The epithelial Na ؉ channel (ENaC) is a tetramer of two ␣-, one ␤-, and one ␥-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone ␣-(95 kDa), ␤-(96 kDa), and ␥-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of ␣␤␥ENaC subunits revealed a second band for each subunit (65 kDa for ␣, 110 kDa for ␤, and 75 kDa for ␥) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [ 3 H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed ␣-and ␥-subunits is also consistent with proteolytic cleavage. By using ␣-and ␥-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the ␣-and ␥-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for ␣ and 18 kDa for ␥) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.The amiloride-sensitive epithelial Na ϩ channel (ENaC) 1 is composed of three structurally related subunits, termed ␣-, ␤-, and ␥-ENaC. The three subunits exhibit limited amino acid sequence identity (30 -40%) but are structurally similar with two membrane-spanning domains and cytosolic amino and carboxyl termini. We and others have shown that ENaC expressed in Xenopus oocytes has a subunit stoichiometry of two ␣-, one ␤-, and one ␥-subunit (1, 2

Proteolytic processing of epithelial sodium channel (ENaC) subunits occurs as channels mature within the biosynthetic pathway. The proteolytic processing events of the ␣ and ␥ subunits are associated with channel activation. Furin cleaves the ␣ subunit ectodomain at two sites, releasing an inhibitory tract and activating the channel. However, furin cleaves the ␥ subunit ectodomain only once. A second distal cleavage in the ␥ subunit induced by other proteases, such as prostasin and elastase, is required to release a second inhibitory tract and further activate the channel. We found that the serine protease plasmin activates ENaC in association with inducing cleavage of the ␥ subunit at ␥Lys 194 , a site distal to the furin site. A ␥K194A mutant prevented both plasmin-dependent activation of ENaC and plasmin-dependent production of a unique 70-kDa carboxyl-terminal ␥ subunit cleavage fragment. Plasmin-dependent cleavage and activation of ENaC may have a role in extracellular volume expansion in human disorders associated with proteinuria, as filtered plasminogen may be processed by urokinase, released from renal tubular epithelium, to generate active plasmin.The epithelial sodium channel (ENaC) 3 transports Na ϩ across the apical membrane of principal cells in the aldosterone-sensitive distal nephron (1). Alterations in ENaC activity disrupt Na ϩ balance, leading to changes in both extracellular volume and blood pressure. Expansion of extracellular volume occurs in a variety of clinical disorders, including nephrotic syndrome. The role of ENaC activation in many of the clinical disorders associated with extracellular volume expansion remains to be defined.ENaC activity in the cells that line the distal nephron depends on the number of channels in the apical membrane and on channel open probability (P o ) (1). ENaC subunits undergo post-translational processing by specific proteases (2-9). Cleavage of the ␣ and ␥ subunits by proteases has a key role in activating ENaC, presumably by releasing inhibitory domains within the ectodomains of the ␣ and ␥ subunits (3, 5, 10, 11). We have proposed that multiple proteolytic cleavage events lead to a stepwise activation of ENaC, reflected in a stepwise increase in channel P o (3,5,10,12). Channels that lack proteolytic processing have a low P o (3, 10, 12, 13). Channels that have been cleaved solely by furin, where an ␣ subunit inhibitory tract has been released, exhibit an intermediate P o (3,10,12). Furinprocessed channels likely represent the channels that are observed in Xenopus oocytes at a single channel level. Channels that have released both ␣ and ␥ subunit inhibitory tracts exhibit a high P o , as we observed in oocytes co-expressing ENaC and prostasin (3, 5). Both non-cleaved channels and furin-processed channels at the plasma membrane provide a reservoir of channels that can be activated by extracellular proteases (3,14).One potential activator of ENaC is the serine protease plasmin. Although known for its involvement in fibrinolysis, plasmin has been implicate...

Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-lra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular I jection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for -5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1p3. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by genetransfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy.Arthritis is a chronic, debilitating condition affecting over 30 million Americans (1). Presently incurable, it remains the agent of considerable suffering and economic loss. Therapeutic intervention in arthritis is hindered by a number of factors, including difficulties in targeting drugs to joints. Proteins are particularly vulnerable to this restriction, which is of special concern, as many new agents with considerable antiarthritic potential are proteins. As an alternative to traditional methods of drug delivery, we have suggested the transfer oftherapeutic genes to the synovial lining ofjoints (2,3). Expression of these genes would overcome proteindelivery problems and lead to the intraarticular accumulation of the gene products at the site of disease, with reduced exposure of nontarget organs.Using the rabbit knee joint as a model system, we are therefore developing in vivo and ex vivo methods for delivering genes to joints. This model takes advantage of the similarity in size between the knee joint of the rabbit and the human proximal interphalangeal joint, a common site of rheumatoid arthritis. Moreover, there exists a large body of literature on the biology of the rabbit's knee, including well-established methods for synovial cell culture (e.g., refs. 4-6). Here we report the transfer to synovium of two marker genes and one potentially therapeutic gene by an ex vivo approach. Intraarticular expression of the interleukin 1-re-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. (rhIL-1,8). These results demonstrate the feasibility of ...

