Structural Basis for The Function and Regulation of the Epithelial Sodium Channel

Epithelial sodium channels (ENaC) mediate sodium transport across epithelia. Functional channels are assembled from three homologous α, β and γ subunits with ~30% similarity in amino acid sequence. Mutations in different subunits of this channel are responsible for diseases including Liddle's syndrome and type I pseudohypoaldosteronism. ENaC is synthesized on the ER membrane, aquires complex N-linked glycosylation in the Golgi and is trafficked to the plasma membrane where it is activated upon cleavage by numerous membrane-anchored and/or soluble serine proteases secreted into the extracellular milieu. Although it has been established that exogenous expression of all three subunits in oocytes is required for robust channel activity, the number and stoichiometry of subunits comprising one functional channel remains unclear. Different biophysical and electrophysiological studies have concluded that ENaC assembles as a trimer or a tetramer with possible larger molecular weight oligomers arising from higher order assembly of trimers or tetramers. Due to the lack of structural information on ENaC, the molecular aspects of channel activation and regulation of function remain less well understood. In the current study, using a battery of computational and experimental techniques, we address specific questions concerning the structural aspects of regulation of channel activation and function by constructing a structural model of the channel. Significant advances through this study include determination of oligomerization state of ENaC using native gel electrophoresis and identification of allosteric communication within the channel and modulating channel activity by rational mutagenesis of the identified allosteric sites. In this study, we conclude that ENaC assembles as both trimers and tetramers in the same cell. The amount of tetramers correlates well with increase in function and more importantly, the gamma subunit plays a crucial role in the formation of tetramers in oocytes. We believe that the results presented here would be immensely helpful in the future for understanding the cellular aspects of channel regulation and function at the molecular level.Doctor of Philosoph

Feedback Consolidation Algorithms for ABR Point-to-Multipoint Connections in ATM Networks

ABR traffic management for point-to-multipoint connections controls the source rate to the minimum rate supported by all the branches of the multicast tree. A number of algorithms have been developed for extending ABR congestion avoidance algorithms to perform feedback consolidation at the branch points. This paper discusses various design options and implementation alternatives for the consolidation algorithms, and proposes a number of new algorithms. The performance of the proposed algorithms and the previous algorithms is compared under a variety of conditions. Results indicate that the algorithms we propose eliminate the consolidation noise (caused if the feedback is returned before all branches respond), while exhibiting a fast transient response.Comment: Proceedings of IEEE INFOCOM 1998, March 1998, volume 3, pp. 1004-101

Approaches for probing the sequence space of substrates recognized by molecular chaperones

Neurodegeneration, the progressive loss of function in neurons that eventually leads to their death, is the cause of many neurodegenerative disorders including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Protein aggregation is a hallmark of most neurodegenerative diseases, where unfolded proteins form intranuclear, cytosolic, and extracellular insoluble aggregates in neurons. Mounting evidence from studies in neurodegenerative disease models shows that molecular chaperones, key regulators of protein aggregation and degradation, play critical roles in the progression of neurodegeneration. Although chaperones exhibit promiscuity in their substrate specificity, specific molecular features are required for substrate recognition. Understanding the basis for substrate recognition by chaperones will aid in the development of therapeutic strategies that regulate chaperone expression levels in order to combat neurodegeneration. Many experimental techniques, including alanine scanning mutagenesis and phage display library screening, have been developed and applied to understand the basis of substrate recognition by chaperones. Here, we present computational algorithms that can be applied to rapidly screen the sequence space of potential substrates to determine the sequence and structural requirements for substrate recognition by chaperones

Automated minimization of steric clashes in protein structures

Molecular modeling of proteins including homology modeling, structure determination, and knowledge-based protein design requires tools to evaluate and refine three-dimensional protein structures. Steric clash is one of the artifacts prevalent in low-resolution structures and homology models. Steric clashes arise due to the unnatural overlap of any two non-bonding atoms in a protein structure. Usually, removal of severe steric clashes in some structures is challenging since many existing refinement programs do not accept structures with severe steric clashes. Here, we present a quantitative approach of identifying steric clashes in proteins by defining clashes based on the Van der Waals repulsion energy of the clashing atoms. We also define a metric for quantitative estimation of the severity of clashes in proteins by performing statistical analysis of clashes in high-resolution protein structures. We describe a rapid, automated and robust protocol, Chiron, which efficiently resolves severe clashes in low-resolution structures and homology models with minimal perturbation in the protein backbone. Benchmark studies highlight the efficiency and robustness of Chiron compared to other widely used methods. We provide Chiron as an automated web server to evaluate and resolve clashes in protein structures that can be further used for more accurate protein design

Gaia: automated quality assessment of protein structure models

Motivation: Increasing use of structural modeling for understanding structure–function relationships in proteins has led to the need to ensure that the protein models being used are of acceptable quality. Quality of a given protein structure can be assessed by comparing various intrinsic structural properties of the protein to those observed in high-resolution protein structures

