Aquaglyceroporine: Einfache Glyceroporen?: Evaluation der Leitfähigkeit ausgewählter Aquaglyceroporine für geladene Solute

In Aquaporinen wird ein Ausschluss von Kationen durch ein Zusammenspiel des ar/R-Selektivitätsfilters und der NPA-Region gewährleistet. Im Rahmen dieser Arbeit wurde die Leitfähigkeit des T. brucei AQP2 (TbAQP2) für Pentamidin als organisches Kation und des humanen AQP9 (AQP9) für Monocarboxylate als organische Anionen untersucht. In TbAQP2 befindet sich anstelle eines konservierten Arginins im ar/R-Selektivitätsfilter an Position 264 ein Leucin, welches dafür sorgt, dass das benachbarte Aspartat265 für eine Wechselwirkung freisteht. Leitfähigkeitsmessungen zeigten, dass organische Kationen, die aufgrund ihrer Größe die Kanalpore von TbAQP2 überwinden könnten, aufgrund ihrer Ladung ausgeschlossen wurden und damit eine Passage des 340 Da großen und zweifach positiven Pentamidins unwahrscheinlich ist. Pentamidin agiert vielmehr als nanomolarer Inhibitor der Glycerolleitfähigkeit von TbAQP2 mit einem IC50 von 130 nM. Das Aspartat265 wurde als essentielle Aminosäure identifiziert, die mit der Amidinfunktion des Pentamidins eine elektrostatische Wechselwirkung eingeht. AQP9 hat einen unauffälligen Selektivitätsfilter, zeigt hingegen ein ungewöhnlich breites Solutspektrum, u.a. für Monocarbonsäuren/Monocarboxylate. Es konnte gezeigt werden, dass nicht das Anion, sondern die neutrale Säure die Kanalpore von AQP9 unabhängig vom Protonengradienten überquert. Es wurden zwei Arginine an Position 51 und 53 in der Poreneingangsregion als für die Monocarbonsäureleitfähigkeit essentiell identifiziert. Diese sorgen für eine Anziehung des Monocarboxylat-Anions und eine erhöhte Aufenthaltswahrscheinlichkeit dessen. Somit wurde gezeigt, dass die Elektrostatik von Aquaglyceroporinen einen entscheidenden Einfluss auf ihre Funktion hat. In AQP9 führt eine positive Elektrostatik zu einer Spezialisierung auf Monocarbonsäuren und in TbAQP2 ist es eine elektrostatische Interaktion mit der Carboxylgruppe einer einzelnen Aminosäure, die zur nanomolaren Affinität von Pentamidin beiträgt.In aquaporins the ar/R-selectivity filter and NPA-region form a positive electrostatic barrier that strictly excludes cations. In this thesis the conductivity of T. brucei AQP2 (TbAQP2) for the positively charged pentamidine and of human AQP9 (AQP9) for negatively charged monocarboxylates was evaluated. The canonical amino acid composition of the AQP selectivity filter is an arginine in an aromatic environment (ar/R). TbAQP2, however, carries a Leu264 instead of arginine exposing an Asp265 to the channel lumen. Stopped-flow measurements showed the selectivity filter is functional and excludes cations despite its unconventional layout. Organic cations that could theoretically pass because of their size are detained due to their positive charge. The di-cationic, 340 Da seized pentamidine is not a permeant but a nanomolar inhibitor of TbAQP2´s glycerol permeability in yeast with an IC50 of 130 nM. The Asp265 was found to be responsible for the electrostatic interaction with the amidine moiety of pentamidine. In AQP9, the selectivity filter is inconspicuous but the protein shows conductance for a remarkable variety of solutes including monocarboxylic acids/monocarboxylates. Stopped-flow measurements in the framework of this thesis revealed that the neutral acid is the channeled species independent of the proton gradient. Two arginines at positions 51 and 53 in loop a of AQP9 were found to be crucial for conductance for monocarboxylic acids. The postulated mechanism of AQP9 channeling monocarboxylic acids bases on attraction of the monocarboxylate anion leading to a higher probability of protonation via the solvent depending on pH and pKs followed by a passage of the neutral acid. TbAQP2 and AQP9 both represent examples for the influence of protein electrostatics on solute selectivity of aquaglyceroporins

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis

Inhibition of TbAQP2 glycerol permeability by pentamidine and derivatives.

<p>(A) Shown are example traces of protoplast light scattering in 300 mM isotonic glycerol gradients in the presence of pentamidine. Red traces indicate uninhibited glycerol influx via TbAQP2 (left) and TbAQP3 (right), effects of pentamidine concentrations from 50 nM to 50 μM are colored light blue, and of 500 μM pentamidine in dark blue. (B) Structure-function evaluation of TbAQP2 inhibition by pentamidine. The chemical structures of the used compounds are shown on the left and respective dose-response curves on the right. (C) Effect of pH titration of TbAQP2 Asp265 and replacement by mutation to alanine on pentamidine inhibition. The dashed lines show the pentamidine inhibition of wild-type TbAQP2 at pH 7.2 (data taken from Fig 2B). The left panel shows dose-response curves of pentamidine pH 3.5 (closed symbols; IC₅₀ 450 nM), and pH 2.5 (open symbols; IC₅₀ 1.1 μM). The effect of the Asp265Ala point mutation (expression confirming Western blot in the inset) on the inhibition by pentamidine is depicted on the right. Data taken at pH 7.2 (IC₅₀ 4.5 μM) are indicated by closed symbols, those at pH 2.5 (IC₅₀ 5.8 μM) by open symbols. Each data point is an average of 5–9 light scattering traces each from at least two independent experiments.</p

Model of the pentamidine binding mode to TbAQP2 and proposed uptake by endocytosis in the flagellar pocket.

<p>(A) Shown are the crystal structure of the prototypical aquaglyceroporin GlpF and a model of TbAQP2. GlpF Arg206 and TbAQP2 Leu264 mark the position of the ar/R selectivity filter. In TbAQP2, the Asp265 sidechain carboxylate binds to an amidine moiety of pentamidine (light blue), whereas in GlpF the space is occupied by the guanidine sidechain of Arg206. The location of the ‘NPA/NPA’ region (white bar) and sequence deviations in TbAQP2 are indicated. (B) Proposed uptake mechanism of pentamidine via high-affinity binding to TbAQP2, endocytosis of the complex, and release of pentamidine in the acidic lysosome due to pH shift or TbAQP2 degradation.</p