Identification of the Intracellular Na+ Sensor in Slo2.1 Potassium Channels.

Slo2 potassium channels have a very low open probability under normal physiological conditions, but are readily activated in response to an elevated [Na(+)]i (e.g. during ischemia). An intracellular Na(+) coordination motif (DX(R/K)XXH) was previously identified in Kir3.2, Kir3.4, Kir5.1, and Slo2.2 channel subunits. Based loosely on this sequence, we identified five potential Na(+) coordination motifs in the C terminus of the Slo2.1 subunit. The Asp residue in each sequence was substituted with Arg, and single mutant channels were heterologously expressed in Xenopus oocytes. The Na(+) sensitivity of each of the mutant channels was assessed by voltage clamp of oocytes using micropipettes filled with 2 M NaCl. Wild-type channels and four of the mutant Slo2.1 channels were rapidly activated by leakage of NaCl solution into the cytoplasm. D757R Slo2.1 channels were not activated by NaCl, but were activated by the fenamate niflumic acid, confirming their functional expression. In whole cell voltage clamp recordings of HEK293 cells, wild-type but not D757R Slo2.1 channels were activated by a [NaCl]i of 70 mM. Thus, a single Asp residue can account for the sensitivity of Slo2.1 channels to intracellular Na(+). In excised inside-out macropatches of HEK293 cells, activation of wild-type Slo2.1 currents by 3 mM niflumic acid was 14-fold greater than activation achieved by increasing [NaCl]i from 3 to 100 mM. Thus, relative to fenamates, intracellular Na(+) is a poor activator of Slo2.1.

Concerted all-or-none subunit interactions mediate slow deactivation of human ether-à-go-go-related gene K+ channels.

During the repolarization phase of a cardiac action potential, hERG1 K(+) channels rapidly recover from an inactivated state then slowly deactivate to a closed state. The resulting resurgence of outward current terminates the plateau phase and is thus a key regulator of action potential duration of cardiomyocytes. The intracellular N-terminal domain of the hERG1 subunit is required for slow deactivation of the channel as its removal accelerates deactivation 10-fold. Here we investigate the stoichiometry of hERG1 channel deactivation by characterizing the kinetic properties of concatenated tetramers containing a variable number of wild-type and mutant subunits. Three mutations known to accelerate deactivation were investigated, including R56Q and R4A/R5A in the N terminus and F656I in the S6 transmembrane segment. In all cases, a single mutant subunit induced the same rapid deactivation of a concatenated channel as that observed for homotetrameric mutant channels. We conclude that slow deactivation gating of hERG1 channels involves a concerted, fully cooperative interaction between all four wild-type channel subunits.

Mechanistic insight into human ether-à-go-go-related gene (hERG) K+ channel deactivation gating from the solution structure of the EAG domain.

Human ether-à-go-go-related gene (hERG) K(+) channels have a critical role in cardiac repolarization. hERG channels close (deactivate) very slowly, and this is vital for regulating the time course and amplitude of repolarizing current during the cardiac action potential. Accelerated deactivation is one mechanism by which inherited mutations cause long QT syndrome and potentially lethal arrhythmias. hERG deactivation is highly dependent upon an intact EAG domain (the first 135 amino acids of the N terminus). Importantly, deletion of residues 2-26 accelerates deactivation to a similar extent as removing the entire EAG domain. These and other experiments suggest the first 26 residues (NT1-26) contain structural elements required to slow deactivation by stabilizing the open conformation of the pore. Residues 26-135 form a Per-Arnt-Sim domain, but a structure for NT1-26 has not been forthcoming, and little is known about its site of interaction on the channel. In this study, we present an NMR structure for the entire EAG domain, which reveals that NT1-26 is structurally independent from the Per-Arnt-Sim domain and contains a stable amphipathic helix with one face being positively charged. Mutagenesis and electrophysiological studies indicate that neutralizing basic residues and breaking the amphipathic helix dramatically accelerate deactivation. Furthermore, scanning mutagenesis and molecular modeling studies of the cyclic nucleotide binding domain suggest that negatively charged patches on its cytoplasmic surface form an interface with the NT1-26 domain. We propose a model in which NT1-26 obstructs gating motions of the cyclic nucleotide binding domain to allosterically stabilize the open conformation of the pore.

Assessment of soybean injury from glyphosate using airborne multispectral remote sensing.

