Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride Transport

To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO₃ transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO₃ transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO₃⁻ currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO₃-dependent pH recovery from a CO₂-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO₂/HCO₃⁻-free solution that was abolished by Cl⁻ removal from the bath. In the presence of CO₂/HCO₃⁻, however, the outward current produced by D555E decreased only slightly after Cl⁻ removal. Starting from a Cl⁻-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO₂/HCO₃⁻. The substitution of Asp⁵⁵⁵ with an asparagine also produced a Cl⁻ current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO₃⁻. Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp⁵⁵⁵ plays a role in HCO₃⁻ selectivity.The electrogenic Na/HCO₃ cotransporter NBCe1 (SLC4A4) is one of the SLC4A gene family members transporting HCO₃⁻ across the plasma membrane (1–3). NBCe1 plays a role in transepithelial HCO₃⁻ movement and pH_i regulation in many tissues (4–6). NBCe1 is responsible for HCO₃⁻ reabsorption in the proximal tubules of the kidney (7). The proximal tubule cells reclaim HCO₃⁻ from the lumen through a series of reactions involving titration of HCO₃⁻ by H⁺ secretion via the apical Na/H exchanger, production of CO₂, and regeneration of HCO₃⁻ and H⁺ in the tubule cells. HCO₃⁻ then moves to the interstitium via the basolateral NBCe1. The essential feature driving this basolateral Na⁺/HCO₃⁻ exit is the stoichiometry of 1:3 Na⁺:HCO₃⁻, which makes the equilibrium potential for NBCe1 more positive than the resting membrane potential of the proximal tubule cells (8). The stoichiometry of 1Na⁺:1HCO₃⁻ or 1Na⁺:2HCO₃⁻ causes both ions to move into cells in other tissues such as pancreas, brain, and cardiovascular tissues (9, 10).Despite the importance of NBCe1 for basolateral HCO₃⁻ reabsorption in the proximal tubules, the mechanism of electrogenic Na/HCO₃ transport via the transporter is not well understood. Ion movement depends on loading ions at their translocation or binding sites that likely reside within the membrane field at some distance from the bath solution (11). This implies that the transmembrane domains (TMs)2 of NBCe1 and amino acid residues within TMs play critical roles in ion transport.Sequence analysis of different SLC4A proteins shows similar hydropathy plots, predicting that these proteins share structural elements of transport function (12). Such similarities have facilitated structure/function studies to define molecular domains or motifs responsible for conferring Na/HCO₃ transport of NBCe1. Abuladze et al. (13) performed a large scale mutagenesis on acidic and basic amino acids in non-TMs and found many residues affecting Na⁺-dependent base flux. McAlear et al. (14) identified amino acids in TM8 involving ion translocation. By a systematic approach of chimeric transporters between NBCe1 and the electroneutral Na/HCO₃ cotransporter NBCn1 (SLC4A7) (15), we and our colleagues (16) demonstrated that electrogenic Na/HCO₃ transport of NBCe1 requires interactions between the regions TM1–5 and TM6–13 of the protein. Zhu et al. (17) recently proposed TM1 as a domain lining the ion translocation pathway. On the other hand, Chang et al. (18) reported that the cytoplasmic N-terminal domain might contribute to HCO₃⁻ permeation.In the present study, we searched amino acid residues that are highly conserved among electrogenic Na/HCO₃ transporters but not among electroneutral Na/HCO₃ transporters and examined their role in electrogenic Na/HCO₃ transport. Nine candidate residues in human renal NBCe1-A (5, 19) were selected and mutated by replacement with the amino acids at the corresponding sites of NBCn1. Mutant transporters were expressed in Xenopus oocytes and assessed via two-electrode voltage clamp. Our data show that Asp⁵⁵⁵ of NBCe1 plays an important role in HCO₃⁻ selectivity.     

Determinants of Secretion and Intracellular Localization of Human Herpesvirus 8 Interleukin-6

