Use of voltage clamp fluorimetry in understanding potassium channel gating: a review of Shaker fluorescence data.

Voltage clamp fluorimetry (VCF) utilizes fluorescent probes that covalently bind to cysteine residues introduced into proteins and emit light as a function of their environment. Measurement of this emitted light during membrane depolarization reveals changes in the emission level as the environment of the labelled residue changes. This allows for the correlation of channel gating events with movement of specific protein moieties, at nanosecond time resolution. Since the pioneering use of this technique to investigate Shaker potassium channel activation movements, VCF has become an invaluable technique used to understand ion channel gating. This review summarizes the theory and some of the data on the application of the VCF technique. Although its usage has expanded beyond voltage-gated potassium channels and VCF is now used in a number of other voltage- and ligand-gated channels, we will focus on studies conducted in Shaker potassium channels, and what they have told us about channel activation and inactivation gating.

Copper chelation in discharged groundwater via EDTA addition: a real world application.

The Water Effects Ratio allows regulators to modify the water quality criteria of metals based on the knowledge that complexing a metal generally reduces its toxicity. Using this logic, the Alameda Corridor Transportation Authority was granted a temporary variance based on bioavailable metal concentration for groundwater discharged into the L.A.-Long Beach Harbor in California. Chelex-labile copper, used as a surrogate for bioavailable copper, was measured using Chelex-100 resin combined with graphite furnace-atomic absorption spectrophotometry (CRC-GFAAS). Ethylendiaminetetraacetic acid (EDTA) was added on-site to a concentration of 10-60 mg L(-1) in order to reduce the bioavailable fraction to non-detectable levels, however unexpectedly only approximately 50% of the available copper was chelated. This partial complexation of the copper was due to high iron concentrations in the industrial mixture of EDTA used for the project, whereas pure EDTA was shown to fully chelate the copper. This technique may hold promise for similar short-term projects.

Mercury toxicity in the aquatic oligochaete Sparganophilus pearsei: I. Variation in resistance among populations.

Mercury contamination has become a problem in many San Francisco Bay Area watersheds due to its elevated presence in sediments and aquatic organisms. The present study used laboratory lethal toxicity (LC50) tests to examine the mercury tolerance of aquatic oligochaete worms, Sparganophilus pearsei, from contaminated and uncontaminated areas. The oligochaetes were collected in the following fresh water reservoirs: Sandy Wool (reference area), San Pablo, Lake Anza, Lake Herman, and Guadalupe. These last four reservoirs were contaminated with levels of mercury that ranged from 1.5 to 2 mg/kg (wet weight). Mercury concentrations in sediment and tissue from Sandy Wool were below detection limits and worms from this site were the least tolerant of mercury in laboratory exposures (LC50 = 0.22 mg/L). Worms from the other, more contaminated, reservoirs contained elevated tissue mercury concentrations and were more tolerant in laboratory tests (LC50 = 1.48-2.19 mg/L). The present study demonstrates that different populations of the aquatic oligochaete S. pearsei have developed different tolerances to mercury depending on their previous history of exposure to mercury contamination.

Mercury toxicity in the aquatic oligochaete Sparganophilus pearsei. II: Autotomy as a novel form of protection.

Aquatic oligochaetes are commonly used for toxicity testing and for assessment of sediment impairment; some species can be relatively tolerant of sediment contaminants. However, there have been few studies of tolerance mechanisms; most work has focused on behavioral changes. The aquatic oligochaete worm, Sparganophilus pearsei, can be extremely tolerant to mercury in sediments depending on its prior history of exposure. Three S. pearsei populations, differing in their history of mercury exposure and in their tolerance to mercury, were assessed to determine tolerance mechanisms. In mercury-contaminated sediments, tolerant worms accumulated this contaminant in their caudal segments (i.e., their tails), which were then jettisoned via the process of autotomy, thus providing a mechanism of detoxification. This detoxification process appears to require a certain level of tolerance and may represent a novel exposure route for other organisms via feeding on discarded tails or release of accumulated contaminants as the tails decompose. Measurements of tissue mercury concentrations as contaminant body residues for this species (CBRs) are compared to other aquatic invertebrates.

Acquisition of a conditioned reflex in New Zealand white rabbits from three sources.

In studies of learning using rabbits, there has been standardization of behavioural procedures across laboratories. Less attention has been paid to variation that may arise from genetic differences and/or differences in rearing conditions. The present experiment revealed that acquisition of a conditioned reflex can be affected dramatically by such differences. Specifically, the acquisition of a conditioned reflex in New Zealand White (NZW) rabbits from 3 different suppliers was compared. All rabbits received behavioural training in which a tone or a light signalled an electrotactile stimulation of the trigeminal nerve near the rabbits' right eye. This tactile stimulus reliably elicited an eyeblink. Repeated presentations of the auditory and visual signals followed by the tactile stimulus yielded the acquisition of a conditioned response (CR), namely closure of the eyelids during the warning period provided by the signal stimuli. Two of the groups showed steady CR acquisition at a rate that matched previous results in other laboratories as well as in the senior author's laboratory. However, the third group of rabbits showed very slow acquisition, and some rabbits failed to show any CR acquisition.

Real-time processing of serial stimuli in classical conditioning of the rabbit's nictitating membrane response.

Real-time models contend that a conditioned stimulus (CS) gives rise to a cascade of hypothetical stimuli that govern conditioned responses (CRs) on a moment-by-moment basis. Experiments with the rabbit nictitating membrane response successfully extended these models to external stimuli. CSs were trained in sequence with an unconditioned stimulus (CSA-CSB-US). When the CSA-CSB interval was shortened, the CR was compressed; when the CSA-CSB interval was lengthened, the CR was broadened. Peaks appeared at 2 places, namely, 1 following CSA by a period equal to its CS-US interval and another following CSB by its CS-US interval. Outside the sequence, the individual CSs evoked CRs located between their respective CS-US intervals. When, however, the 2 CSs were trained separately, the CRs were appropriate to their respective CS-US intervals when tested alone or in sequence. The results are discussed in terms of the J.E. Desmond and J.W. Moore (1988) and S. Grossberg and N.A. Schmajuk (1989) models.

Physiological Studies of Oxygen Protection Mechanisms in the Heterocysts of Anabaena cylindrica.

The mechanism of O(2) protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O(2) inhibition of nitrogenase activity correlated with the O(2) tension of the medium in which heterocyst formation was induced. O(2) resistance also correlated with the apparent K(m) for acetylene, indicating that O(2) tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O(2) that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O(2) sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O(2) but not in its absence. After 3 h of aerobic incubation in the presence of 20 mM azide, 75% of cross-reactive component I (Fe-Mo protein) in nitrogenase was lost; less than 35% was lost under microaerophilic conditions. Sodium malonate and monofluoroacetate, inhibitors of Krebs cycle activity, had only small inhibitory effects on nitrogenase activity in the light and on cross-reactive material. The results suggest that oxygen protection is dependent on both an O(2) diffusion barrier and active respiration by the heterocyst.

Suppression of nitrogen fixation by blue-green algae in a eutrophic lake with trace additions of copper.

Nitrogen fixation by blue-green algae in highly eutrophic Clear Lake, California, was severely inhibited by trace amounts of copper. The chelation capacity of the lake is probably saturated by indigenous copper. Additions were only 1/200 of those normally used in algal control. Since nitrogen fixation provides half of the lake's annual nitrogen budget, economical eutrophication control appears possible.