Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against Clostridium difficile infection.

The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile. Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab')2 fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens.

Interhelical hydrogen bonds in the CFTR membrane domain.

Critical mutations in the membrane-spanning domains of proteins cause many human diseases. We report the expression in Escherichia coli of helix-loop-helix segments of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel domain in milligram quantities. Analysis of gel migration patterns of these constructs, in conjunction with circular dichroism spectroscopy, demonstrate that a neutral-to-charged, CF-phenotypic point mutation of a hydrophobic residue (V232D) in the CFTR transmembrane (TM) helix 4 induces a hydrogen bond with neighboring wild type Gln 207 in TM helix 3. As an electrostatic crosslink within a hydrocarbon phase, such a hydrogen bond could alter the normal assembly and alignment of CFTR TM helices and/or impede their movement in response to substrate transport. Our results imply that membrane proteins may be vulnerable to loss of function through formation of membrane-buried interhelical hydrogen bonds by partnering of proximal polar side chains.

Molecular and functional studies of the gamma subunit of the sodium pump.

This article reviews our studies of the gamma subunit of the sodium pump. Gamma is a member of the FXYD family of small, single transmembrane proteins and is expressed predominantly in the kidney tubule. There are two major variants of gamma which function similarly to bring about two distinct effects, one on K'(ATP) and the other, on K(K), the affinity of the pump for K+ acting as a competitor of cytoplasmic Na+. In this way, gamma is believed to provide a self-regulatory mechanism for maintaining the steady-state activity of the pump in the kidney. Our studies also suggest that K+ antagonism of cytoplasmic Na+ activation of the pump is relevant not only to the presence of gamma in the kidney, but probably some hitherto undefined factor(s) in other tissues, most notably heart. The interesting possibility that not only gamma but other members of the FXYD family regulate ion transport in a tissue-specific manner is discussed.

Mechanisms of sodium pump regulation.

The Na(+)-K(+)-ATPase, or sodium pump, is the membrane-bound enzyme that maintains the Na(+) and K(+) gradients across the plasma membrane of animal cells. Because of its importance in many basic and specialized cellular functions, this enzyme must be able to adapt to changing cellular and physiological stimuli. This review presents an overview of the many mechanisms in place to regulate sodium pump activity in a tissue-specific manner. These mechanisms include regulation by substrates, membrane-associated components such as cytoskeletal elements and the gamma-subunit, and circulating endogenous inhibitors as well as a variety of hormones, including corticosteroids, peptide hormones, and catecholamines. In addition, the review considers the effects of a range of specific intracellular signaling pathways involved in the regulation of pump activity and subcellular distribution, with particular consideration given to the effects of protein kinases and phosphatases.

K(+)/Na(+) antagonism at cytoplasmic sites of Na(+)-K(+)-ATPase: a tissue-specific mechanism of sodium pump regulation.

Tissue-distinct interactions of the Na(+)-K(+)-ATPase with Na(+) and K(+), independent of isoform-specific properties, were reported previously (A. G. Therien, N. B. Nestor, W. J. Ball, and R. Blostein. J. Biol. Chem. 271: 7104-7112, 1996). In this paper, we describe a detailed analysis of tissue-specific kinetics particularly relevant to regulation of pump activity by intracellular K(+), namely K(+) inhibition at cytoplasmic Na(+) sites. Our results show that the order of susceptibilities of alpha(1) pumps of various rat tissues to K(+)/Na(+) antagonism, represented by the ratio of the apparent affinity for Na(+) binding at cytoplasmic activation sites in the absence of K(+) to the affinity constant for K(+) as a competitive inhibitor of Na(+) binding at cytoplasmic sites, is red blood cell < axolemma approximately rat alpha(1)-transfected HeLa cells < small intestine < kidney < heart. In addition, we have carried out an extensive analysis of the kinetics of K(+) binding and occlusion to the cytoplasmic cation binding site and find that, for most tissues, there is a relationship between the rate of K(+) binding/occlusion and the apparent affinity for K(+) as a competitive inhibitor of Na(+) activation, the order for both parameters being heart >/= kidney > small intestine approximately rat alpha(1)-transfected HeLa cells. The notion that modulations in cytoplasmic K(+)/Na(+) antagonism are a potential mode of pump regulation is underscored by evidence of its reversibility. Thus the relatively high K(+)/Na(+) antagonism characteristic of kidney pumps was reduced when rat kidney microsomal membranes were fused into the dog red blood cell.

Expression and functional role of the gamma subunit of the Na, K-ATPase in mammalian cells.

The functional role of the gamma subunit of the Na,K-ATPase was studied using rat gamma cDNA-transfected HEK-293 cells and an antiserum (gammaC33) specific for gamma. Although the sequence for gamma was verified and shown to be larger (7237 Da) than first reported, it still comprises a single initiator methionine despite the expression of a gammaC33-reactive doublet on immunoblots. Kinetic analysis of the enzyme of transfected compared with control cells and of gammaC33-treated kidney pumps shows that gamma regulates the apparent affinity for ATP. Thus, gamma-transfected cells have a decreased K'ATP as shown in measurements of (i) K'ATP of Na,K-ATPase activity and (ii) K+ inhibition of Na-ATPase at 1 microM ATP. Consistent with the behavior of gamma-transfected cells, gammaC33 pretreatment increases K'ATP of the kidney enzyme and K+ inhibition (1 microM ATP) of both kidney and gamma-transfected cells. These results are consistent with previous findings that an antiserum raised against the pig gamma subunit stabilizes the E2(K) form of the enzyme (Therien, A. G., Goldshleger, R., Karlish, S. J., and Blostein, R. (1997) J. Biol. Chem. 272, 32628-32634). Overall, our data demonstrate that gamma is a tissue (kidney)-specific regulator of the Na,K-ATPase that can increase the apparent affinity of the enzyme for ATP in a manner that is reversible by anti-gamma antiserum.

Tissue-specific distribution and modulatory role of the gamma subunit of the Na,K-ATPase.

The Na,K-ATPase comprises a catalytic alpha subunit and a glycosylated beta subunit. Another membrane polypeptide, gamma, first described by Forbush et al. (Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. (1978) Biochemistry 17, 3667-3676) associates with alpha and beta in purified kidney enzyme preparations. In this study, we have used a polyclonal anti-gamma antiserum to define the tissue specificity and topology of gamma and to address the question of whether gamma has a functional role. The trypsin sensitivity of the amino terminus of the gamma subunit in intact right-side-out pig kidney microsomes has confirmed that it is a type I membrane protein with an extracellular amino terminus. Western blot analysis shows that gamma subunit protein is present only in membranes from kidney tubules (rat, dog, pig) and not those from axolemma, heart, red blood cells, kidney glomeruli, cultured glomerular cells, alpha1-transfected HeLa cells, all derived from the same (rat) species, nor from three cultured cell lines derived from tubules of the kidney, namely NRK-52E (rat), LLC-PK (pig), or MDCK (dog). To gain insight into gamma function, the effects of the anti-gamma serum on the kinetic behavior of rat kidney sodium pumps was examined. The following evidence suggests that gamma stabilizes E1 conformation(s) of the enzyme and that anti-gamma counteracts this effect: (i) anti-gamma inhibits Na,K-ATPase, and the inhibition increases at acidic pH under which condition the E2(K) --> E1 phase of the reaction sequence becomes more rate-limiting, (ii) the oligomycin-stimulated increase in the level of phosphoenzyme was greater in the presence of anti-gamma indicating that the antibody shifts the E1 left and right arrow left and right arrow E2P equilibria toward E2P, and (iii) when the Na+-ATPase reaction is assayed with the Na+ concentration reduced to levels ( --> E2P transition, anti-gamma is stimulatory. These observations taken together with evidence that the pig gamma subunit, which migrates as a doublet on polyacrylamide gels, is sensitive to digestion by trypsin, and that Rb+ ions partially protect it against this effect, indicate that the gamma subunit is a tissue-specific regulator which shifts the steady-state equilibria toward E1. Accordingly, binding of anti-gamma disrupts alphabeta-gamma interactions and counteracts these modulatory effects of the gamma subunit.

Tissue-specific versus isoform-specific differences in cation activation kinetics of the Na,K-ATPase.

The experiments described in this report reconcile some of the apparent differences in isoform-specific kinetics of the Na,K-ATPase reported in earlier studies. Thus, tissue-specific differences in Na+ and K+ activation kinetics of Na,K-ATPase activity of the same species (rat) were observed when the same isoform was assayed in different tissues or cells. In the case of alpha1, alpha1-transfected HeLa cell, rat kidney, and axolemma membranes were compared. For alpha3, the ouabain-insensitive alpha3*-transfected HeLa cell (cf. Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930), pineal gland, and axolemma (mainly alpha3) membranes were compared. The order of apparent affinities for Na+ of alpha1 pumps was axolemma approximately rat alpha1-transfected HeLa > kidney, and for K+, kidney approximately alpha1-transfected HeLa > axolemma. For alpha3, the order of apparent affinities for Na+ was pineal gland approximately axolemma > alpha3*-transfected HeLa, and for K+, alpha3*-transfected HeLa > axolemma approximately pineal gland. In addition, the differences in apparent affinities for Na+ of either kidney alpha1 or HeLa alpha3* as compared to the same isoform in other tissues were even greater when the K+ concentration was increased. A kinetic analysis of the apparent affinities for Na+ as a function of K+ concentration indicates that isoform-specific as well as tissue-specific differences are related to the apparent affinities for both Na+ and K+, the latter acting as a competitive inhibitor at cytoplasmic Na+ activation sites. Although the nature of the tissue-specific modulation of K+/Na+ antagonism remains unknown, an analysis of the nature of the beta isoform associated with alpha1 or alpha3 using isoform-specific immunoprecipitation indicates that the presence of distinct beta subunits does not account for differences of alpha1 of kidney, axolemma, and HeLa, and of alpha3 of axolemma and HeLa; in both instances beta1 is the predominant beta isoform present or associated with either alpha1 or alpha3. However, a kinetic difference in K+/Na+ antagonism due to distinct betas may apply to alpha3 of axolemma (alpha3beta1) and pineal gland ( alpha3beta2).