Polyunsaturated fatty acids (PUFAs) have beneficial effects on epileptic seizures and cardiac arrhythmia. We report that omega-3 and omega-6 all-cis-PUFAs affected the voltage dependence of the Shaker K channel by shifting the conductance versus voltage and the gating charge versus voltage curves in negative direction along the voltage axis. Uncharged methyl esters of the PUFAs did not affect the voltage dependence, whereas changes of pH and charge mutations on the channel surface affected the size of the shifts. This suggests an electrostatic effect on the channel's voltage sensors. Monounsaturated and saturated fatty acids, as well as trans-PUFAs did not affect the voltage dependence. This suggests that fatty acid tails with two or more cis double bonds are required to place the negative carboxylate charge of the PUFA in a position to affect the channel's voltage dependence. We propose that charged lipophilic compounds could play a role in regulating neuronal excitability by electrostatically affecting the channel's voltage sensor. We believe this provides a new approach for pharmacological treatment that is voltage sensor pharmacology.

Voltage-gated ion channels regulate the electric activity of excitable tissues, such as the heart and brain. Therefore, treatment for conditions of disturbed excitability is often based on drugs that target ion channels. In this study of a voltage-gated K channel, we propose what we believe to be a novel pharmacological mechanism for how to regulate channel activity. Charged lipophilic substances can tune channel opening, and consequently excitability, by an electrostatic interaction with the channel's voltage sensors. The direction of the effect depends on the charge of the substance. This was shown by three compounds sharing an arachidonyl backbone but bearing different charge: arachidonic acid, methyl arachidonate, and arachidonyl amine. Computer simulations of membrane excitability showed that small changes in the voltage dependence of Na and K channels have prominent impact on excitability and the tendency for repetitive firing. For instance, a shift in the voltage dependence of a K channel with -5 or +5 mV corresponds to a threefold increase or decrease in K channel density, respectively. We suggest that electrostatic tuning of ion channel activity constitutes a novel and powerful pharmacological approach with which to affect cellular excitability.

The leukotrienes are a family of biologically active molecules, formed by leukocytes, mastocytoma cells, macrophages, and other tissues and cells in response to immunological and nonimmunological stimuli. They exhibit a number of biological effects such as contraction of bronchial smooth muscles, stimulation of vascular permeability, and attraction and activation of leukocytes. Compared to histamine, which causes constriction of airways and edema formation, the leukotrienes are three to four orders of magnitude more potent and the effects have longer duration. The leukotrienes were discovered in 1938 as a smooth muscle-contracting factor in lung perfusates. It was referred to as "slow reacting substance" (SRS) or "slow reacting substance of anaphylaxis" (SRS-A) until 1979 when its structure was reported. The term "leukotriene" was introduced at that time as a trivial name for the new type of compound. Leukotrienes C4 and D4 are glutathione and cysteinylglycine conjugates, respectively, of arachidonic acid. After hydrolytic release from phospholipids of the cell membrane, arachidonic acid is oxygenated by a lipoxygenase to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. This product is further converted to leukotrienes by elimination of the 10-pro-R hydrogen and OH from the hydroperoxy group to give 5,6-oxido-7,9,11, 14-eicosatetraenoic acid (leukotriene A4). Nucleophilic opening of the epoxide at C-6 by the sulfhydryl group of glutathione gives leukotriene C4, which is metabolized to leukotrienes D4 and E4 by sequential elimination of glutamic acid and glycine. The latter reactions are catalyzed by gamma-glutamyl transpeptidase and a particulate dipeptidase from kidney. Alternatively, water may add at C-12 of leukotriene A4, leading also to opening of the epoxide at C-6 with formation of 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B4). Leukotriene B4 is metabolized by omega-hydroxylation to 20-hydroxy and 20-carboxy leukotriene B4. Leukotrienes are also formed from eicosatrienoic acid (n-9) and eicosapentaenoic acid (n-3) after oxygenation at C-5 and from eicosatrienoic acid (n-6) and arachidonic acid after oxygenation at C-8 (eicosatrienoic acid) and C-12 or C-15 (arachidonic acid). Although they are formed from the same and additional fatty acids as prostaglandins and thromboxanes [reviewed in this series in (1)], the structures and the reactions involved in biosynthesis and catabolism of leukotrienes are completely separate from those required for prostaglandin formation and metabolism. The leukotrienes seem to provide a new system of biological regulators that are important in many diseases involving inflammatory or immediate hypersensitivity reactions.

No abstract

Preparation of Labeled CPase. CPase was isolated by affinity chromatography (4) from B. subtilis (strain Porton) membranes obtained by lysozyme DNase treatment (11). In a typical experiment, 10 mg of enzyme was obtained from 300 g of cells. The enzyme in 5 ml of 0.05 M Tris-HCI buffer (pH 7.5) containing 1% (vol/vol) Triton X-100, 0.5 M NaCl and 1 mM 2-mercaptoethanol] was incubated with labeled penicillin G ([8-14C]penicillin G,25 ,uCi, or [35S]penicillin G, 0.8,Ci) for 10 min at 25°. The incubation was stopped by adding 4 volumes of cold acetone containing a 100-fold molar excess of unlabeled penicillin G. The resulting suspension was centrifuged at 11,000 X g for 20 min, and the pellet was dried under a stream of nitrogen.Denaturation, Reduction, Carboxymethylation, and Trypsin or Pronase Digestion. The precipitated carboxypeptidase, labeled with penicillin G (160 nmol, 1.1 X 107 dpm) was dissolved in 2 ml of 6 M guanidine-HCI containing 2 mM EDTA, and 0.2 M Tris-HCI (pH 8.2). After 6 mg of dithiothreitol had been added, the solution was incubated under nitrogen at 370 for 1 hr. Then 20 mg of iodoacetate was added and the solution was incubated under nitrogen and in the dark at room temperature for 1 hr. The solution was dialyzed against distilled water at 40 during which the carboxypeptidase precipitated. The resulting suspension was lyophilized. After lyophilization, the carboxymethylated carboxypeptidase was dissolved in 3 ml of 0.1 M NH4HCO3 and TPCK-trypsin was added to a final concentration of 10% (wt/wt) relative to the carboxypeptidase. The hydrolysis was carried out at 370 for 1 hr, and the reaction was stopped by freezing with dry ice-acetone followed by lyophilization. Pronase digestion was performed under the same conditions as trypsin digestion. If longer incubation times were used, much of the bound penicillin was released.