Gating or switching? Gating is a better model of prospective timing (a response to 'switching or gating?' by Lejeune)(1)

Lejeune (1998) (Switching or gating? The attentional challenge in cognitive models of psychological time. Behav. Process. 44, 127-45) analyzed and compared two models of prospective timing: the classical switching model and the attentional-gate model. Lejeune argued that a modified switch notion, which can be opened and closed in a frequency which reflects the amount of attentional resources allocated for timing can provide a satisfactory explanation for the impact of attention on prospective timing, and therefore the notion of an 'attentional switch' is favored over adding an 'attentional gate.' In the present analysis, the two competing models are compared in terms of correspondence with the nature of attentional processes, as well as in terms of logical analysis and explanatory power. Based on this comparison, it is argued that gating is a better model of prospective timing than switching.     

Gating the bacterial mechanosensitive channels: MscS a new paradigm?

Mechanosensitive channels play major roles in protecting bacteria from hypo-osmotic shock. In the millisecond timescale they must achieve the transition from tightly closed oligomers to large, relatively non-discriminating pores. The crystal structure for MscL, combined with genetic and biochemical analysis, provided the initial insights for the mechanism by which this structural transition might be made. Discovery of the gene for a second class of mechanosensitive channel, MscS, and its subsequent crystallisation, has provided a new paradigm for mechanosensation, enabling a deeper understanding of the mechanisms of sensing membrane tension.     

Crystal Structure of a Yeast Aquaporin at 1.15 ? Reveals a Novel Gating Mechanism

Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.     

