Kisspeptin neuron projections to oxytocin neurons are not necessary for parturition in the mouse.

Oxytocin is synthesized by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) neurons and is released from the posterior pituitary gland to trigger uterine contractions during parturition. In rats, oxytocin neuron innervation by periventricular nucleus (PeN) kisspeptin neurons increases over pregnancy and intra-SON kisspeptin administration excites oxytocin neurons only in late pregnancy. To test the hypothesis that kisspeptin neurons excite oxytocin neurons to trigger uterine contractions during birth in C57/B6J mice, double-label immunohistochemistry for kisspeptin and oxytocin first confirmed that kisspeptin neurons project to the SON and PVN. Furthermore, kisspeptin fibers expressed synaptophysin and formed close appositions with oxytocin neurons in the mouse SON and PVN before and during pregnancy. Stereotaxic viral delivery of caspase-3 into the AVPV/PeN of Kiss-Cre mice before mating reduced kisspeptin expression in the AVPV, PeN, SON and PVN by > 90% but did not affect the duration of pregnancy or the timing of delivery of each pup during parturition. Therefore, it appears that AVPV/PeN kisspeptin neuron projections to oxytocin neurons are not necessary for parturition in the mouse.

The exocyst complex is required for the trafficking and delivery of KCa3.1 to the basolateral membrane of polarized epithelia.

Control of the movement of ions and water across epithelia is essential for homeostasis. Changing the number or activity of ion channels at the plasma membrane is a significant regulator of epithelial transport. In polarized epithelia, the intermediate-conductance calcium-activated potassium channel, KCa3.1 is delivered to the basolateral membrane where it generates and maintains the electrochemical gradients required for epithelial transport. The mechanisms that control the delivery of KCa3.1 to the basolateral membrane are still emerging. Herein, we investigated the role of the highly conserved tethering complex exocyst. In epithelia, exocyst is involved in the tethering of post-Golgi secretory vesicles with the basolateral membrane, which is required before membrane fusion. In our Fisher rat thyroid cell line that stably expresses KCa3.1, siRNA knockdown of either of the exocyst subunits Sec3, Sec6, or Sec8 significantly decreased KCa3.1-specific current. In addition, knockdown of exocyst complex subunits significantly reduced the basolateral membrane protein level of KCa3.1. Finally, co-immunoprecipitation experiments suggest associations between Sec6 and KCa3.1, but not between Sec8 and KCa3.1. Collectively, based on these data and our previous studies, we suggest that components of exocyst complex are crucially important in the tethering of KCa3.1 to the basolateral membrane. After which, Soluble N-ethylmaleimide-sensitive factor (SNF) Attachment Receptors (SNARE) proteins aid in the insertion of KCa3.1-containing vesicles into the basolateral membrane of polarized epithelia.NEW & NOTEWORTHY Our Ussing chamber and immunoblot experiments demonstrate that when subunits of the exocyst complex were transiently knocked down, this significantly reduced the basolateral population and functional expression of KCa3.1. These data suggest, combined with our protein association experiments, that the exocyst complex regulates the tethering of KCa3.1-containing vesicles to the basolateral membrane prior to the SNARE-dependent insertion of channels into the basolateral membrane of epithelial cells.

Role of SNARE Proteins in the Insertion of KCa3.1 in the Plasma Membrane of a Polarized Epithelium.