Joints are difficult organs to target therapeutically. Intravenous, intramuscular, and oral routes of drug delivery provide poor access to the joint, and expose the body systemically to the therapeutic agent. Although intraarticular injection provides direct access to the joint, most injected materials have a short intraarticular half-life. We propose to circumvent these problems by introducing into the synovium gene(s) coding for proteins with antiarthritic properties. Two methods of gene delivery to synovium are under development. In the direct approach, in situ transduction of synoviocytes follows the injection of suitable vectors into the joint. In the indirect approach, synovium is removed from the joint, its synoviocytes are isolated, and the cells transduced in vitro. Genetically modified cells are subsequently transplanted back into the synovium. Using retroviral vectors, we have been able to express the lacZ and neo genes in lapine synovial fibroblasts in vitro. Following neoselection, all cells became LacZ+. Neo-selected cells carrying the lacZ marker gene were transplanted back into the knees of recipient rabbits to examine the persistence and expression of these genes in vivo. Islands of LacZ+, transplanted cells persisted in the recipient joints for at least 3 months. Furthermore, Neo+ cells could be grown from synovia recovered from these joints. Initial attempts to use retroviruses for the direct, in situ transduction of synovium have failed, probably because synoviocytes in the normal synovium are mitotically inactive. Present efforts are directed towards further development of our techniques for transferring genes to joints, and using these techniques to antagonize the intraarticular actions of interleukin-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies were raised against the InaW protein, the product of the ice nucleation gene of Pseudomonas fluorescens MS1650, after protein isolation from an Escherichia coli clone. On Western blots (immunoblots), these antibodies recognized InaW protein and InaZ protein (the ice nucleation gene product of Pseudomonas syringae S203), produced by both E. coli clones and the source organisms. The InaZ protein appeared in P. syringae S203 during stationary phase; its appearance was correlated with the appearance of the ice nucleation-active phenotype. In contrast, the InaW protein occurred at relatively constant levels throughout the growth phases of P. fluorescens MS1650; the ice nucleation activity was also constant. Western analyses of membrane preparations ofP. syringae PS31 and Erwinia herbicola MS3000 with this antibody revealed proteins which were synthesized with development of the nucleating phenotype. In these species the presence or absence of the nucleating phenotype was controlled by manipulation of culture conditions. In all nucleation-positive cultures examined, cross-reacting low-molecular-weight bands were observed; these bands appeared to be products of proteolytic degradation of ice nucleation proteins. The proteolysis pattern of InaZ protein seen on Western blots showed a periodic pattern of fragment sizes, suggesting a highly repetitive site for protease action. A periodic primary structure is predicted by the DNA sequence of the inaZ gene.The ability of some species of gram-negative bacteria from the genera Pseudomonas (1, 2, 21, 22), Erwinia (19), and Xanthomonas (23) to nucleate the crystallization of ice demonstrates a unique manipulation of the environment by bacteria. Such bacteria are the major nucleating agents found on the leaves and flowers of many plants (16,17,20) and initiate much of the damage done to crops by frost. The ice nucleation-active (Ina') phenotype may confer a selective advantage on bacteria which express it; whether this advantage exists has been the subject of speculation (17,18 formed E. coli (32); these nuclei are active only at temperatures below -8°C.The precise relationship between ice nucleation proteins and the Ina' phenotype remains unclear. Many models for the role of ice nucleation proteins have been proposed (3,14,29). Such models are premature when two basic questions remain unanswered. Are ice nucleation proteins detectable in the organisms which are sources of ice nucleation genes, and are these proteins present only if the Ina' phenotype is present? This work reports results obtained with a polyclonal antibody raised against one ice nucleation protein. This antibody was used to answer the questions posed above, to identify two previously uncharacterized ice nucleation proteins, and to discover phenomena which indicate that the secondary structure of at least one nucleation protein is periodic.MATERIALS AND METHODS Chemicals, bacterial strains, and culture conditions. All chemicals were reagent grade, purchased from Sigma Chemical Co. unless otherwise note...

