No abstract

1. The presence and properties of K+ channels activated by arachidonic acid were studied in neuronal cells cultured from the mesencephalic and hypothalamic areas of rat brain.2. Arachidonic acid produced a concentration-dependent (5-50 #UM) and reversible activation of whole-cell currents. 3. In excised membrane patches, arachidonic acid applied to the cytoplasmic or extracellular side of the membrane caused opening of three types of channels whose current-voltage relationships were slightly outwardly rectifying, inwardly rectifying and linear, and whose single channel slope conductances at +60 mV were 143, 45 and 52 pS, respectively.4. All three currents were K+ selective and blocked by 2 mm Ba2+ but not by other K+ channel blockers such as tetraethylammonium chloride, 4-aminopyridine and quinidine. The outwardly and inwardly rectifying currents were slightly voltage dependent with higher channel activity at more depolarized potentials.5. Arachidonic acid activated the K+ channels in cells treated with cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and nordihydroguaiaretic acid), indicating that arachidonic acid itself can directly activate the channels. Alcohol and methyl ester derivatives of arachidonic acid failed to activate the K+ channels, indicating that the charged carboxyl group is important for activation. 6. Certain unsaturated fatty acids (linoleic, linolenic and docosahexaenoic acids), but not saturated fatty acids (myristic, palmitic, stearic acids), also reversibly activated all three types of K+ channel. 7. All three K+ channels were activated by pressure applied to the membrane (i.e. channels were stretch sensitive) with a half-maximal pressure of -18 mmHg. The K+ channels were not blocked by 100 ,UM GdCl3. 8. A decrease in intracellular pH (over the range 5 6-7 2) caused a reversible, pH-dependent increase in channel activity whether the channel was initially activated by arachidonic acid or stretch.9. Glutamate, a neurotransmitter reported to generate arachidonic acid in striatal neurons, did not cause activation of the K+ channels when applied extracellularly in cell-attached patches. 10. It is suggested that the K+ channels described here belong to a distinct family of ion channels that are activated by either fatty acids or membrane stretch. Although the physiological roles of these K+ channels are not yet known, they may be involved in cellular processes such as cell volume regulation and ischaemia-induced elevation of K+ loss.

KB cells are know to respond to epidermal growth factor (EGF) by producing prodigious ruffles in the plasma membrane within minutes. The signal transduction pathway underlying this effect in fibroblasts is mediated through Rac, a member of the Ras-like family of GTPases. As ruffles are rich in components of the cytoskeleton--particularly in actin and ezrin--it has been suggested that ruffles arise when activated Rac modulates the actin cytoskeleton to push out a membrane protrusion. We set out to see whether the surface of new ruffles arises from neighbouring membrane, or whether it comes from an intracellular pool of endocytosed membrane. If it arose by exocytosis from endosomes, it would be expected to be enriched in those recycling receptors that are concentrated in coated pits in the endocytic side of the cycle. On the other hand, if it arose passively from the adjacent plasma membrane, a uniform distribution of these receptors would be expected. Here, we show that as soon as ruffles appear on KB cells in response to EGF, their membrane surfaces are enriched in both transferrin and low density lipoprotein (LDL) receptors. Both these proteins are known to be selectively concentrated into endosomal membranes by clathrin-mediated endocytosis. Our results reveal that the surfaces of ruffles arise by exocytosis of internal membrane from the endocytic cycle and, therefore, that a primary action of Rac is to redirect the exocytosis of recycling membrane into just those specific sites where ruffles form.

We have developed a fluorimetric assay with the use of the dye FM1-43 to determine the rate at which Dictyostelium amoebae endocytose their surface membrane. Our results show that they do so about once each 4 -10 min. A clathrin null mutant takes its surface up only ϳ30% more slowly, showing that this membrane uptake cannot be caused by clathrin-coated vesicles. Surprisingly, Ax2 and its parent, NC4, which differ in their rates of fluid-phase internalization by ϳ60-fold, take up their surfaces at the same rates. These results show that, in axenic cells, the uptake of fluid and of surface area are separate processes. The large activity of this new endocytic cycle in both Ax2 and NC4 amoebae appears capable of delivering sufficient new surface area to advance the cells' fronts during migration.