Targeting proteins to a specific membrane is crucial for proper epithelial cell function. KCa3.1, a calcium-activated, intermediate-conductance potassium channel, is targeted to the basolateral membrane (BLM) in epithelial cells. Surprisingly, the mechanism of KCa3.1 membrane targeting is poorly understood. We previously reported that targeting of KCa3.1 to the BLM of epithelial cells is Myosin-Vc-, Rab1-and Rab8-dependent. Here, we examine the role of the SNARE proteins VAMP3, SNAP-23 and syntaxin 4 (STX-4) in the targeting of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We carried out immunoblot, siRNA and Ussing chamber experiments on FRT cells, stably expressing KCa3.1-BLAP/Bir-A-KDEL, grown as high-resistance monolayers. siRNA-mediated knockdown of VAMP3 reduced BLM expression of KCa3.1 by 57 ± 5% (p ≤ 0.05, n = 5). Measurements of BLM-localized KCa3.1 currents, in Ussing chambers, demonstrated knockdown of VAMP3 reduced KCa3.1 current by 70 ± 4% (p ≤ 0.05, n = 5). Similarly, siRNA knockdown of SNAP-23 reduced the expression of KCa3.1 at the BLM by 56 ± 7% (p ≤ 0.01, n = 6) and reduced KCa3.1 current by 80 ± 11% (p ≤ 0.05, n = 6). Also, knockdown of STX-4 lowered the BLM expression of KCa3.1 by 54 ± 6% (p ≤ 0.05, n = 5) and reduced KCa3.1 current by 78 ± 11% (p ≤ 0.05, n = 5). Finally, co-immunoprecipitation experiments demonstrated associations between KCa3.1, VAMP3, SNAP-23 and STX-4. These data indicate that VAMP3, SNAP-23 and STX-4 are critical for the targeting KCa3.1 to BLM of polarized epithelial cells.

Epithelial Sodium Channel δ Subunit Is Expressed in Human Arteries and Has Potential Association With Hypertension.

BACKGROUND: Elevated expression and increased activity of vascular epithelial sodium channel (ENaC) can result in vascular dysfunction in small animal models. However, there is limited or no knowledge on expression and function of ENaC channels in human vasculature. Hence, this study explored the expression and function of ENaC in human arteries and their association with hypertension. METHODS: Human internal mammary artery (IMA) and aorta were obtained from cardiovascular patients undergoing coronary artery bypass graft surgery. Expression of the ENaC subunit was analyzed by polymerase chain reaction, Western blot, and immunohistochemistry. ENaC function was observed by patch-clamp electrophysiology in endothelial cells isolated from IMA. Levels of ENaC subunit expression levels were compared between arteries from normotensive, uncontrolled hypertensive, and controlled hypertensive patients. RESULTS: For the first time, expression of α, ß, γ, and δ was detected at mRNA and protein levels in human IMA and aorta. Single-channel patch-clamp recordings identified both αßγ- and δßγ-like channel conductance in primary endothelial cells isolated and cultured from IMA. Reduced expression of the δ subunit was observed in controlled hypertensive IMA, whereas reduced expression of γ-ENaC was observed in controlled hypertensive aorta. CONCLUSIONS: These data suggest that functional ENaC channels are expressed in human arteries and their expression levels are associated with hypertension.

The epithelial sodium channel has a role in breast cancer cell proliferation.

PURPOSE: Breast cancer is the most common cancer affecting women worldwide with half a million associated deaths annually. Despite a huge global effort, the pathways of breast cancer progression are not fully elucidated. Ion channels have recently emerged as novel regulators of cancer cell proliferation and metastasis. The epithelial sodium channel, ENaC, made up of α, ß and γ subunits is well known for its role in Na+ reabsorption in epithelia, but a number of novel roles for ENaC have been described, including potential roles in cancer. A role for ENaC in breast cancer, however, has yet to be described. Therefore, the effects of ENaC level and activity on breast cancer proliferation were investigated. METHODS: Through the publicly available SCAN-B dataset associations between αENaC mRNA expression and breast cancer subtypes, proliferation markers and epithelial-mesenchymal transition markers (EMT) were assessed. αENaC expression, through overexpression or siRNA-mediated knockdown, and activity, through the ENaC-specific inhibitor amiloride, were altered in MCF7, T47D, BT549, and MDAMB231 breast cancer cells. MTT and EdU cell proliferation assays were used to determine the effect of these manipulations on breast cancer cell proliferation. RESULTS: High αENaC mRNA expression was associated with less aggressive and less proliferative breast cancer subtypes and with reduced expression of proliferation markers. Decreased αENaC expression or activity, in the mesenchymal breast cancer cell lines BT549 and MDAMB231, increased breast cancer cell proliferation. Conversely, increased αENaC expression decreased breast cancer cell proliferation. CONCLUSION: αENaC expression is associated with a poor prognosis in breast cancer and is a novel regulator of breast cancer cell proliferation. Taken together, these results identify ENaC as a potential future therapeutic target.