Voltage Sensor Gating Charge Transfer in a hERG Potassium Channel?Model

Relaxation of a hERG K⁺ channel model during molecular-dynamics simulation in a hydrated POPC bilayer was accompanied by transitions of an arginine gating charge across a charge transfer center in two voltage sensor domains. Inspection of the passage of arginine side chains across the charge transfer center suggests that the unique hydration properties of the arginine guanidine cation facilitates charge transfer during voltage sensor responses to changes in membrane potential, and underlies the preference of Arg over Lys as a mobile charge carrier in voltage-sensitive ion channels.The response of voltage-sensitive ion channels to changes in membrane potential is mediated by voltage sensor domains (VSD) containing a transmembrane helical segment (S4) with a repeating motif of positively charged and hydrophobic amino acids (Fig. 1) (1,2). Changes in membrane potential drive the S4 helix through the membrane plane with the charged side chains (largely arginine) on S4 swapping Glu/Asp carboxylate partners that lie on less mobile elements of the VSD (2). Movement of S4 is coupled to the ion-conducting pore to transmit changes in membrane potential to channel gating (3).Open in a separate windowFigure 1Structures of the VSD of membrane domains before MD in a POPC bilayer. The S2 (pink) and S4 (blue) helices of the VSD of the hERG model (A) and Kv1.2/2.1 chimera structure (B) are highlighted. (C) Sequence alignment of S2 and S4 among homologous voltage-sensitive K⁺ channels.The VSD charge-pairing motif of K⁺ and Na⁺ channels is best represented in VSD states at zero membrane potential (S4 helix up) for which crystal structures exist for Kv1.2 (4), Kv1.2/2.1 chimera (5), and Na_v channels (6,7). In these states, positively charged residues on the intra- and extracellular sections of the S4 helix are separated by a hydrophobic charge-transfer center (CTC) (1) or plug (8) containing a highly conserved Phe residue (Fig. 1). This plug restricts water incursion across the VSD, focusing the electric field across a narrow region near the bilayer center. In voltage-driven transitions between S4 down- and up-states, positively charged S4 side chains move across the CTC.The ether-à-go-go (eag) and eag-related family of voltage-sensitive K⁺ channels likely share similar charge pairing interactions with VSDs in other channels (9,10). However, eag VSDs contain an extra negative charge on S2 (underlined in Fig. 1 C) so that in hERG, Asp residues (D460 and D466) lie approximately one helical turn above and below the conserved charge-transfer center Phe (F463) (Fig. 1). This eag-specific motif might be expected to facilitate transfer of Arg side chains through the CTC and to stabilize the voltage sensor (VS) in the up state. We recently described an open state (VS-up) hERG model built on the crystal structure template of the Kv1.2/2.1 chimera and molecular-dynamics (MD) simulation of this model in a hydrated POPC bilayer (11). We have inspected an extended version of this simulation and identified transitions of a gating charge into the CTC despite the absence of a membrane potential change. These transitions are absent in equivalent MD simulations of the chimera structure in a POPC bilayer.Fig. 1 shows a single VS from starting structures of the hERG model and the chimera structure in a hydrated POPC bilayer, after restrained MD to anneal the protein-lipid interface (see Methods in the Supporting Material). Because the hERG model is constructed on the chimera structure according to the alignment in Fig. 1 the pattern of pairing between S4 charges and acidic VS side chains is equivalent in the hERG model and chimera structure.The arrangement of charge-paired side chains remains constant during MD in all subunits of the chimera (e.g., Fig. 2 E and see Fig. S2 in the Supporting Material). However, in two subunits of the hERG model the R534 side chain moves toward the extracellular side of the bilayer, sliding into the CTC to form a charge interaction with the extra Asp residue (D460 in hERG) that lies just above F463 (Fig. 2, A–C). This transition is facilitated by changes in side-chain rotamers of R534 and F463 as the planar Arg guanidine group rotates past the F463 ring, and the availability of D460 as a counterion for the R534 guanidine (Fig. 2). Movement of an Arg guanidine past the Phe side chain of the CTC is similar to that described in steered MD of an isolated VS domain (12).Open in a separate windowFigure 2Movement of the R534 side chain across the CTC in chain a of the hERG model simulation (A). Similar transitions are observed in chains a and b (panels B and C), but not chains c (D) or d (not shown), where the R534 side chain remains close to D466. In all subunits of the Kv1.2/2.1 chimera simulation, charge pairing of the starting structure (Fig. 1B) was maintained throughout (e.g., panel E and see Fig. S2 in the Supporting Material). (Black and blue lines) Distances from the Arg CZ or Lys ε atom to the two O atoms, respectively, of Asp or Glu.Mason et al. (13) have shown, using neutron scattering, that the low charge density guanidine cation (Gdm⁺) corresponding to the Arg side chain is poorly hydrated above and below the molecular plane. This property may underlie the universal