ channels. The attention of investigators is currently focused on the properties and molecular basis of channels involved in Ca2+ entry into nonexcitable cells. According to current views, mammalian TRP proteins are involved in receptor-and store-dependent influx of Ca2+; however, little is known about the linkage between specific TRP proteins and endogenous channels responsible for Ca2+ entry. The aim of the present study was to elucidate the role of TRPC3 in the formation of store-dependent or receptor-operated pathways of Ca2+ entry into A431 cells. Registration of Ca2+ influx based on fluorescence measurements of intracellular Ca2+ concentrations and analysis of integral membrane currents revealed that partial inhibition of TRPC3 expression by small interfering RNA (siRNA) results in suppression of store-dependent Ca2+ entry without any effect on receptor-operated Ca2+ influx. In-depth studies of single channels revealed that TRPC3 suppression in A431 cells results in the disappearance of one type of store-operated channels and formation of a novel type of store-independent Ca2+-permeable channels. This, in turn, testifies to the crucial role of TRPC3 in normal functioning of store-operated Ca2+ channels in A431 cells.

Discretionary control of Na(+) excretion is a key component of the regulation of arterial blood pressure in mammals. Sodium excretion is fine-tuned in the aldosterone-sensitive distal nephron by the activity of the epithelial Na(+) channel (ENaC). Here, ENaC functions as a final effector of the renin-angiotensin-aldosterone system (RAAS) during negative feedback control of blood pressure. Mutations affecting ENaC activity and abnormal regulation of this channel affect blood pressure through pathological changes to Na(+) excretion. Recent evidence demonstrates that powerful signalling pathways function in parallel with the RAAS to modulate ENaC activity and blood pressure. An inclusive paradigm is emerging with respect to regulation of blood pressure where ENaC serves as a critical point of convergence for several important signalling systems that affect renal Na(+) excretion. A robust inhibitory purinergic signalling system intrinsic to the distal nephron dynamically regulates ENaC through paracrine ATP signalling via the metabotropic P2Y2 purinergic receptor to properly match urinary Na(+) excretion to dietary Na(+) intake. This enables blood pressure to be maintained within a normal range despite broad changes in dietary Na(+) consumption. Loss of purinergic inhibition of ENaC increases blood pressure by causing inappropriate Na(+) excretion. In contrast, stimulation of the P2Y2 receptor promotes natriuresis and a decrease in blood pressure. Such observations identify purinergic signalling in the distal nephron as possibly causative, when dysfunctional, for certain forms of elevated blood pressure, and as a possible therapeutic target for the treatment of elevated blood pressure particularly that associated with salt sensitivity.

Vasopressin modulates sodium reabsorption in the collecting duct through adenylyl cyclase-stimulated cyclic AMP, which exists as multiple isoforms; the specific isoform involved in vasopressin-stimulated sodium transport is unknown. To assess this, we studied mice deficient in adenylyl cyclase type VI specifically in the principal cells of the collecting duct. Knockout mice had increased urine volume and reduced urine sodium concentration, but regardless of the level of sodium intake, they did not exhibit significant alterations in urinary sodium excretion, arterial pressure, or pulse rate. Plasma renin concentration was elevated in knockout mice, however, suggesting a compensatory response. Valsartan significantly reduced arterial pressure in knockout mice but not in controls. Knockout mice had decreased renal cortical mRNA content of all three epithelial sodium channel (ENaC) isoforms, and total cell sodium channel isoforms α and γ were reduced in these animals. Patch-clamp analysis of split-open cortical collecting ducts revealed no difference in baseline activity of sodium channels, but knockout mice had abolished vasopressin-stimulated ENaC open probability and apical membrane channel number. In summary, these data suggest that adenylyl cyclase VI mediates vasopressin-stimulated ENaC activity in the kidney.

