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No description available.

Voltage-dependent K+ channels can undergo a gating process known as C-type inactivation. This type of gating consists of entry into a nonconducting state that may involve conformational changes near the channel's selectivity filter. However, the details of the underlying mechanisms are not clear. Here, I report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K+ channel that lacks a canonical voltage sensor. In single-channel recordings, I observed that open probability (Po) decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca2+ or Ba2+, suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K+] (47 mV per 10-fold increase in [K+]), suggesting that K+ binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K+ ≈ Rb+ > Cs+ > Na+ > Li+ ≈ NMG+. Selectivity of the stabilizing site is somewhat weaker than that of sites that determine permeability of these ions, consistent with the idea that the site may lie toward the external end of the MthK selectivity filter. MthK gating was described over a wide range of positive voltages and external [K+] using kinetic schemes in which the open-conductive state is stabilized by K+ binding to a site that is not deep within the electric field, with the voltage-dependence of inactivation arising from both voltage-dependent K+ dissociation and transitions between nonconducting (inactivated) states. Studies of C-type inactivation in voltage-gated K+ channels have demonstrated that inactivation can be enhanced by quaternary ammonium (QA) derivatives, which block current through the channel by binding to a site at the cytoplasmic side of the pore. Enhancement of inactivation is thought to occur through a mechanism in which QA blockade leads to depletion of K+ ions in the pore, thus driving the channel toward the inactivated state. I tested this model by using divalent cations to block the current through the MthK channel, and then quantifying the effects on inactivation. I observed that the voltage-dependence of blockade by Ca2+, Mg2+, and Sr2+ was approximately equal (zδ ≈ 0.4 e0 for blockade by each of the divalent cations), suggesting a similar location for the site of blockade. However, Ca2+ and Sr2+ were found to enhance inactivation, whereas Mg2+ does not. Molecular dynamics (MD) simulations suggested that Ca2+ and Sr2+ bind to a site (S5) closer to the selectivity filter than Mg2+, consistent with the idea that binding of a divalent cation to S5 enhances inactivation; the bound cation may in turn electrostatically interact with K+ ions in the selectivity filter to break the K+ conduction cycle. Previous studies on inactivation in KcsA have identified a critical residue involved in the mechanism of C-type inactivation in this channel. This residue, E71, is located in a region known as the pore helix, and is involved in a hydrogen bonding network involving a tryptophan residue also in the pore helix, as well as an aspartic acid residue in the selectivity filter, which drives the channel toward the inactivated state. However, mutation to alanine breaks the hydrogen bonding network and effectively prevents inactivation. To determine whether a similar mechanism may enhance inactivation in MthK, I performed mutagenesis at the MthK residue analogous to KcsA E71 (V55). In single channel recordings, I observed that mutation to glutamate (V55E) destabilized the open state of the channel, consistent with the idea that a hydrogen bonding network that drives the channel toward the inactivated state may be formed in MthK to enhance inactivation, similar to the mechanism proposed for KcsA. These results, along with previous findings, suggest that inactivation gating is linked to the selectivity filter of the channel. In most K+ selective channels, the selectivity filter is composed of a sequence of highly-conserved residues (TVGYG). Within this sequence, the sidechain of the conserved threonine residue determines the entry to the selectivity filter, and may thus be a key regulator of the K+ conduction cycle. Interestingly, the rapidly inactivating voltage-gated K+ channel, HERG, contains a serine at this position instead of a threonine. To determine the impact of a change from threonine to serine, I quantified effects of the mutation T59S in MthK on conduction and inactivation, and further probed these effects using blockade by divalent cations. I observed that this mutation reduces channel conductance and enhances inactivation, compared to the wild type channel, and enhanced blockade by Sr2+. MD simulations suggested an increased energy barrier for K+ ions to enter the selectivity filter, which may account for the decreased conductance. In addition, the serine sidechain may effect a redistribution of K+ within the selectivity filter, which may impact stability of the conducting state. Overall, my results suggest that several mechanisms contribute to K+ channel inactivation, involving a combination of ion-ion interactions in the pore, structural interactions among residues in the selectivity filter that may affect the stability of the conducting state, and interactions between ions and a key sidechain at the entry to the selectivity filter. Further understanding of these components of the inactivation process may provide a clearer picture of the mechanisms that generate diversity in gating properties among K+ channels. / Biochemistry

Thesis. 1975. Ph.D.--Massachusetts Institute of Technology. Dept. of Earth and Planetary Sciences. / Vita. / Includes bibliographies. / Ph.D.