Explosion in the complexity of membrane protein recycling.

For decades, recycling of membrane proteins has been represented in figures by arrows between the "endosome" and the plasma membrane, but recently there has been an explosion in the understanding of the mechanisms and protein complexes required to facilitate protein recycling. Here, some key discoveries will be introduced, including assigning function to a number of recently recognized protein complexes and linking their function to protein recycling. Furthermore, the importance of lipid interactions and links to diseases and epithelial polarity will be summarized.

The δ subunit of epithelial sodium channel in humans-a potential player in vascular physiology.

Vascular epithelial sodium channels (ENaCs) made up of canonical α, ß, and γ subunits have attracted more attention recently owing to their physiological role in vascular health and disease. A fourth subunit, δ-ENaC, is expressed in various mammalian species, except mice and rats, which are common animal models for cardiovascular research. Accordingly, δ-ENaC is the least understood subunit. However, the recent discovery of δ subunit in human vascular cells indicates that this subunit may play a significant role in normal/pathological vascular physiology in humans. Channels containing the δ subunit have different biophysical and pharmacological properties compared with channels containing the α subunit, with the potential to alter the vascular function of ENaC in health and disease. Hence, it is important to investigate the expression and function of δ-ENaC in the vasculature to identify whether δ-ENaC is a potential new drug target for the treatment of cardiovascular disease. In this review, we will focus on the existing knowledge of δ-ENaC and implications for vascular physiology and pathophysiology in humans.

Retromer is involved in epithelial Na+ channel trafficking.

The epithelial Na+ channel (ENaC) located at the apical membrane in many epithelia is the rate-limiting step for Na+ reabsorption. Tight regulation of the plasma membrane population of ENaC is required, as hypertension or hypotension may result if too many or too few ENaCs are present. Endocytosed ENaC travels to the early endosome and is then either trafficked to the lysosome for degradation or recycled back to the plasma membrane. Recently, the retromer recycling complex, located at the early endosome, has been implicated in plasma membrane protein recycling pathways. We hypothesized that the retromer is required for recycling of ENaC. Stabilization of retromer function with the retromer stabilizing chaperone R55 increased ENaC current, whereas knockdown or overexpression of individual retromer and associated proteins altered ENaC current and cell surface population of ENaC. KIBRA was identified as an ENaC-binding protein allowing ENaC to link to sorting nexin 4 to alter ENaC trafficking. Knockdown of the retromer-associated cargo-binding sorting nexin 27 protein did not alter ENaC current, whereas CCDC22, a CCC-complex protein, coimmunoprecipitated with ENaC, and CCDC22 knockdown decreased ENaC current and population at the cell surface. Together, our results confirm that retromer and the CCC complex play a role in recycling of ENaC to the plasma membrane.

Membrane trafficking pathways regulating the epithelial Na+ channel.

Renal Na+ reabsorption, facilitated by the epithelial Na+ channel (ENaC), is subject to multiple forms of control to ensure optimal body blood volume and pressure through altering both the ENaC population and activity at the cell surface. Here, the focus is on regulating the number of ENaCs present in the apical membrane domain through pathways of ENaC synthesis and targeting to the apical membrane as well as ENaC removal, recycling, and degradation. Finally, the mechanisms by which ENaC trafficking pathways are regulated are summarized.

Annexin II Light Chain p11 Interacts With ENaC to Increase Functional Activity at the Membrane.

