Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism

Paxilline, a tremorogenic fungal alkaloid, potently inhibits large conductance Ca(2+)- and voltage-activated K(+) (BK)-type channels, but little is known about the mechanism underlying this inhibition. Here we show that inhibition is inversely dependent on BK channel open probability (Po), and is fully relieved by conditions that increase Po, even in the constant presence of paxilline. Manipulations that shift BK gating to more negative potentials reduce inhibition by paxilline in accordance with the increase in channel Po. Measurements of Po times the number of channels at negative potentials support the idea that paxilline increases occupancy of closed states, effectively reducing the closed–open equilibrium constant, L(0). Gating current measurements exclude an effect of paxilline on voltage sensors. Steady-state inhibition by multiple paxilline concentrations was determined for four distinct equilibration conditions, each with a distinct Po. The IC(50) for paxilline shifted from around 10 nM when channels were largely closed to near 10 µM as maximal Po was approached. Model-dependent analysis suggests a mechanism of inhibition in which binding of a single paxilline molecule allosterically alters the intrinsic L(0) favoring occupancy of closed states, with affinity for the closed conformation being >500-fold greater than affinity for the open conformation. The rate of inhibition of closed channels was linear up through 2 µM paxilline, with a slope of 2 × 10(6) M(−1)s(−1). Paxilline inhibition was hindered by either the bulky cytosolic blocker, bbTBA, or by concentrations of cytosolic sucrose that hinder ion permeation. However, paxilline does not hinder MTSET modification of the inner cavity residue, A313C. We conclude that paxilline binds more tightly to the closed conformation, favoring occupancy of closed-channel conformations, and propose that it binds to a superficial position near the entrance to the central cavity, but does not hinder access of smaller molecules to this cavity

Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homology to dolichyl phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochemical evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.National Institutes of Health (U.S.) (Grant GM039334

Glycine311, a determinant of paxilline block in BK channels: a novel bend in the BK S6 helix

The tremorogenic fungal metabolite, paxilline, is widely used as a potent and relatively specific blocker of Ca2+- and voltage-activated Slo1 (or BK) K+ channels. The pH-regulated Slo3 K+ channel, a Slo1 homologue, is resistant to blockade by paxilline. Taking advantage of the marked differences in paxilline sensitivity and the homology between subunits, we have examined the paxilline sensitivity of a set of chimeric Slo1/Slo3 subunits. Paxilline sensitivity is associated with elements of the S5–P loop–S6 module of the Slo1 channel. Replacement of the Slo1 S5 segment or the second half of the P loop results in modest changes in paxilline sensitivity. Replacing the Slo1 S6 segment with the Slo3 sequence abolishes paxilline sensitivity. An increase in paxilline affinity and changes in block kinetics also result from replacing the first part of the Slo1 P loop, the so-called turret, with Slo3 sequence. The Slo1 and Slo3 S6 segments differ at 10 residues. Slo1-G311S was found to markedly reduce paxilline block. In constructs with a Slo3 S6 segment, S300G restored paxilline block, but most effectively when paired with a Slo1 P loop. Other S6 residues differing between Slo1 and Slo3 had little influence on paxilline block. The involvement of Slo1 G311 in paxilline sensitivity suggests that paxilline may occupy a position within the central cavity or access its blocking position through the central cavity. To explain the differences in paxilline sensitivity between Slo1 and Slo3, we propose that the G311/S300 position in Slo1 and Slo3 underlies a structural difference between subunits in the bend of S6, which influences the occupancy by paxilline

Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels

Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K(+) channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the "helix-bundle crossing". However, in the inwardly rectifying (Kir) potassium channel family, the role of this "hinge" residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper "hinge" residues are in close contact with the base of the pore alpha-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the "lower" gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue

Gating of a pH-Sensitive K2P Potassium Channel by an Electrostatic Effect of Basic Sensor Residues on the Selectivity Filter

K+ channels share common selectivity characteristics but exhibit a wide diversity in how they are gated open. Leak K2P K+ channels TASK-2, TALK-1 and TALK-2 are gated open by extracellular alkalinization. The mechanism for this alkalinization-dependent gating has been proposed to be the neutralization of the side chain of a single arginine (lysine in TALK-2) residue near the pore of TASK-2, which occurs with the unusual pKa of 8.0. We now corroborate this hypothesis by transplanting the TASK-2 extracellular pH (pHo) sensor in the background of a pHo-insensitive TASK-3 channel, which leads to the restitution of pHo-gating. Using a concatenated channel approach, we also demonstrate that for TASK-2 to open, pHo sensors must be neutralized in each of the two subunits forming these dimeric channels with no apparent cross-talk between the sensors. These results are consistent with adaptive biasing force analysis of K+ permeation using a model selectivity filter in wild-type and mutated channels. The underlying free-energy profiles confirm that either a doubly or a singly charged pHo sensor is sufficient to abolish ion flow. Atomic detail of the associated mechanism reveals that, rather than a collapse of the pore, as proposed for other K2P channels gated at the selectivity filter, an increased height of the energetic barriers for ion translocation accounts for channel blockade at acid pHo. Our data, therefore, strongly suggest that a cycle of protonation/deprotonation of pHo-sensing arginine 224 side chain gates the TASK-2 channel by electrostatically tuning the conformational stability of its selectivity filter

On the simple random-walk models of ion-channel gate dynamics reflecting long-term memory

Several approaches to ion-channel gating modelling have been proposed. Although many models describe the dwell-time distributions correctly, they are incapable of predicting and explaining the long-term correlations between the lengths of adjacent openings and closings of a channel. In this paper we propose two simple random-walk models of the gating dynamics of voltage and Ca2+-activated potassium channels which qualitatively reproduce the dwell-time distributions, and describe the experimentally observed long-term memory quite well. Biological interpretation of both models is presented. In particular, the origin of the correlations is associated with fluctuations of channel mass density. The long-term memory effect, as measured by Hurst R/S analysis of experimental single-channel patch-clamp recordings, is close to the behaviour predicted by our models. The flexibility of the models enables their use as templates for other types of ion channel

Studying the Public in the Pandemic

The paper discusses studying the art audience dur-ing the period of self-isolation. Author’s researches of the audience of theaters, concert halls and muse-ums, carried out prior and during the period of mas-sive self-isolation, suggest the need to develop new forms of active consumption of art for the elderly audience, deprived of the opportunity to personally attend concerts and performances. The study ex-plains the main reasons why remote consumption of art does not replace visiting live performances and museums. In addition, it is suggested that leisure time should be returned to the budget structure as an independent category that allows avoiding role conflicts in working conditions at home and dis-tance learning.</jats:p

The Potential of Ballet Performances in the Development of Situational Cultural Tourism, Based on the Study of the Audience of the St. Petersburg state Academic Boris Eifman Ballet Theater in St. Petersburg

The paper presents data on the results of the re-search on the potential of performances of the St. Petersburg state Academic Boris Eifman ballet thea-ter (Boris Eifman Theater) as a tourist destination, using the example of studying the cultural prefer-ences and practices of visitors to this theater. The main data on the theater audience was obtained during a visit to the performances of the Boris Eif-man Theater in St. Petersburg, in March – April 2019. We can note the significant specifics of the audience of a particular theater, which is due to both the crea-tive method of the theater's artistic Director B. E. Eifman, and the organization of the theater's theatri-cal, stage and tour process. The identified character-istics of the theater audience, as well as the study of the tourist market of St. Petersburg, allowed us to conclude that it is possible to develop a certain segment of situational cultural tourism in St. Peters-burg, both for domestic and outbound tourism.</jats:p