Abstract
Transport activity through the mutant D44A of the M2 proton channel from influenza virus A was measured in excised inside-out macro-patches of Xenopus laevis oocytes at cytosolic pH values of 5.5, 7.5 and 8.2. The current-voltage relationships reveal some peculiarities: 1. “Transinhibition”, i.e., instead of an increase of unidirectional outward current with increasing cytosolic H+ concentration, a decrease of unidirectional inward current was found. 2. Strong inward rectification. 3. Exponential rise of current with negative potentials. In order to interpret these findings in molecular terms, different kinetic models have been tested. The transinhibition basically results from a strong binding of H+ to a site in the pore, presumably His37. This assumption alone already provides inward rectification and exponential rise of the IV curves. However, it results in poor global fits of the IV curves, i.e., good fits were only obtained for cytosolic pH of 8.2, but not for 7.5. Assuming an additional transport step as e.g. caused by a constriction zone at Val27 resulted in a negligible improvement. In contrast, good global fits for cytosolic pH of 7.5 and 8.2 were immediately obtained with a cyclic model. A “recycling step” implies that the protein undergoes conformational changes (assigned to Trp41 and Val27) during transport which have to be reset before the next proton can be transported. The global fit failed at the low currents at pHcyt = 5.5, as expected from the interference of putative transport of other ions besides H+. Alternatively, a regulatory effect of acidic cytosolic pH may be assumed which strongly modifies the rate constants of the transport cycle.
Highlights
M2 is a homo-oligomeric protein from the membrane of the influenza virus A with ion transport activity
In order to study the influence of the pH on that side of the M2 protein, which is exposed to the interior of the viral particle, current-voltage relationships (IV curves) were measured in excised patches from Xenopus oocytes expressing the mutant M2D44A of the influenza A/Udorn/72 strain
The analysis below shows that M2D44A is a suitable model for wt as the measured IV curves can be explained in terms of the structural data known from wild type M2
Summary
M2 is a homo-oligomeric protein from the membrane of the influenza virus A with ion transport activity. After endocytosis by the host cell, the virus particle is transported into the endosome. In this acidic environment, the viral membrane fuses with the membrane of the endosome, and an acid-stimulated opening of the M2 channel catalyzes influx of H+ into the virus particle [3,4]. Influx of protons causes uncoating of the viral RNP (ribonucleoprotein) and release into the host cell [5,6]. From a pharmacological point of view, the interest in the M2 protein arises from the fact that a block of this channel can inhibit viral replication in host cells. Amino-adamantyls [7] or related derivatives [8] are inhibitors of the M2 channel, and they were already successfully used to terminate RNA replication of influenza A [9,10]
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