The activation mechanism of alpha 1 homomeric glycine receptors

The glycine receptor mediates fast synaptic inhibition in the spinal cord and brainstem. Its activation mechanism is not known, despite the physiological importance of this receptor and the fact that it can serve as a prototype for other homopentameric channels. We analyzed single-channel recordings from rat recombinant alpha1 glycine receptors by fitting different mechanisms simultaneously to sets of sequences of openings at four glycine concentrations (10-1000 muM). The adequacy of the mechanism and the rate constants thus fitted was judged by examining how well these described the observed dwell-time distributions, open-shut correlation, and single-channel P-open dose-response curve. We found that gating efficacy increased as more glycine molecules bind to the channel, but maximum efficacy was reached when only three (of five) potential binding sites are occupied. Successive binding steps are not identical, implying that binding sites can interact while the channel is shut. These interactions can be interpreted in the light of the topology of the binding sites within a homopentamer

Openings of the rat recombinant alpha1 homomeric glycine receptor as a function of the number of sgonist molecules bound

The functional properties of rat homomeric {alpha}1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat {alpha}1 subunit cDNA. Whole-cell dose-response curves gave EC50 estimates between 30 and 120 µM and a Hill slope of ~3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 µM) to outside-out patches. Single channel conductances were mostly 60–90 pS, but smaller conductances of ~40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency

Single-channel behavior of heteromeric α1β glycine receptors: an attempt to detect a conformational change before the channel opens

The α1β heteromeric receptors are likely to be the predominant synaptic form of glycine receptors in the adult. Their activation mechanism was investigated by fitting putative mechanisms to single-channel recordings obtained at four glycine concentrations (10-1000 µM) from rat {alpha}1{beta} receptors, expressed in human embryonic kidney 293 cells. The adequacy of each mechanism, with its fitted rate constants, was assessed by comparing experimental dwell time distributions, open-shut correlations, and the concentration-open probability (Popen) curve with the predictions of the model. A good description was obtained only if the mechanism had three glycine binding sites, allowed both partially and fully liganded openings, and predicted the presence of open-shut correlations. A strong feature of the data was the appearance of an increase in binding affinity as more glycine molecules bind, before the channel opens. One interpretation of this positive binding cooperativity is that binding sites interact, each site sensing the state of ligation of the others. An alternative, and novel, explanation is that agonist binding stabilizes a higher affinity form of the receptor that is produced by a conformational change ("flip") that is separate from, and precedes, channel opening. Both the "interaction" scheme and the flip scheme describe our data well, but the latter has fewer free parameters and above all it offers a mechanism for the affinity increase. Distinguishing between the two mechanisms will be important for our understanding of the structural dynamics of activation in the nicotinic superfamily and is important for our understanding of mutations in these receptors

Intracellular Chloride Ions Regulate the Time Course of GABA-Mediated Inhibitory Synaptic Transmission

The time-dependent integration of excitatory and inhibitory synaptic currents is an important process for shaping the input - output profiles of individual excitable cells, and therefore the activity of neuronal networks. Here, we show that the decay time course of GABAergic inhibitory synaptic currents is considerably faster when recorded with physiological internal Cl- concentrations than with symmetrical Cl- solutions. This effect of intracellular Cl- is due to a direct modulation of the GABA(A) receptor that is independent of the net direction of current flow through the ion channel. As a consequence, the time window during which GABAergic inhibition can counteract coincident excitatory inputs is much shorter, under physiological conditions, than that previously measured using high internal Cl-. This is expected to have implications for neuronal network excitability and neurodevelopment, and for our understanding of pathological conditions, such as epilepsy and chronic pain, where intracellular Cl- concentrations can be altered

Chloride Ions in the Pore of Glycine and GABA Channels Shape the Time Course and Voltage Dependence of Agonist Currents