Human herpesvirus 8 (HHV-8) interleukin-6 (vIL-6) is distinct from human and other cellular IL-6 proteins in that it does not require the nonsignaling α-receptor subunit for the formation of gp130-based signal transducing complexes and also is largely retained intracellularly rather than being secreted. We and others have reported that vIL-6 is retained and is active in the endoplasmic reticulum (ER) compartment, and data from our laboratory have demonstrated that intracellular vIL-6 is functional in the autocrine promotion of proliferation and survival of HHV-8 latently infected primary effusion lymphoma cells. It has also been reported that vIL-6 secretion in gp130-deficient cells can be enhanced by introduced gp130, thereby implicating the signal transducer in vIL-6 trafficking to the cell surface. We examine here the requirements for intracellular retention and localization of vIL-6. Using vIL-6-hIL-6 chimeric and point-mutated vIL-6 proteins, we identified regions and residues of vIL-6 influencing vIL-6 secretion. However, there was no correlation between vIL-6 secretion and gp130 interaction. We found that vIL-6, but not hIL-6, could associate stably with ER-resident chaperone protein calnexin. Glycosylation-dependent interaction of vIL-6 with calnexin correlated with proper protein folding, but there was no direct relationship between vIL-6-calnexin interaction and intracellular retention. While calnexin depletion had little influence on absolute amounts of secreted vIL-6, it led to markedly reduced levels of intracellular cytokine. This was reversed by gp130 transduction, which had no detectable effect on vIL-6 secretion, but redistributed vIL-6 into ER-distinct locations in calnexin-depleted cells, specifically. Our data reveal that calnexin plays a role in ER localization of vIL-6 and that gp130 promotes ER exit, but not secretion, of the viral cytokine.The viral homologue of interleukin-6, vIL-6, specified by human herpesvirus 8 (HHV-8) shows only 25% amino acid identity to human IL-6 (hIL-6) but is highly related structurally (2, 5). Despite the high degree of conservation of three-dimensional structure and equivalence of receptor interaction interfaces (1, 6), the viral cytokine can associate functionally with the gp130 signal transducer in the absence of the gp80 α-subunit, absolutely required for cellular IL-6 signaling through gp130. The nonsignaling gp80 subunit can be incorporated into vIL-6-induced signaling complexes and indeed seems to have a stabilizing effect that enhances signal transduction (1, 3, 11). Another major difference between vIL-6 and cellular IL-6 proteins, including hIL-6, is that the viral cytokine is very inefficiently secreted, retained largely within the endoplasmic reticulum (ER) compartment, where it is able to transduce signal via gp80-deficient vIL-6₂/gp130₂ tetrameric complexes, exclusively (4, 15). Thus, the unique ability of vIL-6 to signal intracellularly may be explained by its gp80 independence; hIL-6 cannot signal in the ER even when targeted to this compartment (4). The biological significance of intracellular, strictly autocrine signaling by vIL-6 was demonstrated recently in primary effusion lymphoma (PEL) cells, which are latently infected with HHV-8; these cells grew with markedly reduced kinetics and displayed higher rates of apoptosis upon shRNA-mediated vIL-6 depletion relative to cocultured untransduced cells (4). Thus, vIL-6 appears not only to be expressed in latently infected PEL cultures but also to be biologically active in this setting via intracrine signaling.Despite these findings and other mechanistic studies of vIL-6, the means by which the viral cytokine is retained in the ER and secreted so inefficiently is unknown. The elegant work of Meads and Medveczky (15) demonstrated the slow secretion kinetics of vIL-6 relative to hIL-6 and implicated gp130 as a necessary cofactor for vIL-6 secretion. Thus, vIL-6 expressed in gp130-negative Ba/F3 cells was able to be secreted only if gp130 was supplied via expression vector transduction. However, most cell types express gp130; thus, while the signal transducer may be involved in vIL-6 trafficking, the underlying explanation for the very slow rate of vIL-6 secretion must involve other factors.We report here investigations of the structural requirements for vIL-6 intracellular retention, the influence of gp130 on this process, and the possible involvement of ER-resident chaperon proteins for retention of vIL-6 in the ER. Our data identify effects of structural alterations and point mutations of vIL-6 on secretion efficiency, the lack of gp130 involvement in these observed effects, mechanistically relevant interactions of calnexin with the viral cytokine, and the influence of gp130 on vIL-6 subcellular localization and stability in the context of calnexin depletion. The results presented thus further advance our understanding of vIL-6-cellular protein interactions that impact upon its intracellular function.     

Classical swine fever virus can remain virulent after specific elimination of the interferon regulatory factor 3-degrading function of Npro

Pestiviruses prevent alpha/beta interferon (IFN-alpha/beta) production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3) by means of the viral N(pro) nonstructural protein. N(pro) is also an autoprotease, and its amino-terminal coding sequence is involved in translation initiation. We previously showed with classical swine fever virus (CSFV) that deletion of the entire N(pro) gene resulted in attenuation in pigs. In order to elaborate on the role of the N(pro)-mediated IRF3 degradation in classical swine fever pathogenesis, we searched for minimal amino acid substitutions in N(pro) that would specifically abrogate this function. Our mutational analyses showed that degradation of IRF3 and autoprotease activity are two independent but structurally overlapping functions of N(pro). We describe two mutations in N(pro) that eliminate N(pro)-mediated IRF3 degradation without affecting the autoprotease activity. We also show that the conserved standard sequence at these particular positions is essential for N(pro) to interact with IRF3. Surprisingly, when these two mutations are introduced independently in the backbones of highly and moderately virulent CSFV, the resulting viruses are not attenuated, or are only partially attenuated, in 8- to 10-week-old pigs. This contrasts with the fact that these mutant viruses have lost the capacity to degrade IRF3 and to prevent IFN-alpha/beta induction in porcine cell lines and monocyte-derived dendritic cells. Taken together, these results demonstrate that contrary to previous assumptions and to the case for other viral systems, impairment of IRF3-dependent IFN-alpha/beta induction is not a prerequisite for CSFV virulence.     

Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers

AimsUnder normal conditions, the intestinal mucosa acts as a local barrier to prevent the influx of luminal contents. The intestinal epithelial tight junction is comprised of several membrane associated proteins, including zonula occludens-1 (ZO-1) and occludin. Disruption of this barrier can lead to the production of pro-inflammatory mediators and ultimately multiple organ failure. We have previously shown that Pentoxifylline (PTX) decreases histologic gut injury and pro-inflammatory mediator synthesis. We hypothesize that PTX prevents the breakdown of ZO-1 and occludin in an in vitro model of immunostimulated intestinal cell monolayers.Main methodsCaco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-α, IFN-γ, IL-1), or Cytomix + PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin.Key findingsCytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix + PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization.SignificanceTreatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown.