NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia.

BACKGROUND AND PURPOSE: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia. EXPERIMENTAL APPROACH: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC). KEY RESULTS: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action. CONCLUSIONS AND IMPLICATIONS: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.

Pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) nicotinic acetylcholine receptors: subunit arrangement determines functional expression.

BACKGROUND AND PURPOSE: alpha4 and beta2 nicotinic acetylcholine (ACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixed population of (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors. In order to express these receptors separately in heterologous systems, we have engineered pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors. EXPERIMENTAL APPROACH: alpha4 and beta2 subunits were concatenated by synthetic linkers into pentameric constructs to produce either (alpha4)(2)(beta2)(3) or (alpha4)(3)(beta2)(2) receptors. Using two-electrode voltage-clamp techniques, we examined the ability of the concatenated constructs to produce functional expression in Xenopus oocytes. Functional constructs were further characterized in respect to agonists, competitive antagonists, Ca2+ permeability, sensitivity to modulation by Zn2+ and sensitivity to up-regulation by chaperone protein 14-3-3. KEY RESULTS: We found that pentameric concatamers with a subunit arrangement of beta2_alpha4_beta2_alpha4_beta2 or beta2_alpha4_beta2_alpha4_alpha4 were stable and functional in Xenopus oocytes. By comparison, when alpha4 and beta2 were concatenated with a subunit order of beta2_beta2_alpha4_beta2_alpha4 or beta2_alpha4_alpha4_beta2_alpha4, functional expression in Xenopus oocytes was very low, even though the proteins were synthesized and stable. Both beta2_alpha4_beta2_alpha4_beta2 and beta2_alpha4_beta2_alpha4_alpha4 concatamers recapitulated the ACh concentration response curve, the sensitivity to Zn2+ modulation, Ca2+ permeability and the sensitivity to up-regulation by chaperone protein 14-3-3 of the corresponding non-linked (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors respectively. Using these concatamers, we found that most alpha4beta2-preferring compounds studied, including A85380, 5I-A85380, cytisine, epibatidine, TC2559 and dihydro-beta-erythroidine, demonstrate stoichiometry-specific potencies and efficacies. CONCLUSIONS AND IMPLICATIONS: We concluded that the alpha4beta2 nicotinic ACh receptors produced with beta2_alpha4_beta2_alpha4_beta2 or beta2_alpha4_beta2_alpha4_alpha4 pentameric constructs are valid models of non-linked (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors respectively.

The guinea-pig tracheal potential difference as an in vivo model for the study of epithelial sodium channel function in the airways.

BACKGROUND AND PURPOSE: The epithelial sodium channel (ENaC) is a key regulator of airway mucosal hydration and mucus clearance. Negative regulation of airway ENaC function is predicted to be of clinical benefit in the cystic fibrosis lung. The aim of this study was to develop a small animal model to enable the direct assessment of airway ENaC function in vivo. EXPERIMENTAL APPROACH: Tracheal potential difference (TPD) was utilized as a measure of airway epithelial ion transport in the guinea-pig. ENaC activity in the trachea was established with a dose-response assessment to a panel of well-characterized direct and indirect pharmacological modulators of ENaC function, delivered by intra-tracheal (i.t.) instillation. KEY RESULTS: The TPD in anaesthetized guinea-pigs was attenuated by the direct ENaC blockers: amiloride, benzamil and CF552 with ED(50) values of 16, 14 and 0.2 microg kg(-1) (i.t.), respectively. 5-(N-Ethyl-N-isopropyl) amiloride, a structurally related compound but devoid of activity on ENaC, was without effect on the TPD. Intra-tracheal dosing of the Kunitz-type serine protease inhibitors aprotinin and placental bikunin, which have previously been demonstrated to inhibit proteolytic activation of ENaC, likewise potently attenuated TPD in guinea-pigs, whereas alpha(1)-antitrypsin and soya bean trypsin inhibitor were without effect. CONCLUSIONS AND IMPLICATIONS: The pharmacological sensitivity of the TPD to amiloride analogues and also to serine protease inhibitors are both consistent with that of ENaC activity in the guinea-pig trachea. The guinea-pig TPD therefore represents a suitable in vivo model of human airway epithelial ion transport.

Formation of functional alpha3beta4alpha5 human neuronal nicotinic receptors in Xenopus oocytes: a reporter mutation approach.

1. The alpha5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related beta3 subunit, alpha5 forms functional recombinant receptors if expressed together with a pair of typical alpha and beta subunits, but its effect on the properties of the resulting alphabetaalpha5 receptor depends on the alpha and beta subunits chosen and on the expression system. We used a reporter mutation approach to test whether alpha5, like beta3, is incorporated as a single copy in human alpha3beta4alpha5 receptors expressed in oocytes. 2. As previously reported, the main indication of the presence of alpha5 in alpha3beta4alpha5(wt) was an increase in apparent receptor desensitization (compared with alpha3beta4 receptors). If the alpha3beta4alpha5 receptor bore a 9'T mutation in the second transmembrane domain of either alpha3 or beta4, alpha5 incorporation produced a decrease in ACh sensitivity (by 4 fold for alpha3(LT)beta4alpha5 vs. alpha3(LT)beta4 and by 40 fold for alpha3beta4(LT)alpha5 vs. alpha3beta4(LT)). The much greater effect observed in alpha3beta4(LT)alpha5 receptors accords with the hypothesis that alpha5 takes the place of a beta subunit in the receptor. 3. Introducing a 9'T mutation in alpha5 had no effect on the agonist sensitivity of alpha3beta4alpha5 receptors, but reduced apparent desensitisation, as judged by the sag in the current response to high agonist concentrations. 4. Introducing the 9'T mutation in alpha3 or beta4 in the triplet receptor reduced the EC(50) for ACh by a similar extent (7 and 9 fold, respectively), suggesting that alpha3beta4alpha5 receptors contain two copies each of alpha and beta and therefore only one copy of alpha5.