Cotton (Gossipium hiristum, L) is a warm season perennial with indeterminant growth habit. In 1995, 42,500 and 300,000 hectares were grown in Virginia and North Carolina, respectively. Soil physical and chemical properties may limit cotton yields. The objective of this study was to; 1) determine influences of soil physical and chemical properties on yield, 2) validate existing preharvest yield estimators, and 3) determine the effect of subsoiling and/or subsurface liming on cotton development and root growth. Two hundred sites were sampled across the Coastal Plain of Virginia and North Carolina to a depth of 92 cm representing 5 major soil series. Soil samples were analyzed for selected physical and chemical properties from each horizon. Boll and plant counts were obtained while harvesting a 3-meter length of row at each site to determine yield for the 1996 and 1997 growing season. Cotton was grown in the greenhouse on 30 cm diameter cores of a soil with low subsoil pH and a hard pan to determine the effects of subsoiling and/or subsurface liming. Ninety days after planting, the cotton plants were harvested and the above ground biomass and rootmass were analyzed. Physical and chemical properties explained 52% of yield variability in 1996 and 27% in 1997. Physical and chemical properties that were significant to yield were surface bulk density, available water holding capacity, depth of the water table and Bt horizon, Mg, K, Ca, and Al content. Soil analysis for nutrient status at depths up to 45 cm were better indicators of cotton yield. Subsoiling with or without subsurface liming increased rooting depth over the untreated check. The subsurface liming reached first flower 11 days prior to the other treatments. The additional period for flowering and boll set in Virginia and North Carolina could increase potential yield. / Ph. D.

Biologically-mediated subsurface remediation by biofilm communities is a poorly understood process that is spatially and temporally dynamic. Two microbial responses, catabolism and the stress response glutathione-gated potassium efflux (GGKE), to benzene, pentachlorophenol (PCP), or Cd exposure were studied in up-flow sand columns to examine the contribution of each response to the overall functional response of a subsurface biofilm. Benzene was catabolized in the aerobic zone, and did not activate the GGKE response, and exhibited the highest biomass concentrations of all columns. PCP was not catabolized during this study, but was found to elicit two responses, oxidative phosphorylation uncoupling and GGKE, that appeared to be concentration dependent. Oxidative uncoupling was the controlling metabolic response up to 10 mg/L PCP, while the GGKE stress response was activated near 20 mg/L PCP. PCP column biomass did not show long-term biomass detachment, although immediate detachment occurred during initial GGKE activation. Cd column biomass activated the GGKE response as perturbing Cd concentrations increased. Extracellular polymeric substance (EPS)-Cd complexation was a possible detoxification mechanism, as biomass concentrations did not decrease with increasing Cd concentration, and increased as Cd concentrations decreased. Results of this study suggested that the increased exposure of electrophilic contaminants to sand column biomass did not cause biomass detachment. / Master of Science