BACKGROUND: Glyphosate drift onto off-target sensitive crops can reduce growth and yield and is of great concern to growers and pesticide applicators. Detection of herbicide injury using biological responses is tedious, so more convenient and rapid detection methods are needed. The objective of this research was to determine the effects of glyphosate on biological responses of non-glyphosate-resistant (non-GR) soybean and to correlate vegetation indices (VIs) derived from aerial multispectral imagery. RESULTS: Plant height, shoot dry weight and chlorophyll (CHL) content decreased gradually with increasing glyphosate rate, regardless of weeks after application (WAA). Accordingly, soybean yield decreased by 25% with increased rate from 0 to 0.866 kg AI ha(-1) . Similarly to biological responses, the VIs derived from aerial imagery - normalized difference vegetation index, soil adjusted vegetation index, ratio vegetation index and green NDVI - also decreased gradually with increasing glyphosate rate, regardless of WAA. CONCLUSION: The VIs were highly correlated with plant height and yield but poorly correlated with CHL, regardless of WAA. This indicated that indices could be used to determine soybean injury from glyphosate, as indicated by the difference in plant height, and to predict the yield reduction due to crop injury from glyphosate.

Glyphosate-resistant and glyphosate-susceptible Palmer amaranth (Amaranthus palmeri S. Wats.): hyperspectral reflectance properties of plants and potential for classification.

BACKGROUND: Palmer amaranth (Amaranthus palmeri S. Wats.) is a troublesome agronomic weed in the southern United States, and several populations have evolved resistance to glyphosate. This paper reports on spectral signatures of glyphosate-resistant (GR) and glyphosate-sensitive (GS) plants, and explores the potential of using hyperspectral sensors to distinguish GR from GS plants. RESULTS: GS plants have higher light reflectance in the visible region and lower light reflectance in the infrared region of the spectrum compared with GR plants. The normalized reflectance spectrum of the GR and GS plants had best separability in the 400-500 nm, 650-690 nm, 730-740 nm and 800-900 nm spectral regions. Fourteen wavebands from within or near these four spectral regions provided a classification of unknown set of GR and GS plants, with a validation accuracy of 94% for greenhouse-grown plants and 96% for field-grown plants. CONCLUSIONS: GR and GS Palmer amaranth plants have unique hyperspectral reflectance properties, and there are four distinct regions of the spectrum that can separate the GR from GS plants. These results demonstrate that hyperspectral imaging has potential application to distinguish GR from GS Palmer amaranth plants (without a glyphosate treatment), with future implications for glyphosate resistance management. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Biological responses to glyphosate drift from aerial application in non-glyphosate-resistant corn.

BACKGROUND: Glyphosate drift from aerial application onto susceptible crops is inevitable, yet the biological responses to glyphosate drift in crops are not well characterized. The objectives of this research were to determine the effects of glyphosate drift from a single aerial application (18.3 m swath, 866 g AE ha(-1)) on corn injury, chlorophyll content, shikimate level, plant height and shoot dry weight in non-glyphosate-resistant (non-GR) corn. RESULTS: One week after application (WAA), corn was killed at 3 m from the edge of the spray swath, with injury decreasing to 18% at 35.4 m downwind. Chlorophyll content decreased from 78% at 6 m to 22% at 15.8 m, and it was unaffected beyond 25.6 m at 1 WAA. Shikimate accumulation in corn decreased from 349% at 0 m to 93% at 15.8 m, and shikimate levels were unaffected beyond 25.6 m downwind. Plant height and shoot dry weight decreased gradually with increasing distance. At a distance of 35.4 m, corn height was reduced by 14% and shoot dry weight by 10% at 3 WAA. CONCLUSIONS: Corn injury and other biological responses point to the same conclusion, that is, injury from glyphosate aerial drift is highest at the edge of the spray swath and decreases gradually with distance. The LD(50) (the lethal distance that drift must travel to cause a 50% reduction in biological response) ranged from 12 to 26 m among the biological parameters when wind speed was 11.2 km h(-1) and using a complement of CP-09 spray nozzles on spray aircraft.

Agronomic and environmental implications of enhanced s-triazine degradation.

Novel catabolic pathways enabling rapid detoxification of s-triazine herbicides have been elucidated and detected at a growing number of locations. The genes responsible for s-triazine mineralization, i.e. atzABCDEF and trzNDF, occur in at least four bacterial phyla and are implicated in the development of enhanced degradation in agricultural soils from all continents except Antarctica. Enhanced degradation occurs in at least nine crops and six crop rotation systems that rely on s-triazine herbicides for weed control, and, with the exception of acidic soil conditions and s-triazine application frequency, adaptation of the microbial population is independent of soil physiochemical properties and cultural management practices. From an agronomic perspective, residual weed control could be reduced tenfold in s-triazine-adapted relative to non-adapted soils. From an environmental standpoint, the off-site loss of total s-triazine residues could be overestimated 13-fold in adapted soils if altered persistence estimates and metabolic pathways are not reflected in fate and transport models. Empirical models requiring soil pH and s-triazine use history as input parameters predict atrazine persistence more accurately than historical estimates, thereby allowing practitioners to adjust weed control strategies and model input values when warranted.