Gain-of-Function Mutation W493R in the Epithelial Sodium Channel Allosterically Reconfigures Intersubunit Coupling

Sodium absorption in epithelial cells is rate-limited by the epithelial sodium channel (ENaC) activity in lung, kidney, and the distal colon. Pathophysiological conditions, such as cystic fibrosis and Liddle syndrome, result from water-electrolyte imbalance partly due to malfunction of ENaC regulation. Because the quaternary structure of ENaC is yet undetermined, the bases of pathologically linked mutations in ENaC subunits α, β, and γ are largely unknown. Here, we present a structural model of heterotetrameric ENaC α1βα2γ that is consistent with previous cross-linking results and site-directed mutagenesis experiments. By using this model, we show that the disease-causing mutation αW493R rewires structural dynamics of the intersubunit interfaces α1β and α2γ. Changes in dynamics can allosterically propagate to the channel gate. We demonstrate that cleavage of the γ-subunit, which is critical for full channel activation, does not mediate activation of ENaC by αW493R. Our molecular dynamics simulations led us to identify a channel-activating electrostatic interaction between α2Arg-493 and γGlu-348 at the α2γ interface. By neutralizing a sodium-binding acidic patch at the α1β interface, we reduced ENaC activation of αW493R by more than 2-fold. By combining homology modeling, molecular dynamics, cysteine cross-linking, and voltage clamp experiments, we propose a dynamics-driven model for the gain-of-function in ENaC by αW493R. Our integrated computational and experimental approach advances our understanding of structure, dynamics, and function of ENaC in its disease-causing state

Regulation of the epithelial Na+ channel and airway surface liquid volume by serine proteases

Mammalian airways are protected from infection by a thin film of airway surface liquid (ASL) which covers airway epithelial surfaces and acts as a lubricant to keep mucus from adhering to the epithelial surface. Precise regulation of ASL volume is essential for efficient mucus clearance and too great a reduction in ASL volume causes mucus dehydration and mucus stasis which contributes to chronic airway infection. The epithelial Na+ channel (ENaC) is the rate-limiting step that governs Na+ absorption in the airways. Recent in vitro and in vivo data have demonstrated that ENaC is a critical determinant of ASL volume and hence mucus clearance. ENaC must be cleaved by either intracellular furin-type proteases or extracellular serine proteases to be active and conduct Na+, and this process can be inhibited by protease inhibitors. ENaC can be regulated by multiple pathways, and once proteolytically cleaved ENaC may then be inhibited by intracellular second messengers such as cAMP and PIP2. In the airways, however, regulation of ENaC by proteases seems to be the predominant mode of regulation since knockdown of either endogenous serine proteases such as prostasin, or inhibitors of ENaC proteolysis such as SPLUNC1, has large effects on ENaC activity in airway epithelia. In this review, we shall discuss how ENaC is proteolytically cleaved, how this process can regulate ASL volume, and how its failure to operate correctly may contribute to chronic airway disease

Energetic and Structural Basis for Activation of the Epithelial Sodium Channel by Matriptase

Limited proteolysis, accomplished by endopeptidases, is a ubiquitous phenomenon underlying the regulation and activation of many enzymes, receptors and other proteins synthesized as inactive precursors. Serine proteases are one of the largest and conserved families of endopeptidases involved in diverse cellular activities including wound healing, blood coagulation and immune responses. Heteromeric α,β,γ-epithelial sodium channels (ENaC) associated with diseases like cystic fibrosis and Liddle’s syndrome, are irreversibly stimulated by membrane-anchored proteases (MAPs) and furin-like convertases. Matriptase/Channel activating protease-3 (CAP3) is one of the several MAPs that potently activate ENaC. Despite identification of protease cleavage sites, the basis for enhanced susceptibility of α- and γ-ENaC to proteases remains elusive. Here, we elucidate the energetic and structural bases for activation of ENaC by CAP3. We find a region near the γ-ENaC furin site that is previously unidentified as a critical cleavage site for CAP3-mediated stimulation. We also report that CAP3 mediates cleavage of ENaC at basic residues downstream of the furin site. Our results indicate that surface proteases alone are sufficient to fully activate uncleaved ENaC, and explain how ENaC in epithelia expressing surface-active proteases can appear refractory to soluble proteases. Our results support a model in which proteases prime ENaC for activation by cleaving at the furin site, and cleavage at downstream sites is accomplished by membrane surface proteases or extracellular soluble proteases. Based on our results, we propose a dynamics-driven “anglerfish” mechanism that explains less stringent sequence requirements for substrate recognition and cleavage by matriptase compared to furin

Rational coupled dynamics network manipulation rescues disease-relevant mutant cystic fibrosis transmembrane conductance regulator

A novel approach identifying networks of residues involved in trans -protein dynamic coupling is applied to rescue mutant CFTR