The epithelial Na+ channel (ENaC) provides for Na+ absorption in various types of epithelia including the kidney, lung, and colon where ENaC is localized to the apical membrane to enable Na+ entry into the cell. The degree of Na+ entry via ENaC largely depends on the number of active channels localized to the cell membrane, and is tightly controlled by interactions with ubiquitin ligases, kinases, and G-proteins. While regulation of ENaC endocytosis has been well-studied, relatively little is understood of the proteins that govern ENaC exocytosis. We hypothesized that the annexin II light chain, p11, could participate in the transport of ENaC along the exocytic pathway. Our results demonstrate that all three ENaC channel subunits interacted with p11 in an in vitro binding assay. Furthermore, p11 was able to immunoprecipitate ENaC in epithelial cells. Quantitative mass spectrometry of affinity-purified ENaC-p11 complexes recovered several other trafficking proteins including HSP-90 and annexin A6. We also report that p11 exhibits a robust protein expression in cortical collecting duct epithelial cells. However, the expression of p11 in these cells was not influenced by either short-term or long-term exposure to aldosterone. To determine whether the p11 interaction affected ENaC function, we measured amiloride sensitive Na+ currents in Xenopus oocytes or mammalian epithelia co-expressing ENaC and p11 or a siRNA to p11. Results from these experiments showed that p11 significantly augmented ENaC current, whereas knockdown of p11 decreased current. Further, knockdown of p11 reduced ENaC cell surface population suggesting p11 promotes membrane insertion of ENaC. Overall, our findings reveal a novel protein interaction that controls the number of ENaC channels inserted at the membrane via the exocytic pathway.

Structural insights into the architecture and membrane interactions of the conserved COMMD proteins.

The COMMD proteins are a conserved family of proteins with central roles in intracellular membrane trafficking and transcription. They form oligomeric complexes with each other and act as components of a larger assembly called the CCC complex, which is localized to endosomal compartments and mediates the transport of several transmembrane cargos. How these complexes are formed however is completely unknown. Here, we have systematically characterised the interactions between human COMMD proteins, and determined structures of COMMD proteins using X-ray crystallography and X-ray scattering to provide insights into the underlying mechanisms of homo- and heteromeric assembly. All COMMD proteins possess an α-helical N-terminal domain, and a highly conserved C-terminal domain that forms a tightly interlocked dimeric structure responsible for COMMD-COMMD interactions. The COMM domains also bind directly to components of CCC and mediate non-specific membrane association. Overall these studies show that COMMD proteins function as obligatory dimers with conserved domain architectures.

Epithelial Na+ Channel: Reciprocal Control by COMMD10 and Nedd4-2.

Optimal function of the epithelial sodium channel (ENaC) in the distal nephron is key to the kidney's long-term control of salt homeostasis and blood pressure. Multiple pathways alter ENaC cell surface populations, including correct processing and trafficking in the secretory pathway to the cell surface, and retrieval from the cell surface through ubiquitination by the ubiquitin ligase Nedd4-2, clathrin-mediated endocytosis, and sorting in the endosomal system. Members of the Copper Metabolism Murr1 Domain containing (COMMD) family of 10 proteins are known to interact with ENaC. COMMD1, 3 and 9 have been shown to down-regulate ENaC, most likely through Nedd4-2, however, the other COMMD family members remain uncharacterized. To investigate the effects of the COMMD10 protein on ENaC trafficking and function, the interaction of ENaC and COMMD10 was confirmed. Stable COMMD10 knockdown in Fischer rat thyroid epithelia decreased ENaC current and this decreased current was associated with increased Nedd4-2 protein, a known negative regulator of ENaC. However, inhibition of Nedd4-2's ubiquitination of ENaC was only able to partially rescue the observed reduction in current. Stable COMMD10 knockdown results in defects both in endocytosis and recycling of transferrin suggesting COMMD10 likely interacts with multiple pathways to regulate ENaC and therefore could be involved in the long-term control of blood pressure.

Inhibition of calcium/calmodulin-dependent kinase II restores contraction and relaxation in isolated cardiac muscle from type 2 diabetic rats.