preference for Arg (over Lys) in voltage sensor charge transfer. Although the poorly-hydrated surfaces of Gdm⁺ interact favorably with nonpolar (especially planar) surfaces (14,15), Gdm⁺ retains in-plane hydrogen bonding (13). In the transition of R534 across the CTC, in-plane solvation of the guanidine side chain is provided initially by D466, D501, and water molecules below the CTC, and during and after the transition by D501 and D460 side chains and waters above the CTC (Fig. 3, A and B). Complete transfer of the R534 side chain across the CTC was not observed, but would be expected to involve movement of the guanidine group away from H-bonding distance with D501.Open in a separate windowFigure 3In-plane solvation of R534 guanidine in the charge transfer center during the hERG model MD (A). (Dotted lines) H-bond distances of <2.5 Å. The right-hand group consists of top-down (B) and end-on (C) views of the distribution of oxygen atoms around the side chain of hERG R534 at 20-ns intervals during MD (subunit a). (D) End-on view of equivalent atom distributions around the K302 side chain during the Kv1.2/2.1 chimera MD (subunit c). (Red spheres, water O; pink, Asp OD1 and OD2; purple:, Glu OE1 and OE2.)The atom distribution around the R534 side chain during MD (Fig. 3, B and C) conforms to the experimental Gdm⁺ hydration structure (13), with H-bonding to waters and side-chain Asp O atoms exclusively in the guanidine plane. The passage of Gdm⁺ through the CTC is facilitated by the hydrophobic nature of Gdm⁺ above and below the molecular plane (13), which allows interaction with the nonpolar groups (especially F463) in the CTC (Fig. 3 A and see Fig. S3). This contrasts with the solvation properties of the Lys amino group (e.g., K302 of the Kv1.2/2.1 chimera (Fig. 1), which has a spherical distribution of H-bonding and charge-neutralizing oxygen atoms (Fig. 3 D and see Fig. S4).To further test these interpretations, we ran additional MD simulations of the isolated hERG VS domain model and an R534K mutant in a hydrated POPC bilayer. Again, the R534 side chain entered the CTC in the wild-type model simulation whereas the K534 side chain did not (see Fig. S5). Inspection of the atom distributions in Fig. 3 D (and see Fig. S4) indicates that the pocket below the conserved Phe of the CTC is particularly favorable for a Lys side chain, with waters and acidic side chains that satisfy the spherical solvation requirements of the terminal amino group, and nonpolar side chains that interact with the aliphatic part of the side chain.The occurrence of transitions of the R534 side chain through the CTC in the hERG model, in the absence of a change in membrane potential, indicates a relaxation from a less-stable starting structure. However, the path of the R534 side chain provides useful molecular-level insight into the nature of charge transfer in voltage sensors. How do these observations accord with broader evidence of charge transfer in voltage-sensitive channels in general, and hERG in particular? Studies with fluorinated analogs of aromatic side chains equivalent to F463 of hERG or F233 of the chimera indicate the absence of a significant role for cation-π interactions involving the CTC aromatic group in K⁺ and Na_v channels, although a planar side chain is preferred in some cases (1,16). In hERG, F463 can be replaced by M, L, or V with small effects on channel gating (17), indicating that the hERG CTC requires only a bulky nonpolar side chain to seal the hydrophobic center of the VS and allow passage of the Arg side chain through the CTC. Both absence of requirement for cation-π interactions, and accommodation of nonplanar hydrophobic side chains in a functional hERG CTC, are broadly consistent with the interpretation that it is the poorly-hydrated nature of the Arg guanidine group above and below the molecular plane (together with its tenacious proton affinity (18)) that governs its role in carrying gating charge in voltage sensors.While the simulations suggest that R534 may interact with D460 in the open channel state, the possibility that the extra carboxylate side chain above the CTC might facilitate gating charge transfer is seemingly inconsistent with the slow activation of hERG, although hERG D460C does activate even more slowly than the WT channel (9). However, S4 movement in hERG occurs in advance of channel opening (19), and slow gating is partly mediated by interactions involving hERG cytoplasmic domains (20); thus, slow S4 movement may not be an inherent property of the hERG voltage sensor. Recent studies show that when hERG gating is studied at very low [Ca²⁺] (50 μM) and low [H⁺] (pH 8.0), the channel is strongly sensitized in the direction of the open state; this effect is reduced in hERG D460C (and hERG D509C) (10). These observations support a role for the extra hERG Asp residues in binding Ca²⁺ (and H⁺) (10), allowing the channel to be allosterically responsive to changes in pH and [Ca²⁺]. A true comparison of a hERG model with experimental channel gating might involve studies on a channel lacking cytoplasmic domains that modulate gating, and using conditions (high pH and low [Ca²⁺]) that leave the eag-specific Asp residues unoccupied. This could reveal the inherent current-voltage relationships and kinetics of the hERG voltage sensor.     