The epithelial Na(+) channel (ENaC) is modulated by membrane lipid composition. However, the effect of an in vivo change of membrane composition is unknown. We examined the effect of a 70-day enhanced cholesterol diet (ECD) on ENaC and renal Na(+) handling. Rats were fed a standard chow or one supplemented with 1% cholesterol and 0.5% cholic acid (ECD). ECD animals exhibited marked anti-diuresis and anti-natriuresis (40 and 47%), which peaked at 1-3 weeks. Secondary compensation returned urine output and urinary Na(+) excretion to control levels by week 10. During these initial changes, there were no accompanying effects on systolic blood pressure, serum creatinine, or urinary creatinine excretion, indicating that the these effects of ECD preceded those which modify renal filtration and blood pressure. The effects of ECD on ENaC were evaluated by measuring the relative protein content of α, β, and γ subunits. α and γ blots were further examined for subunit cleavage (a process that activates ENaC). No significant changes were observed in α and β levels throughout the study. However, levels of cleaved γ were elevated, suggesting that ENaC was activated. The changes of γ persisted at week 10 and were accompanied by additional subunit fragments, indicating potential changes of γ-cleaving proteases. Enhanced protease activity, and specifically that which could act on the second identified cleavage site in γ, was verified in a newly developed urinary protease assay. These results predict enhanced ENaC activity, an effect that was confirmed in patch clamp experiments of principal cells of split open collecting ducts, where ENaC open probability was increased by 40% in the ECD group. These data demonstrate a complex series of events and a new regulatory paradigm that is initiated by ECD prior to the onset of elevated blood pressure. These events lead to changes of renal Na(+) handling, which occur in part by effects on extracellular γ-ENaC cleavage.

The mechanisms underlying "aldosterone escape," which refers to the excretion of sodium (Na(+)) during high Na(+) intake despite inappropriately increased levels of mineralocorticoids, are incompletely understood. Because local purinergic tone in the aldosterone-sensitive distal nephron downregulates epithelial Na(+) channel (ENaC) activity, we tested whether this mechanism mediates aldosterone escape. Here, urinary ATP concentration increased with dietary Na(+) intake in mice. Physiologic concentrations of ATP decreased ENaC activity in a dosage-dependent manner. P2Y(2)(-/-) mice, which lack the purinergic receptor, had significantly less increased Na(+) excretion than wild-type mice in response to high-Na(+) intake. Exogenous deoxycorticosterone acetate and deletion of the P2Y(2) receptor each modestly increased the resistance of ENaC to changes in Na(+) intake; together, they markedly increased resistance. Under the latter condition, ENaC could not respond to changes in Na(+) intake. In contrast, as a result of aldosterone escape, wild-type mice had increased Na(+) excretion in response to high-Na(+) intake regardless of the presence of high deoxycorticosterone acetate. These data suggest that control of ENaC by purinergic signaling is necessary for aldosterone escape.

We used patch-clamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron Na+ reabsorption during water conservation vs. maintenance of Na+ balance. We find that vasopressin significantly increases ENaC activity within 2–3 min by increasing open probability ( Po). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18–24 h) and pretreatment of isolated CD with vasopressin (∼30 min) resulted in a similar increase in Po. In addition, this also increased the number ( N) of active ENaC in the apical membrane. Similar to Po, increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and Na+ each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron Na+ reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC.

Purpose of review Epithelial sodium channel (ENaC) activity is limiting for sodium reabsorption in the distal nephron. Humans regulate blood pressure by fine-tuning sodium balance through control of ENaC. ENaC dysfunction causes some hypertensive and renal salt wasting diseases. Thus, it is critical to understand the cellular mechanisms controlling ENaC activity. Recent findings ENaC is sensitive to phosphatidylinositol 4,5-bisphosphate (PIP2), the target of phospholipase C-mediated metabolism, and phosphatidylinositiol 3,4,5-trisphosphate (PIP3), the product of phosphatidylinositide 3-OH kinase (PI3-K). PIP2 is permissive for ENaC gating possibly interacting directly with the channel. Activation of distal nephron P2Y receptors tempers ENaC activity by promoting PIP2 metabolism. This is important because gene deletion of P2Y2 receptors causes hypertension associated with hyperactive ENaC. Aldosterone, the final hormone in a negative-feedback cascade activated by decreases in blood pressure, increases ENaC activity. PIP3 sits at a critical bifurcation in the aldosterone-signaling cascade, increasing ENaC open probability and number. PIP3-effectors mediate increases in ENaC number by suppressing channel retrieval. PIP3 binds ENaC, at a site distinct from that important to PIP2 regulation, to modulate directly open probability. Summary Phosphoinositides play key roles in physiologic control of ENaC and perhaps dysregulation plays a role in disease associated with abnormal renal sodium handling.

We used patch-clamp electrophysiology to investigate regulation of the epithelial Na+channel (ENaC) by endothelin-1 (ET-1) in isolated, split-open rat collecting ducts. ET-1 significantly decreases ENaC open probability by about threefold within 5 min. ET-1 decreases ENaC activity through basolateral membrane ETBbut not ETAreceptors. In rat collecting duct, we find no role for phospholipase C or protein kinase C in the rapid response of ENaC to ET-1. ET-1, although, does activate src family tyrosine kinases and their downstream MAPK1/2 effector cascade in renal principal cells. Both src kinases and MAPK1/2 signaling are necessary for ET-1-dependent decreases in ENaC open probability in the split-open collecting duct. We conclude that ET-1 in a physiologically relevant manner rapidly suppresses ENaC activity in native, mammalian principal cells. These findings may provide a potential mechanism for the natriuresis observed in vivo in response to ET-1, as well as a potential cause for the salt-sensitive hypertension found in animals with impaired endothelin signaling.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na(+) channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P(3) and PI(4,5)P(2) to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in beta- and gamma- but not alpha-ENaC as necessary for PI(3,4,5)P(2) but not PI(4,5)P(2) modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in beta- and gamma-ENaC are critical to PI(3,4,5)P(3) augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of beta- and gamma-ENaC were identified as being critical to down-regulation of ENaC activity and P(o) in response to depletion of membrane PI(4,5)P(2). These regions of the channel played no identifiable role in a PI(3,4,5)P(3) response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P(2) to increase open probability. We conclude that beta and gamma subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P(3) and PI(4,5)P(2). This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.

In most non-excitable cells, calcium (Ca(2+)) release from the inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx through the plasma membrane Ca(2+) channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca(2+) influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.

The epithelial Na(+) channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is set, in part, by its membrane levels. The small G protein RhoA increases ENaC activity by increasing the membrane levels of this channel. We hypothesize that RhoA increases ENaC activity by promoting channel trafficking to the plasma membrane. Few experimental methods are available to directly visualize trafficking of ion channels to the plasma membrane. Here we combine electrophysiology with two complementary imaging methods, total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching, to study the mechanistic basis of RhoA actions on ENaC. Patch clamp results demonstrate that RhoA increases ENaC activity in an additive manner with dominant-negative dynamin. This is consistent with a mechanism of increased ENaC trafficking to the membrane. Direct visualization of ENaC movement near the plasma membrane with total internal reflection fluorescence-fluorescence recovery after photobleaching revealed that RhoA accelerates ENaC trafficking toward the membrane. RhoA-facilitated movement of the channel was sensitive to disrupting the endomembrane system. Moreover, facilitating retrieval decreased ENaC activity but not trafficking toward the membrane. ENaC at the plasma membrane clustered and was laterally immobile suggesting that the cytoskeleton tethers or corrals membrane resident channels or membrane-directed vesicles containing ENaC. Disrupting microtubules but not microfilaments led to reorganization of ENaC clusters and slowed trafficking toward the membrane. The cytoskeleton is an established target for RhoA signaling. We conclude that RhoA, likely through effects on the cytoskeleton, promotes ENaC trafficking to the plasma membrane to increase channel membrane levels and activity.

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.

The regulation of the heartbeat by the two largest neurons, d-VLN and d-RPLN, on the dorsal surface of visceral and right parietal ganglia of Giant African snail, Achatina fulica, was examined. Using the new method of animal preparation, for the first time, discrete biphasic inhibitory-excitatory junction potentials (I-EJPs) in the heart and several muscles of the visceral sac were recorded. The duration of hyperpolarizing phase (H-phase) of biphasic I-EJPs was 269+/-5.6 ms (n=5), which is 2-3 times less than that of the cholinergic inhibitory JPs (682+/-68.5 ms, n=5). The H-phase of I-EJPs was not altered by the application of atropine, picrotoxine, succinylcholinchloride, D-tubocurarine and tetraethylammonium or substitution of Cl(-) ions. Even the low-frequency neuronal discharges (1-2 imp/s) evoked significant facilitation and potentiation of the H-phase. Between the multimodal neurons d-VLN/d-RPLN and mantle or visceral organs there is evidence of direct synaptic connections. These neurons were found to have no axonal branches in the intestinal nerve as once suspected but reach the heart through several other nerves. New giant heart motoneurons do not interact with previously identified cardioregulatory neurons.