Demand for potassium (K) increases with increasing soybean yield. Little research has been conducted on soybean response to K on coastal plain and piedmont soils of Virginia, especially in double-crop systems. Nineteen full-season and 14 double-crop soybean experiments were conducted in 2013 and 2014 in Virginia and northeastern North Carolina to determine full-season and double-crop soybean (with wheat straw remaining or removed) response to soil test K and K fertilizer application rates. Field moist, air dried, and oven dry soil test K extraction techniques were also compared to predict responsiveness of sites to K applications. Potassium fertilizer increased yield in five of 19 full-season experiments and one of 14 double-crop experiments. Full-season soybean yield plateaued at 88% relative yield and soil test K value of 38.8 mg K kg-1. Full-season plant K critical concentrations were 18.2 g K kg-1 for V5 and 24.6 g K kg-1 for R2. Although critical concentrations could not be determined for double-crop soybean, V5 and R2 concentrations ranged from 17.6 to 35.6 g K kg-1 or 13.2 to 28.1 K kg-1, respectively, most of which were within or above accepted sufficiency levels. Eight of 13 sites resulted in greater soil K concentrations when alternative soil drying methods were compared to air-dry methods. However, differences were not consistent and no single method was superior for these soils. More data is needed for double-crop soybean systems due to lack of response and lack of low soil test K sites in these experiments. / Master of Science

Master of Science / Department of Agronomy / David B. Mengel / Over the last decade low (< 130 mg kg -1) soil test potassium (K) levels and increased crop K deficiency have become a major concern in the clay-pan soils of southeast Kansas. The use of more intense crop rotations and the increased production of high K extracting crops (e.g. soybeans (Glycine max L.)) has significantly increased K removal from these soils. In addition, the traditional use of the nutrient sufficiency-based fertilizer recommendations has resulted in K application rates being substantially lower than removal rates. Because of these practices, many soils that had naturally elevated K availability 25 years ago have declined in K content. More troubling is the extreme yearly variation of soil test exchangeable K levels reported in the region, which has many producers and consultants concerned about proper K management. This study was initiated to examine the extent of K soil test variation and to determine if the variability is impacting plant K availability by analyzing soybean leaf K content and crop yield. A major objective of our research is to identify the mechanism(s) driving these changes in soil test K levels and K availability to crops during the growing season. The long-term goal is to be able to design a soil sampling system and develop alternative K fertilizer recommendation strategies that could alleviate K deficiency impacts on crop yield. Evaluation of different K fertilizer application practices including rate of application and broadcast or surface band methods of application were studied as tools to correct soybean K deficiency. The direct and residual impacts of K fertilization and placement were also evaluated on corn (Zea mays L.) grown in the rotation with the soybeans. Results observed from this research showed that monthly soil samples taken during three crop years at multiple locations have ammonium acetate exchangeable K levels that indeed change dramatically. The data we collected together with data accumulated by farmers and crop consultants showed significant fluctuation in exchangeable K levels of up to 50% on a yearly and even on a monthly basis. Levels seem to demonstrate seasonal changes: higher in the spring months and then decline in the summer and fall. Potassium soil test levels also appear to follow a similar trend as monthly precipitation and soil moisture status. During wet months soil levels tend to increase and then decline during drier months, however, this is not a perfect relationship and other factors are likely to be involved in regulating soil test K levels. No clear effect of K fertilization or method of placement on soybean or corn yields was observed during the study. However, soybean leaf samples revealed that on very low (< 90 mg kg -1) soil test sites surface band applied fertilizer increased leaf K concentrations compared to broadcasted applications. Furthermore, the corn study revealed no distinct difference between using a split annual or biannual fertilizer application system. Maintaining soil test K levels above 130 mg kg -1 using a spring soil test appears to be a successful strategy for avoiding K deficiency. Traditionally most soil sampling occurs in late summer or fall when soil conditions are dry. Our data has demonstrated that during this period one should expect to encounter low soil test results that may not be true indicators of soil K levels during the spring planting months. With that said, spring soil sampling can be difficult to do in a timely fashion due to weather, as well as potential labor restrictions. Another critical point is to not switch back and forth between spring and fall sampling dates. Staying consistent with your sample timing will minimize the seasonal variability that is frequently experienced. Additionally, adopting a build and maintain fertilizer recommendation philosophy rather than a nutrient sufficiency-based recommendation approach is a better nutrient budgeting method to avoid having removal rates exceeding nutrient additions. The best K management proposal would be to consider using a build and maintain approach in combination with basing fertilizer rates on spring soil test K levels.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.