BACKGROUND: Calcium/calmodulin-dependent kinase II-delta (CaMKIIδ) activity is enhanced during hyperglycemia and has been shown to alter intracellular calcium handling in cardiomyocytes, ultimately leading to reduced cardiac performance. However, the effects of CaMKIIδ on cardiac contractility during type 2 diabetes are undefined. METHODS: We examined the expression and activation of CaMKIIδ in right atrial appendages from non-diabetic and type 2 diabetic patients (n = 7 patients per group) with preserved ejection fraction, and also in right ventricular tissue from Zucker Diabetic Fatty rats (ZDF) (n = 5-10 animals per group) during early diabetic cardiac dysfunction, using immunoblot. We also measured whole heart function of ZDF and control rats using echocardiography. Then we measured contraction and relaxation parameters of isolated trabeculae from ZDF to control rats in the presence and absence of CaMKII inhibitors. RESULTS: CaMKIIδ phosphorylation (at Thr287) was increased in both the diabetic human and animal tissue, indicating increased CaMKIIδ activation in the type 2 diabetic heart. Basal cardiac contractility and relaxation were impaired in the cardiac muscles from the diabetic rats, and CaMKII inhibition with KN93 partially restored contractility and relaxation. Autocamtide-2-related-inhibitor peptide (AIP), another CaMKII inhibitor that acts via a different mechanism than KN93, fully restored cardiac contractility and relaxation. CONCLUSIONS: Our results indicate that CaMKIIδ plays a key role in modulating performance of the diabetic heart, and moreover, suggest a potential therapeutic role for CaMKII inhibitors in improving myocardial function during type 2 diabetes.

Epithelial Na+ channel differentially contributes to shear stress-mediated vascular responsiveness in carotid and mesenteric arteries from mice.

A potential "new player" in arteries for mediating shear stress responses is the epithelial Na+ channel (ENaC). The contribution of ENaC as shear sensor in intact arteries, and particularly different types of arteries (conduit and resistance), is unknown. We investigated the role of ENaC in both conduit (carotid) and resistance (third-order mesenteric) arteries isolated from C57Bl/6J mice. Vessel characteristics were determined at baseline (60 mmHg, no flow) and in response to increased intraluminal pressure and shear stress using a pressure myograph. These protocols were performed in the absence and presence of the ENaC inhibitor amiloride (10 µM) and after inhibition of endothelial nitric oxide synthase (eNOS) by Nω-nitro-l-arginine methyl ester (l-NAME; 100 µM). Under no-flow conditions, amiloride increased internal and external diameters of carotid (13 ± 2%, P < 0.05) but not mesenteric (0.5 ± 0.9%, P > 0.05) arteries. In response to increased intraluminal pressure, amiloride had no effect on the internal diameter of either type of artery. However, amiloride affected the stress-strain curves of mesenteric arteries. With increased shear stress, ENaC-dependent effects were observed in both arteries. In carotid arteries, amiloride augmented flow-mediated dilation (9.2 ± 5.3%) compared with control (no amiloride, 6.2 ± 3.3%, P < 0.05). In mesenteric arteries, amiloride induced a flow-mediated constriction (-11.5 ± 6.6%) compared with control (-2.2 ± 4.5%, P < 0.05). l-NAME mimicked the effect of ENaC inhibition and prevented further amiloride effects in both types of arteries. These observations indicate that ENaC contributes to shear sensing in conduit and resistance arteries. ENaC-mediated effects were associated with NO production but may involve different (artery-dependent) downstream signaling pathways. NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) contributes to shear sensing in conduit and resistance arteries. In conduit arteries ENaC has a role as a vasoconstrictor, whereas in resistance arteries ENaC contributes to vasodilation. Interaction of ENaC with endothelial nitric oxide synthase/nitric oxide signaling to mediate the effects is supported; however, cross talk with other shear stress-dependent signaling pathways cannot be excluded.

The role of CaMKII in diabetic heart dysfunction.

Diabetes mellitus (DM) is an increasing epidemic that places a significant burden on health services worldwide. The incidence of heart failure (HF) is significantly higher in diabetic patients compared to non-diabetic patients. One underlying mechanism proposed for the link between DM and HF is activation of calmodulin-dependent protein kinase (CaMKIIδ). CaMKIIδ mediates ion channel function and Ca(2+) handling during excitation-contraction and excitation-transcription coupling in the myocardium. CaMKIIδ activity is up-regulated in the myocardium of diabetic patients and mouse models of diabetes, where it promotes pathological signaling that includes hypertrophy, fibrosis and apoptosis. Pharmacological inhibition and knockout models of CaMKIIδ have shown some promise of a potential therapeutic benefit of CaMKIIδ inhibition, with protection against cardiac hypertrophy and apoptosis reported. This review will highlight the pathological role of CaMKIIδ in diabetes and discuss CaMKIIδ as a therapeutic target in DM, and also the effects of exercise on CaMKIIδ.

