Waheed Shabbir

A novel tumor necrosis factor–mediated mechanism of direct epithelial sodium channel activation

RATIONALE Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. OBJECTIVES In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). METHODS We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. MEASUREMENTS AND MAIN RESULTS TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. CONCLUSIONS These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

Mechanism of action of novel lung edema therapeutic AP301 by activation of the epithelial sodium channel.

AP301 [Cyclo(CGQRETPEGAEAKPWYC)], a cyclic peptide comprising the human tumor necrosis factor lectin-like domain (TIP domain) sequence, is currently being developed as a treatment for lung edema and has been shown to reduce extravascular lung water and improve lung function in mouse, rat, and pig models. The current paradigm for liquid homeostasis in the adult mammalian lung is that passive apical uptake of sodium via the amiloride-sensitive epithelial Na+ channel (ENaC) and nonselective cyclic-nucleotide–gated cation channels creates the major driving force for reabsorption of water through the alveolar epithelium in addition to other ion channels such as potassium and chloride channels. AP301 can increase amiloride-sensitive current in A549 cells as well as in freshly isolated type II alveolar epithelial cells from different species. ENaC is expressed endogenously in all of these cell types. Consequently, this study was undertaken to determine whether ENaC is the specific target of AP301. The effect of AP301 in A549 cells as well as in human embryonic kidney cells and Chinese hamster ovary cells heterologously expressing human ENaC subunits (α, β, γ, and δ) was measured in patch clamp experiments. The congener TIP peptide AP318 [Cyclo(4-aminobutanoic acid-GQRETPEGAEAKPWYD)] activated ENaC by increasing single-channel open probability. AP301 increased current in proteolytically activated (cleaved) but not near-silent (uncleaved) ENaC in a reversible manner. αβγ- or δβγ-ENaC coexpression was required for maximal activity. No increase in current was observed after deglycosylation of extracellular domains of ENaC. Thus, our data suggest that the specific interaction of AP301 with both endogenously and heterologously expressed ENaC requires precedent binding to glycosylated extracellular loop(s).

A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes

Background: Membrane attachment of PrP via a GPI anchor is critical for conversion into PrPSc. Results: Semisynthetic membrane-anchored PrP variants are generated and analyzed in different lipid environments. Conclusion: A membrane anchor drastically changes PrP interactions with membranes and enables pore formation. Significance: Membrane-induced conformational changes and pore formation of PrP play a role in the conversion into PrPSc and its toxic effects. Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrPC into pathogenic PrPSc. Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23–231, FL_PrP), N-terminally truncated PrP (residues 90–231, T_PrP), and PrP missing its central hydrophobic region (Δ105–125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit.

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the β and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

Glycosylation-dependent activation of epithelial sodium channel by solnatide

Dysfunction of the epithelial sodium channel (ENaC), which regulates salt and water homeostasis in epithelia, causes several human pathological conditions, including pulmonary oedema. This is a potentially lethal complication of acute lung injury at least partially caused by dysfunctional alveolar liquid clearance, which in turn impairs alveolar gas exchange. Solnatide (named TIP-peptide, AP301), a 17 residue peptide mimicking the lectin-like domain of TNF has been shown to activate ENaC in several experimental animal models of acute lung injury and is being evaluated as a potential therapy for pulmonary oedema. The peptide has recently completed phase 1 and 2a clinical trials. In this study, we identify a glycosylation-dependent mechanism that preserves ENaC function and expression. Since our previous data suggested that the pore-forming subunits of ENaC are essential for maximal current activation by solnatide, we performed single- and multi-N-glycosylation site mutations in αN232,293,312,397,511Q- and δN166,211,384Q-subunits, in order to identify crucial residues for interaction with solnatide within the extracellular loop of the channel. Additionally, we generated αL576X and αN232,293,312,397,511Q,L576X deletion mutants of ENaC-α, since we have previously demonstrated that the carboxy terminal domain of this subunit is also involved in its interaction with solnatide. In cells expressing αN232,293,312,397,511Q,L576Xβγ-hENaC or δN166,311,384Q,D552Xβγ-hENaC activation by solnatide, as measured in whole cell patch clamp mode, was completely abolished, whereas it was attenuated in αL576Xβγ-hENaC- and δD552Xβγ-hENaC-expressing cells. Taken together, our findings delineate an N-glycan dependent interaction between the TIP-peptide and ENaC leading to normalization of both sodium and fluid absorption in oedematous alveoli to non-oedematous levels.

Interaction of diltiazem with an intracellularly accessible binding site on CaV1.2

BACKGROUND AND PURPOSE Diltiazem inhibits CaV1.2 channels and is widely used in clinical practice to treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α1 subunit of CaV1.2. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane‐impermeable quaternary derivative d‐cis‐diltiazem (qDil) and mutant α1 subunits.