In the vertebrate CNS, fast synaptic inhibition is mediated by GABA and glycine receptors. We recently reported that the time course of these synaptic currents is slower when intracellular chloride is high. Here we extend these findings to measure the effects of both extracellular and intracellular chloride on the deactivation of glycine and GABA currents at both negative and positive holding potentials. Currents were elicited by fast agonist application to outside-out patches from HEK-293 cells expressing rat glycine or GABA receptors. The slowing effect of high extracellular chloride on current decay was detectable only in low intracellular chloride (4 mM). Our main finding is that glycine and GABA receptors "sense" chloride concentrations because of interactions between the M2 pore-lining domain and the permeating ions. This hypothesis is supported by the observation that the sensitivity of channel gating to intracellular chloride is abolished if the channel is engineered to become cation selective or if positive charges in the external pore vestibule are eliminated by mutagenesis. The appropriate interaction between permeating ions and channel pore is also necessary to maintain the channel voltage sensitivity of gating, which prolongs current decay at depolarized potentials. Voltage dependence is abolished by the same mutations that suppress the effect of intracellular chloride and also by replacing chloride with another permeant ion, thiocyanate. These observations suggest that permeant chloride affects gating by a foot-in-the-door effect, binding to a channel site with asymmetrical access from the intracellular and extracellular sides of the membrane

Recognition and genotyping of minor germplasm of Friuli Venezia Giulia revealed high diversity

The wealth of vine varieties that used to exist in Friuli Venezia Giulia has been progressively lost. In order to ascertain the current situation regarding vine germplasm in the region, between 2001 and 2008, a wide-ranging study was conducted of recovery, conservation and molecular characterization with microsatellite markers (SSR) of accessions of minor germplasm at risk of erosion or extinction. A total of 178 accessions were analyzed and 93 varieties identified. Of these, 44 are already registered in the Italian Catalogue, 8 have been imported and are well-known foreign varieties even if not registered in the Italian Catalogue, 38 are potentially autochthonous and of these only 15 are described or at least cited in the literature; there are no hypotheses on the remaining three. In order to obtain information on possible genetic similarities, three types of software were used to process the molecular data, but only Structure allowed the existence of two principal groupings to be hypothesized for some of the presumed Friuli autochthons: one that gravitates around ‘Prosecco’ and the other around ‘Refoscone’.

Major threats caused by climate change to grapevine

The main worrying feature of climate change is its rapid evolution, in extent and variation, becoming less and less predictable. In this paper, we have reviewed the available literature and elaborated original data to outline how climate change will affect the grapevine cultivation and wine quality. We start by discussing which features of climate change will impact grapevine production most. The effects of heatwaves, air and soil temperature, extreme rainfall events, atmospheric evaporative demand, wildfires, and smoke are addressed. An increased frequency and intensity of heat waves since 2010 is shown in four grapevine production areas of Northern Italy. The focus then shifts to the impacts of the predicted increase in temperature and drought on frost risks, grapevine phenology, yield, berry quality and water needs as well as vine and vineyard carbon budgets. Climate change will challenge the achievement of current yields and wine quality as well as the ability of vineyards to sequester atmospheric carbon, but such effects will likely depend on the characteristics of the growing environments and on the varieties present. Climate change-related threats to grapevine call for a rapid implementation of adaptation strategies

Influence of vineyard inter-row management and clone on 'Sauvignon Blanc' performance in Friuli Venezia Giulia (north-eastern Italy)

The vineyard inter-row management affects grapevine vegetative and bunch health status, as well as yield and grape quality parameters. Several studies assessed that cover-cropped inter-row in place of soil tillage often reduced plant vigour and yield but positively contributed to vineyard ecosystem services and, to a lower extent, to grape quality. In 2013 and 2014, two inter-row management strategies, i.e. soil tillage and mowing of spontaneous cover crops, were compared in an organic vineyard in north-eastern Italy and cultivated with 'Sauvignon Blanc' (Vitis vinifera L.), clones R3 and 297. In particular, the effects of tillage and mowing treatments on grapevine vegetative and bunch health status, yield and grape quality were evaluated. The vegetative parameters were lower in the mowing treatment than in the tillage one and in clone R3 compared to 297. The incidence of Botrytis cinerea was higher in the tillage treatment than in the mowing one and in clone 297 compared to R3. A significant reduction of the yield and bunch weight was ascertained in the mowing treatment, and these parameters were higher for clone 297 compared to clone R3. Titratable acidity was significantly higher in the tillage treatment than in the mowing one and in clone 297 compared to R3. Moreover, hue of berry skin was qualitatively better in the tillage treatment than in the mowing one. In the pedo-climatic conditions of Friuli Venezia Giulia (north-eastern Italy), the management of the vineyard inter-row with spontaneous cover crops proved to be effective to manage grapevine vigour, reducing yield and improving quality of the grapes during maturation