Functional interaction of COMMD3 and COMMD9 with the epithelial sodium channel.

The epithelial sodium channel (ENaC) plays an important role in controlling Na⁺ homeostasis, extracellular fluid volume, and blood pressure. Copper metabolism Murr1 domain-containing protein 1 (COMMD1) interacts with ENaC and downregulates ENaC. COMMD1 belongs to the COMMD family consisting of COMMD1-10, and all COMMD family members share a C-terminal COMM domain. Here, we report that COMMD2-10 also interacts with ENaC, and COMMD3 and COMMD9 were selected for further study. Amiloride-sensitive current in mammalian epithelia expressing ENaC was significantly reduced by COMMD3 or COMMD9, and ENaC expression at the cell surface was significantly decreased in the presence of COMMD3 or COMMD9. COMMD3 and COMMD9 retained their ability to reduce current when COMMD1 was knocked down. COMMD3 and COMMD9 were widely expressed in kidney and were colocalized with ENaC in renal collecting duct cells. These data suggest that COMMD3 and COMMD9 may be endogenous regulators of ENaC to regulate Na⁺ transport through altering ENaC cell surface expression.

Structure and dynamics of human Nedd4-1 WW3 in complex with the αENaC PY motif.

Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) is an E3 ubiquitin ligase that interacts with and negatively regulates the epithelial Na(+) channel (ENaC). The WW domains of Nedd4-1 bind to the ENaC subunits via recognition of PY motifs. Human Nedd4-1 (hNedd4-1) contains four WW domains with the third domain (WW3*) showing the strongest affinity to the PY motif. To understand the mechanism underlying this binding affinity, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in complex with a peptide comprising the C-terminal tail of the human ENaC α-subunit. The structure reveals that the peptide interacts in a similar manner to other WW domain-ENaC peptide structures. Crucial interactions that likely provide binding affinity are the broad XP groove facilitating additional contacts between the WW3* domain and the peptide, compared to similar complexes, and the large surface area buried (83Å(2)) between R430 (WW3*) and L647' (αENaC). This corroborates the model-free analysis of the (15)N backbone relaxation data, which showed that R430 is the most rigid residue in the domain (S(2)=0.90±0.01). Carr-Purcell-Meiboom-Gill relaxation dispersion analysis identified two different conformational exchange processes on the µs-ms time-scale. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange may play a role in peptide recognition. Thus, both structural and dynamic features of the complex appear to define the high binding affinity. The results should aid interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.

Regulation of the delta and alpha epithelial sodium channel (ENaC) by ubiquitination and Nedd8.

The δ epithelial sodium channel (δENaC) is a proton-activated, sodium-selective, amiloride-sensitive ion channel in the ENaC/degenerin family of ion channels involved in blood pressure regulation and mechanosensation. Other ENaC family members are subject to ubiquitin modification leading to internalization from the cell surface, and degradation of the channel. Here, we show that δENaC is also modified by ubiquitin on three intracellular lysine residues. Absence of these lysines abolished ubiquitin modification of δENaC and increased cell surface levels of δENaC. Although the HECT-domain ubiquitin ligase Nedd4-2 reduced amiloride-sensitive current generated by δßγENaC-containing channels, δENaC does not contain a binding site for Nedd4-2; therefore, this effect is probably mediated by the ßγENaC subunits. Nedd8, a ubiquitin-like protein that regulates RING-domain E3 ubiquitin ligases, promoted δENaC ubiquitination, decreased both the intracellular and cell surface δENaC populations, and decreased δßγENaC amiloride-sensitive short circuit current (Isc -amiloride) in a mammalian epithelium. Nedd8 also promoted α- and γENaC ubiquitination, decreased the cell surface pools, and decreased αßγENaC Isc -amiloride. Conversely, XIAP, a single subunit RING E3 ligase, decreased ubiquitinated δENaC, increased the δENaC cell surface pool and increased δßγENaC Isc -amiloride. Therefore δ- and α - ßγENaC channel function may be influenced by RING-domain E3 ubiquitin ligases.