The epithelial Na ؉ channel (ENaC) is comprised of three homologous subunits (␣, ␤, and ␥) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acidexchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the ␤ and ␥ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant ␤ subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a ␤ C43A,C557A mutant had significantly reduced amiloridesensitive whole cell currents, enhanced Na ؉ self-inhibition, and reduced single channel P o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that ␤ Cys-43 is in proximity to the first transmembrane ␣ helix, whereas ␤ Cys-557 is within an amphipathic ␣-helix contiguous with the second transmembrane domain. We propose that ␤ subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.Epithelial sodium channels (ENaCs) 4 mediate amiloridesensitive Na ϩ transport across the apical membrane of high resistance epithelia and have important roles in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. The ␣, ␤, and ␥ subunits of ENaC are members of the ENaC/Degenerin family of ion channels, which include H ϩ -gated channels (referred to as acid-sensing ion channels (ASICs)) that are expressed in mammalian central and peripheral nervous systems and have a role in nociception and mechanosensation (1). ENaC and other family members are ion channels composed of subunits that have a similar topology with two transmembrane helices, a large extracellular region, including numerous conserved disulfide bridges, and cytoplasmic N and C termini. The recently resolved crystal structure of ASIC1 (2) revealed a homotrimer with highly organized extracellular regions, suggesting that ENaC is an ␣ 1 ␤ 1 ␥ 1 heterotrimer.ENaCs are assembled within the endoplasmic reticulum (ER), where they undergo N-linked glycosylation. Assembly is inefficient, and the majority of newly synthesized subunits undergo ER-associated degradation with a half-life of 1-2 h as determined by metabolic labeling (3-7). A small pool of newly synthesized subunits has a significantly longer half-life (Ͼ4 h) and represents assembled channels that have exited the ER and are present in later compartments. This latter pool contains subunits whose N-glycans have undergone both Golgi-dependent terminal processing and cleavage of ␣ and ␥ by furin, a protease localized primarily to the trans-Golgi network (5, 8, 9). ENaC exit from the ER is regulated by a signal within the C-terminal cytoplasmic domain of the ␣ su...

Integral membrane proteins are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). After being translocated or inserted into the ER, they fold and undergo posttranslational modifications. Within the ER, proteins are also subjected to quality control checkpoints, during which misfolded proteins may be degraded by proteasomes via a process known as ER-associated degradation. Molecular chaperones, including the small heat shock protein ␣A-crystallin, have recently been shown to play a role in this process. We have now found that ␣A-crystallin is expressed in cultured mouse collecting duct cells, where apical Na ؉ transport is mediated by epithelial Na ؉ channels (ENaC). ENaC-mediated Na ؉ currents in Xenopus oocytes were reduced by co-expression of ␣A-crystallin. This reduction in ENaC activity reflected a decrease in the number of channels expressed at the cell surface. Furthermore, we observed that the rate of ENaC delivery to the cell surface of Xenopus oocytes was significantly reduced by co-expression of ␣A-crystallin, whereas the rate of channel retrieval remained unchanged. We also observed that ␣A-crystallin and ENaC coimmunoprecipitate. These data are consistent with the hypothesis that small heat shock proteins recognize ENaC subunits at ER quality control checkpoints and can target ENaC subunits for ER-associated degradation.Like most other integral membrane proteins, newly synthesized epithelial Na ϩ channel (ENaC) 4 subunits translocate cotranslationally into the endoplasmic reticulum (ER), where folding and post-translational modifications occur. Within the ER, proteins are also subjected to quality control checkpoints to ensure that only properly folded proteins mature beyond the ER. If folding is inefficient, the misfolded protein may be degraded by proteasomes via a process known as ER-associated degradation (ERAD). This prevents the accumulation of abnormal proteins in the ER, which, left unchecked, may form toxic protein aggregates. Molecular chaperones, which can bind to exposed, hydrophobic motifs in unfolded proteins, play a key role in selecting substrates for this process.ENaC is expressed at the apical membranes of Na ϩ absorptive epithelia. There, in conjunction with the basolateral Na ϩ /K ϩ ATPase, ENaC facilitates transepithelial Na ϩ transport (1). ENaC is found in a variety of tissues, including the lung airway and alveoli and the distal nephron, where ENaC influences mucociliary clearance and extracellular fluid Na ϩ and volume regulation, respectively (1-3). ENaC is comprised of three homologous subunits, ␣, ␤, and ␥, although the stoichiometry of the functional channel remains controversial (4 -7). Each subunit has short cytoplasmic amino-and carboxyl-terminal domains (50 -110 residues), two transmembrane segments, and a large extracellular domain (ϳ450 residues) (8 -10). Like most other molecular chaperones, small heat shock proteins (sHsps) can bind unfolded, aggregation-prone substrates to retain them in solution. In addition, sHsps have been implicated in pro...

Our experience with 3-D ultrasound suggests that it is an advance in high-quality ultrasound. Its greatest advantage is that it allows the user to view simultaneously the three orthogonal planes.