Stoichiometry of human recombinant neuronal nicotinic receptors containing the b3 subunit expressed in Xenopus oocytes.

The neuronal nicotinic subunit beta3 forms functional receptors when co-expressed with both an alpha and a beta subunit, such as alpha3 and beta4. We examined the subunit stoichiometry of these 'triplet' alpha3beta4beta3 receptors by expression in Xenopus oocytes of the alpha3, beta4 and beta3 subunits, either in wild-type form or after insertion of a reporter mutation. The mutation chosen was the substitution of a conserved hydrophobic residue in the second transmembrane domain of the subunits (leucine or valine 9THORN ) with a hydrophilic threonine. In other ion channels within the nicotinic superfamily, this mutation type consistently increases the potency of agonists. In muscle-type nicotinic receptors, the magnitude of this effect is approximately constant for each mutant subunit incorporated. In alpha3beta4beta3 receptors, the ACh EC50 was decreased by approximately 17-fold when this mutation was in alpha3 alone and only by fourfold when beta3 alone was mutated. Mutating beta4 was equivalent to mutating alpha3, suggesting that the 'triplet' receptor contains one copy of beta3 and two copies each of alpha3 and beta4. Mutating beta3 and alpha3 or beta3 and beta4 reduced the ACh EC50 further, to values two- to threefold lower than those seen when only alpha3 or beta4 carried the mutation. In 'pair' alpha3beta4 receptors (known to contain two alpha and three beta subunits), mutating beta4 had a greater effect on the ACh EC50 than mutating alpha3, in agreement with an alpha:beta ratio of 2:3 and a constant and independent effect of each copy of the mutation. Our results suggest that alpha3beta4beta3 neuronal nicotinic receptors contain one copy of beta3 and two copies each of alpha3 and beta4 and confirm that in pair alpha3beta4 receptors the alpha/beta subunits are present in a 2:3 ratio.

Galantamine is an allosterically potentiating ligand of the human alpha4/beta2 nAChR.

Galantamine (Reminyl) is a novel drug treatment for mild to moderate Alzheimer's disease (AD). Originally established as a reversible inhibitor of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), galantamine also acts as an allosterically potentiating ligand (APL) on nicotinic acetylcholine receptors (nAChR). Having previously established this second mode of action on nAChRs from murine brain, we demonstrate here the same action of galantamine on the most abundant nAChR in the human brain, the alpha4/beta2 subtype. This nAChR-sensitizing action is not a common property of all, or most, AChE inhibitors, as is shown by the absence of this effect for other therapeutically applied AChE inhibitors including tacrine, metrifonate, rivastigmine and donepezil. The possible benefits for therapy of AD of an APL action on nicotinic receptors is discussed.

A reporter mutation approach shows incorporation of the "orphan" subunit beta3 into a functional nicotinic receptor.

We have investigated whether the neuronal nicotinic subunit beta3 can participate in the assembly of functional recombinant receptors. Although beta3 is expressed in several areas of the central nervous system, it does not form functional receptors when expressed heterologously together with an alpha or another beta nicotinic subunit. We inserted into the human beta3 subunit a reporter mutation (V273T), which, if incorporated into a functional receptor, would be expected to increase its agonist sensitivity and maximum response to partial agonists. Expressing the mutant beta3(V273T) in Xenopus oocytes together with both the alpha3 and the beta4 subunits resulted in the predicted changes in the properties of the resulting nicotinic receptor when compared with those of alpha3 beta4 receptors. This indicated that some of the receptors incorporated the mutant beta3 subunit, as part of a "triplet" alpha3 beta4 beta3 receptor. The proportion of triplet receptors was dependent on the ratios of the alpha3:beta4:beta3 cRNA injected. We conclude that, like the related alpha5 subunit, the beta3 subunit can form functional receptors only if expressed together with both alpha and beta subunits.

Cloning and sequence of full-length cDNAs encoding the human neuronal nicotinic acetylcholine receptor (nAChR) subunits beta3 and beta4 and expression of seven nAChR subunits in the human neuroblastoma cell line SH-SY5Y and/or IMR-32.

Using PCR-based techniques, we have isolated and sequenced the full-length cDNAs that encode the human neuronal nAChR subunits alpha3,4,5,7 and beta2,3,4 in the neuroblastoma cell lines SH-SY5Y and/or IMR-32. The predicted nAChR beta3- and beta4-subunit proteins contain 458 and 498 amino acids, respectively. Except for the beta2, all the other cloned cDNAs showed differences with the published sequences. Northern blots show expression of the nAChR subunits alpha3,7 and beta2,4 in the human neuroblastoma cell line SH-SY5Y, with intensity of the hybridisation signal decreasing in the order alpha3 > beta4 > beta2 > alpha7.

Proprotein processing activity and cleavage site selectivity of the Kex2-like endoprotease PACE4.

Proprotein processing activity of the Kex2-like mammalian endoprotease PACE4 and its cleavage selectivity for sites with basic amino acid residues were determined. Using a recombinant vaccinia virus-based expression system, PACE4 was expressed in pig kidney PK(15) cells and, like two other Kex2-like endoproteases furin and PC6A, shown to correctly process the precursor of von Willebrand factor (pro-vWF). Furthermore, characteristics of the cleavage site selectivity of PACE4 were compared to those of furin and PC6A using the vWF cleavage site mutants vWFR-1G, vWFK-2A, and vWFR-4A as substrates. Cleavage site selectivity of PACE4 and PC6A appeared to be similar but they differed from that of furin.