Physicochemical properties of pore residues predict activation gating of CaV1.2: A correlation mutation analysis

Single point mutations in pore-forming S6 segments of calcium channels may transform a high-voltage-activated into a low-voltage-activated channel, and resulting disturbances in calcium entry may cause channelopathies (Hemara-Wahanui et al., Proc Natl Acad Sci U S A 102(21):7553–7558, 16). Here we ask the question how physicochemical properties of amino acid residues in gating-sensitive positions on S6 segments determine the threshold of channel activation of CaV1.2. Leucine in segment IS6 (L434) and a newly identified activation determinant in segment IIIS6 (G1193) were mutated to a variety of amino acids. The induced leftward shifts of the activation curves and decelerated current activation and deactivation suggest a destabilization of the closed and a stabilisation of the open channel state by most mutations. A selection of 17 physicochemical parameters (descriptors) was calculated for these residues and examined for correlation with the shifts of the midpoints of the activation curve (ΔVact). ΔVact correlated with local side-chain flexibility in position L434 (IS6), with the polar accessible surface area of the side chain in position G1193 (IIIS6) and with hydrophobicity in position I781 (IIS6). Combined descriptor analysis for positions I781 and G1193 revealed that additional amino acid properties may contribute to conformational changes during the gating process. The identified physicochemical properties in the analysed gating-sensitive positions (accessible surface area, side-chain flexibility, and hydrophobicity) predict the shifts of the activation curves of CaV1.2.

Restoration of Epithelial Sodium Channel Function by Synthetic Peptides in Pseudohypoaldosteronism Type 1B Mutants

The synthetically produced cyclic peptides solnatide (a.k.a. TIP or AP301) and its congener AP318, whose molecular structures mimic the lectin-like domain of human tumor necrosis factor (TNF), have been shown to activate the epithelial sodium channel (ENaC) in various cell- and animal-based studies. Loss-of-ENaC-function leads to a rare, life-threatening, salt-wasting syndrome, pseudohypoaldosteronism type 1B (PHA1B), which presents with failure to thrive, dehydration, low blood pressure, anorexia and vomiting; hyperkalemia, hyponatremia and metabolic acidosis suggest hypoaldosteronism, but plasma aldosterone and renin activity are high. The aim of the present study was to investigate whether the ENaC-activating effect of solnatide and AP318 could rescue loss-of-function phenotype of ENaC carrying mutations at conserved amino acid positions observed to cause PHA1B. The macroscopic Na+ current of all investigated mutants was decreased compared to wild type ENaC when measured in whole-cell patch clamp experiments, and a great variation in the membrane abundance of different mutant ENaCs was observed with Western blotting experiments. However, whatever mechanism leads to loss-of-function of the studied ENaC mutations, the synthetic peptides solnatide and AP318 could restore ENaC function up to or even higher than current levels of wild type ENaC. As therapy of PHA1B is only symptomatic so far, the peptides solnatide and AP318, which directly target ENaC, are promising candidates for the treatment of the channelopathy-caused disease PHA1B.

Alveolar liquid clearance in lung injury: Evaluation of the impairment of the β2-adrenergic agonist response in an ischemia-reperfusion lung injury model

While alveolar liquid clearance (ALC) mediated by the β2-adrenergic receptor (β2-AR) plays an important role in lung edema resolution in certain models of lung injury, in more severe lung injury models, this response might disappear. Indeed, we have shown that in an ischemia-reperfusion-induced lung injury model, β2-agonists do not enhance ALC. The objective of this study was to determine if downregulation of the β2-AR could explain the lack of response to β2-agonists in this lung injury model. In an in vivo canine model of lung transplantation, we observed no change in β2-AR concentration or affinity in the injured transplanted lungs compared to the native lungs. Furthermore, we could not enhance ALC in transplanted lungs with dcAMP + aminophylline, a treatment that bypasses the β2-adrenergic receptor and is known to stimulate ALC in normal lungs. However, transplantation decreased αENaC expression in the lungs by 50%. We conclude that the lack of response to β2-agonists in ischemia-reperfusion-induced lung injury is not associated with significant downregulation of the β2-adrenergic receptors but is attributable to decreased expression of the ENaC channel, which is essential for sodium transport and alveolar liquid clearance in the lung.

Identification of phosphorylation sites and binding pockets for modulation of NaV1.5 channel by Fyn tyrosine kinase

Cardiac sodium channel NaV1.5 is the predominant form of sodium channels in cardiomyocytes, which exists as a macromolecular complex and interacts with multiple protein partners. Fyn kinase is one of the interacting proteins which colocalize, phosphorylate and modulate the NaV1.5 channel. To elaborate this interaction we created expression vectors for the N‐terminal, intracellular loop, and C‐terminal regions of the NaV1.5 channel, to express in HEK‐293 cells. By co‐immunoprecipitation and anti‐phosphotyrosine blotting, we identified proline‐rich binding sites for Fyn kinase in the N‐terminal, IC‐loopi–ii and C‐terminal. After binding, Fyn kinase phosphorylates tyrosine residues present in the N‐ and C‐terminal, which produce a depolarizing shift of 7 mV in fast inactivation. The functional relevance of these binding and phosphorylation sites was further underpinned by creating full length mutants masking these sites sequentially. An activation and inactivation curves were recorded with or without co‐expressed Fyn kinase which indicates that phosphorylation of tyrosine residues at positions 68, 87, 112 in the N‐terminal and at positions 1811 and 1889 in the C‐terminal creates a depolarizing shift in fast inactivation of NaV1.5 channel.