The epithelial sodium channel (ENaC) is the central component of the sodium absorption pathway in epithelia. It is critical for sodium homeostasis and blood pressure control, which is demonstrated by rare genetic disorders such as Liddle�s syndrome and pseudohypoaldosteronism type I, that are associated with hyper- and hypotension, respectively. ENaC is mainly regulated by mechanisms that control the expression of active channels at the cell surface. Ubiquitin ligases of the Nedd4-like family, such as Nedd4 and Nedd4-2 decrease epithelial sodium absorption by binding to and targeting ENaC for endocytosis and degradation. This is most likely achieved by catalyzing the ubiquitination of ENaC. Conversely the serum- and glucocorticoid regulated kinase (SGK) increases ENaC activity. This effect is partly mediated by the interaction of SGK with the ubiquitin ligases Nedd4 and Nedd4-2. SGK is able to bind to both Nedd4 and Nedd4-2, however only Nedd4-2 is phosphorylated by SGK. The phosphorylation of Nedd4-2 inhibits its interaction with ENaC, thus reducing ENaC ubiquitination, thereby increasing surface expression and sodium absorption. Nedd4-like proteins interact with ENaC via their WW-domains. These domains bind PY-motifs (PPXY) present in ENaC subunits. Nedd4 and Nedd4-2 both have four highly similar WW-domains. Previous studies have shown that interaction between Nedd4 and ENaC is mainly mediated by WW-domain 3. SGK also has a PY-motif; therefore it was analyzed whether the WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK. Here, it is shown that single or tandem WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK and that, despite their high similarity, different WW-domains of Nedd4 and Nedd4-2 are involved. These data also suggest that WW-domains 2 and 3 of Nedd4-2 mediate the interaction with SGK in a concerted manner, and that in vitro the phosphorylation of SGK at serine residue 422 increases its affinity for the WW-domains of Nedd4-2. The stimulatory effect of SGK on ENaC activity is partly mediated via Nedd4-2 and will decrease if competition between Nedd4 and Nedd4-2 for binding to SGK occurs. Here it is shown that Nedd4 and Nedd4-2 are located in the same subcellular compartment and that they compete for binding to SGK. Besides its function in the proteasomal degradation pathway ubiquitination is involved in the regulation of membrane protein trafficking, including their endocytosis. ENaC was shown previously to be ubiquitinated. Here, we provide evidence that ENaC can be ubiquitinated differentially depending on its cellular location. Channels residing in the plasma membrane are multiubiquitinated and we suggest that this serves as an internalization signal for ENaC and a control for further trafficking. Cytosolic ENaC is mainly polyubiquitinated, and therefore probably targeted for proteasomal degradation. However, mono- and multiubiquitination of ENaC located within the cytosol is very likely to occur as well. In addition, it is shown that both proteasomal and lysosomal pathways are involved in the regulation of ENaC.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 213-239). Also available in electronic version.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Hyperkalemic periodic paralysis (HyperKPP) is an autosomal dominant human skeletal muscle channelopathy that causes periods of myotonic discharge and periodic paralysis due to defective Nav1.4 sodium channels. Patients are asymptomatic at birth, attacks become short and frequent during childhood, and more severe during adolescence. Since the Nav1.4 content in the cell membrane is relatively constant during childhood, it was hypothesized that some symptoms start with the defective Nav1.4 channels, while other symptoms start after some changes occur in gene expression affecting other membrane channel content and/or activity. To test the hypothesis, the contractile characteristics of EDL and soleus muscles from HyperKPP mice from the age of 0.5 to 12 months were tested in vitro. For both EDL and soleus, contractile defects, including low force generation, instability and large unstimulated force were observed by two weeks of age. With aging, the defects did not worsen, but muscles actually showed some improvement. Considering that Nav1.4 protein content reaches maximum at three weeks of age, the data suggests that HyperKPP symptoms are solely due to the defective Nav1.4 channels.

Th The kinetics of exocytotic secretion depend not only on the spatial relationship between calcium channels and the exocytotic apparatus, but also on the total amount of Ca2+ influx through Ca2+ channels, the free Ca2+ around the release site and the filling state of the release-ready vesicles. These factors may differ between neurons and endocrine cells. Bovine chromaffin cells (BCCs) are neuroendocrine cells responsible for catecholamine release from the adrenal glands. Ca2+ imaging experiments have shown that localized zones of Ca2+ influx exist on BCC membranes, but how different Ca2+ channel subtypes are distributed, and the mechanisms by which they are targeted, remain to be elucidated. CASK (calcium, calmodulin associated serine kinase) and Mint1 (Munc-18-interacting protein 1), which are modular adaptor proteins involved in synaptic targeting, have recently been found to function in targeting of á1B Ca2+ channels in hippocampal neurons. These data led to the proposal that Ca2+ channels are clustered in BCCs and that CASK and Mint1 play important roles in targeting and/or anchoring channels to their proper location. p*Using RT-PCR and Western blotting, CASK is demonstrated present in isolated BCCs. Mint1 is shown to be present by Western blotting as well. Immunocytochemical experiments and experiments in which BCCs were transfected with plasmids expressing á1A, á1B, and á1C subunits labeled with green fluorescent protein, have shown that á1A and á1B subunits are clustered on the plasma membranes of BCCs, while the á1C subunit is distributed in diffuse patches. With immunoprecipitation, it was determined that CASK interacts biochemically with á1A and á1B Ca2+ channels. Transfection of BCCs with NC3-GFP, which codes for the sequence of the á1B Ca2+ channel that interacts with CASK and Mint1, results in a punctate pattern of fluorescence, which is consistent with the binding of GFP labeled peptide to complexes of CASK and Mint1 at sites of release. Furthermore, immunocytochemical analysis of cells transfected with NC3-GFP showed that á1B Ca2+ channels have a dispersed distribution suggesting that they have been displaced from the binding sites. These data suggest that CASK and Mint1 are important in clustering and targeting Ca2+ channels in the BCC plasma membrane. This study is the first to show the existence and function of CASK and Mint1 in BCCs, and may contribute to our understanding of the exocytotic process in neuroendocrine cells

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are responsible for the synthesis and secretion of vasopressin (VP), which is important for fluid homeostasis, and oxytocin (OT), which is responsible for uterine contraction during parturition and milk let-down during lactation. VP-ergic MNCs undergo a number of structural and functional changes during dehydration, including the adoption of a bursting pattern of firing, the retraction of glial processes from MNC somata and terminals, the translocation of kappa-opioid receptors from internal stores to the plasma membrane, and the somatodendritic release of VP and OT. Since voltage-gated Ca2+ channels have been found on intracellular granules, and since an increase in Ca2+ current could regulate firing patterns and neuropeptide release, the surface expression of Ca2+ channel subtypes in MNCs was tested to determine if it would be altered by 16-24 hours of water deprivation. Using radioligand binding of antagonists of N-type and L-type Ca2+ channels, channel density was measured in the supraoptic nucleus (SON), which is largely composed of MNC somata, and in the neurohypophysis (NH), which is largely composed of MNC terminals. Dehydration caused an increase in the density of L-type channels in the SON, while causing no significant change in the N-type density. No change in density of either channel type was observed in the NH. Electrophysiological measurements in isolated MNC somata showed no change in total Ca2+ current, but a significant increase in the nifedipine-sensitive current following dehydration. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated no increase in messenger RNA levels for L-type channels, suggesting that the increase in channel density is not a consequence of de novo synthesis. These results suggest that L-type Ca2+ channels may be translocated from internal stores to the plasma membrane of MNCs in response to dehydration. Such a process may be important in maximizing secretion of VP when the physiological need is high.

Hyperkalemic periodic paralysis (HyperKPP) is an autosomal dominant human skeletal muscle channelopathy that causes periods of myotonic discharge and periodic paralysis due to defective Nav1.4 sodium channels. Patients are asymptomatic at birth, attacks become short and frequent during childhood, and more severe during adolescence. Since the Nav1.4 content in the cell membrane is relatively constant during childhood, it was hypothesized that some symptoms start with the defective Nav1.4 channels, while other symptoms start after some changes occur in gene expression affecting other membrane channel content and/or activity. To test the hypothesis, the contractile characteristics of EDL and soleus muscles from HyperKPP mice from the age of 0.5 to 12 months were tested in vitro. For both EDL and soleus, contractile defects, including low force generation, instability and large unstimulated force were observed by two weeks of age. With aging, the defects did not worsen, but muscles actually showed some improvement. Considering that Nav1.4 protein content reaches maximum at three weeks of age, the data suggests that HyperKPP symptoms are solely due to the defective Nav1.4 channels.

Today and future wireless networks are facing one of their greatest limiting factors:interference. This is due to the unprecedented increase in the number of connecteddevices. Therefore, in order to meet the ever increasing demand for data rate andquality of services, more advanced techniques than what we have today are requiredto deal with interference. This thesis takes a step towards interference managementin multiuser wireless systems by means of relaying and cooperation. We study fourfundamental building blocks in network information theory, propose new codingschemes, and derive limits on the capacity regions. The first problem we consider is the one-sided interference channel with bidirectional and rate-limited receiver cooperation. We propose a coding scheme that tailors two versions of superposition coding with classical relaying protocols. Theproposed scheme unifies and recovers previous results for the unidirectional coop-eration, yet in simpler forms. Analytical and numerical results confirm the benefitsof cooperation and illuminate the ideas behind the coding strategy. The second problem generalizes the first one by allowing the existence of bothcrossover links in the channel. We propose a coding scheme for this channel byextending noisy network coding to encompass rate-splitting at the encoders. Theachievable rate region is shown to be the same as a region achieved by explicitbinning. As a corollary, we prove that noisy network coding achieves the capacityregion of the Gaussian channel within 1 bit, under strong interference. Our resultis among the first to show constant-gap optimality of noisy network coding for amultiple-unicast problem, and to demonstrate equivalence in terms of achievablerates of two different coding approaches for a noisy interference network. We follow up by introducing a dedicated relay into the interference channelwhich simultaneously helps both receivers. For this third problem, the interferencechannel with a relay, we propose new coding schemes based on layered codes for long- and short-message quantize-forward techniques. The short-message schemesshow improvements in the achievable rates compared to other known coding tech-niques, especially when the channel is asymmetric, while relaxing the excessive delayissue of the long-message scheme. The analysis also reveals the trade-off betweenachievable rates, encoding and decoding delays, and complexity. In the fourth problem, we propose a new model for cooperative communication,the interfering relay channels, which consists of two neighboring relay channelsinducing interference to each other. Each relay, by utilizing a finite-capacity andnoise-free link to its own receiver, helps the receiver decode the desired message.We characterize the exact and approximate capacity region and sum-capacity forvarious classes of channels. The established results generalize and unify severalknown results for the relay and interference channels.The methods and results shown in this thesis aim at providing insight intopotential techniques for cooperative interference management in real-world systems. / <p>QC 20131001</p>

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are responsible for the synthesis and secretion of vasopressin (VP), which is important for fluid homeostasis, and oxytocin (OT), which is responsible for uterine contraction during parturition and milk let-down during lactation. VP-ergic MNCs undergo a number of structural and functional changes during dehydration, including the adoption of a bursting pattern of firing, the retraction of glial processes from MNC somata and terminals, the translocation of kappa-opioid receptors from internal stores to the plasma membrane, and the somatodendritic release of VP and OT. Since voltage-gated Ca2+ channels have been found on intracellular granules, and since an increase in Ca2+ current could regulate firing patterns and neuropeptide release, the surface expression of Ca2+ channel subtypes in MNCs was tested to determine if it would be altered by 16-24 hours of water deprivation. Using radioligand binding of antagonists of N-type and L-type Ca2+ channels, channel density was measured in the supraoptic nucleus (SON), which is largely composed of MNC somata, and in the neurohypophysis (NH), which is largely composed of MNC terminals. Dehydration caused an increase in the density of L-type channels in the SON, while causing no significant change in the N-type density. No change in density of either channel type was observed in the NH. Electrophysiological measurements in isolated MNC somata showed no change in total Ca2+ current, but a significant increase in the nifedipine-sensitive current following dehydration. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated no increase in messenger RNA levels for L-type channels, suggesting that the increase in channel density is not a consequence of de novo synthesis. These results suggest that L-type Ca2+ channels may be translocated from internal stores to the plasma membrane of MNCs in response to dehydration. Such a process may be important in maximizing secretion of VP when the physiological need is high.

Th The kinetics of exocytotic secretion depend not only on the spatial relationship between calcium channels and the exocytotic apparatus, but also on the total amount of Ca2+ influx through Ca2+ channels, the free Ca2+ around the release site and the filling state of the release-ready vesicles. These factors may differ between neurons and endocrine cells. Bovine chromaffin cells (BCCs) are neuroendocrine cells responsible for catecholamine release from the adrenal glands. Ca2+ imaging experiments have shown that localized zones of Ca2+ influx exist on BCC membranes, but how different Ca2+ channel subtypes are distributed, and the mechanisms by which they are targeted, remain to be elucidated. CASK (calcium, calmodulin associated serine kinase) and Mint1 (Munc-18-interacting protein 1), which are modular adaptor proteins involved in synaptic targeting, have recently been found to function in targeting of á1B Ca2+ channels in hippocampal neurons. These data led to the proposal that Ca2+ channels are clustered in BCCs and that CASK and Mint1 play important roles in targeting and/or anchoring channels to their proper location. p*Using RT-PCR and Western blotting, CASK is demonstrated present in isolated BCCs. Mint1 is shown to be present by Western blotting as well. Immunocytochemical experiments and experiments in which BCCs were transfected with plasmids expressing á1A, á1B, and á1C subunits labeled with green fluorescent protein, have shown that á1A and á1B subunits are clustered on the plasma membranes of BCCs, while the á1C subunit is distributed in diffuse patches. With immunoprecipitation, it was determined that CASK interacts biochemically with á1A and á1B Ca2+ channels. Transfection of BCCs with NC3-GFP, which codes for the sequence of the á1B Ca2+ channel that interacts with CASK and Mint1, results in a punctate pattern of fluorescence, which is consistent with the binding of GFP labeled peptide to complexes of CASK and Mint1 at sites of release. Furthermore, immunocytochemical analysis of cells transfected with NC3-GFP showed that á1B Ca2+ channels have a dispersed distribution suggesting that they have been displaced from the binding sites. These data suggest that CASK and Mint1 are important in clustering and targeting Ca2+ channels in the BCC plasma membrane. This study is the first to show the existence and function of CASK and Mint1 in BCCs, and may contribute to our understanding of the exocytotic process in neuroendocrine cells