The selectivity filter is involved in the U-type inactivation process of Kv2.1 and Kv3.1 channels

Voltage-gated potassium (Kv) channels display several types of inactivation processes, including N-, C-, and U-types. C-type inactivation is attributed to a nonconductive conformation of the selectivity filter (SF). It has been proposed that the activation gate and the channel's SF are allosterically coupled because the conformational changes of the former affect the structure of the latter and vice versa. The second threonine of the SF signature sequence (e.g., TTVGYG) has been proven to be essential for this allosteric coupling. To further study the role of the SF in U-type inactivation, we substituted the second threonine of the TTVGYG sequence by an alanine in the hKv2.1 and hKv3.1 channels, which are known to display U-type inactivation. Both hKv2.1-T377A and hKv3.1-T400A yielded channels that were resistant to inactivation, and as a result, they displayed noninactivating currents upon channel opening; i.e., hKv2.1-T377A and hKv3.1-T400A remained fully conductive upon prolonged moderate depolarizations, whereas in wild-type hKv2.1 and hKv3.1, the current amplitude typically reduces because of U-type inactivation. Interestingly, increasing the extracellular K+ concentration increased the macroscopic current amplitude of both hKv2.1-T377A and hKv3.1-T400A, which is similar to the response of the homologous T to A mutation in Shaker and hKv1.5 channels that display C-type inactivation. Our data support an important role for the second threonine of the SF signature sequence in the U-type inactivation gating of hKv2.1 and hKv3.1. SIGNIFICANCE Voltage-dependent K+ (Kv) channels generate cells' repolarizing power, which is consequently regulated by the channel's conductance. Aside from the opening or closure, Kv channels undergo inactivation that drives them into a lower or nonconductive state. Among the different inactivation processes described in Kv channels, the U-type process develops from a preopen but activated state. The molecular determinants of this process are, in contrast to the Ctype mechanism, not well characterized. Our data show that the intracellular part of the K+ selectivity filter within the pore domain is involved. An alanine for threonine substitution results in channels that do not inactivate upon opening, suggesting that an allosteric coupling between the activation gate and selectivity filter exists in U-type inactivation

Pleiotropic effects of levofloxacin, fluoroquinolone antibiotics, against influenza virus-induced lung injury

© 2015 Enoki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Reactive oxygen species (ROS) and nitric oxide (NO) are major pathogenic molecules produced during viral lung infections, including influenza. While fluoroquinolones are widely used as antimicrobial agents for treating a variety of bacterial infections, including secondary infections associated with the influenza virus, it has been reported that they also function as anti-oxidants against ROS and as a NO regulator. Therefore, we hypothesized that levofloxacin (LVFX), one of the most frequently used fluoroquinolone derivatives, may attenuate pulmonary injuries associated with influenza virus infections by inhibiting the production of ROS species such as hydroxyl radicals and neutrophil-derived NO that is produced during an influenza viral infection. The therapeutic impact of LVFX was examined in a PR8 (H1N1) influenza virus-induced lung injury mouse model. ESR spin-trapping experiments indicated that LVFX showed scavenging activity against neutrophil-derived hydroxyl radicals. LVFX markedly improved the survival rate of mice that were infected with the influenza virus in a dose-dependent manner. In addition, the LVFX treatment resulted in a dose-dependent decrease in the level of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative stress) and nitrotyrosine (a nitrative marker) in the lungs of virus-infected mice, and the nitrite/nitrate ratio (NO metabolites) and IFN-? in BALF. These results indicate that LVFX may be of substantial benefit in the treatment of various acute inflammatory disorders such as influenza virus-induced pneumonia, by inhibiting inflammatory cell responses and suppressing the overproduction of NO in the lungs

Inverted allosteric coupling between activation and inactivation gates in K+ channels

The selectivity filter and the activation gate in potassium channels are functionally and structurally coupled. An allosteric coupling underlies C-type inactivation coupled to activation gating in this ion-channel family (i.e., opening of the activation gate triggers the collapse of the channel's selectivity filter). We have identified the second Threonine residue within the TTVGYGD signature sequence of K+ channels as a crucial residue for this allosteric communication. A Threonine to Alanine substitution at this position was studied in three representative members of the K+-channel family. Interestingly, all of the mutant channels exhibited lack of C-type inactivation gating and an inversion of their allosteric coupling (i.e., closing of the activation gate collapses the channel's selectivity filter). A state-dependent crystallographic study of KcsA-T75A proves that, on activation, the selectivity filter transitions from a nonconductive and deep C-type inactivated conformation to a conductive one. Finally, we provide a crystallographic demonstration that closed-state inactivation can be achieved by the structural collapse of the channel's selectivity filter