Principal stratification--a goal or a tool?

Principal stratification has recently become a popular tool to address certain causal inference questions, particularly in dealing with post-randomization factors in randomized trials. Here, we analyze the conceptual basis for this framework and invite response to clarify the value of principal stratification in estimating causal effects of interest.     

Is it a paradox or misinterpretation?

The paradox recently raised by Wang and Yuan (Proteins 2000;38:165-175) in protein structural class prediction is actually a misinterpretation of the data reported in the literature. The Bayes decision rule, which was deemed by Wang and Yuan to be the most powerful method for predicting protein structural classes based on the amino acid composition, and applied by these investigators to derive the upper limit of prediction rate for structural classes, is actually completely the same as the component-coupled algorithm proposed by previous investigators (Chou et al., Proteins 1998;31:97-103). Owing to lack of a complete or near-complete training data set, the upper limit rate thus derived by these investigators might be both invalid and misleading. Clarification of these points will further stimulate investigation of this interesting area.     

Voltage-Dependent Gating in a “Voltage Sensor-Less” Ion Channel

The voltage sensitivity of voltage-gated cation channels is primarily attributed to conformational changes of a four transmembrane segment voltage-sensing domain, conserved across many levels of biological complexity. We have identified a remarkable point mutation that confers significant voltage dependence to Kir6.2, a ligand-gated channel that lacks any canonical voltage-sensing domain. Similar to voltage-dependent Kv channels, the Kir6.2[L157E] mutant exhibits time-dependent activation upon membrane depolarization, resulting in an outwardly rectifying current-voltage relationship. This voltage dependence is convergent with the intrinsic ligand-dependent gating mechanisms of Kir6.2, since increasing the membrane PIP₂ content saturates Po and eliminates voltage dependence, whereas voltage activation is more dramatic when channel Po is reduced by application of ATP or poly-lysine. These experiments thus demonstrate an inherent voltage dependence of gating in a “ligand-gated” K⁺ channel, and thereby provide a new view of voltage-dependent gating mechanisms in ion channels. Most interestingly, the voltage- and ligand-dependent gating of Kir6.2[L157E] is highly sensitive to intracellular [K⁺], indicating an interaction between ion permeation and gating. While these two key features of channel function are classically dealt with separately, the results provide a framework for understanding their interaction, which is likely to be a general, if latent, feature of the superfamily of cation channels.     

Beta-carotene: a cancer chemopreventive agent or a co-carcinogen?

Evidence from both epidemiological and experimental observations have fueled the belief that the high consumption of fruits and vegetables rich in carotenoids may help prevent cancer and heart disease in humans. Because of its well-documented antioxidant and antigenotoxic properties, the carotenoid beta-carotene (betaCT) gained most of the attention in the early 1980s and became one of the most extensively studied cancer chemopreventive agents in population-based trials supported by the National Cancer Institute. However, the results of three randomized lung cancer chemoprevention trials on betaCT supplementation unexpectedly contradicted the large body of epidemiological evidence relating to the potential benefits of dietary carotenoids. Not only did betaCT show no benefit, it was associated with significant increases in lung cancer incidence, cardiovascular diseases, and total mortality. These findings aroused widespread scientific debate that is still ongoing. It also raised the suspicion that betaCT may even possess co-carcinogenic properties. In this review, we summarize the current data on the co-carcinogenic properties of betaCT that is attributed to its role in the induction of carcinogen metabolizing enzymes and the over-generation of oxidative stress. The data presented provide convincing evidence of the harmful properties of this compound if given alone to smokers, or to individuals exposed to environmental carcinogens, as a micronutrient supplement. This has now been directly verified in a medium-term cancer transformation bioassay. In the context of public health policies, while the benefits of a diet rich in a variety of fruits and vegetables should continue to be emphasized, the data presented here point to the need for consideration of the possible detrimental effects of certain isolated dietary supplements, before mass cancer chemoprevention clinical trials are conducted on human subjects. This is especially important for genetically predisposed individuals who are environmentally or occupationally exposed to mutagens and carcinogens, such as those found in tobacco smoke and in industrial settings.     

Nomenclature: or what's in a name?

Transgenic Research -     

Oxidative stress: a theoretical model or a biological reality?

Although oxidative stress has been extensively studied the last fifteen years, many physicians and biologists are still sceptical concerning its interest in biology and medicine. This is probably due, in part, to the fact that this subject is a matter of biophysics, and the first studies reported were written using a physical language that inspired these people used to a more concrete problematic very little. Another problem is the difficulty to detect the species mediating oxidative stress, and to determine their role in biological processes. This review is aimed at presenting oxidative stress, as well as reactive oxygen species and free radicals--the molecules that mediate it--in a clear form able to convince all researchers involved in life sciences that these short-lived intermediates are indissociable from any aerobic organism. Moreover, if reactive oxygen species and free radicals are undoubtedly involved in many pathologies, they have physiological functions too.