A novel peptide was purified from the venom of the scorpion Androctonus australis Garzoni (abbreviated Aa1, corresponding to the systematic number alpha KTX4.4). It contains 37 amino acid residues, has a molecular mass of 3850 Da, is closely packed by three disulfide bridges and a blocked N-terminal amino acid. This peptide selectively affects the K(+) currents recorded from cerebellum granular cells. Only the fast activating and inactivating current, with a kinetics similar to I(A)-type current, is completely blocked by the addition of low micromolar concentrations (K(i) value of 150 nM) of peptide Aa1 to the external side of the cell preparation. The blockade is partially reversible in our experimental conditions. Aa1 blocks the channels in both the open and the closed states. The blockage is test potential independent and is not affected by changes in the holding potential. The kinetics of the current are not affected by the addition of Aa1 to the preparation; it means that the block is a simple 'plugging mechanism', in which a single toxin molecule finds a specific receptor site in the external vestibule of the K(+) channel and thereby occludes the outer entry to the K(+) conducting pore.
By using the patch-clamp technique we have shown that, in hypotonic extracellular solutions, the mouse neuroblastoma cells Neuro2A (N2A) develop ionic currents mediated by a chloride-selective channel which is also permeable to other anions in accordance with the permeability sequence: I->Br->Cl->gluconate->glutamate-. The currents persist for several hours when Mg-ATP is present in the recording pipette but occur only transiently in the absence of Mg-ATP. Typical blockers of anions channels such as La3+ and Zn2+ do not affect the hypotonicity-activated channel; conversely, the stilbene sulfonate-derivatives, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), reversibly inhibit the channel in a voltage-dependent manner. Also intact cells exposed to hyposmotic solutions activate volume-regulation mechanisms which decrease the transient volume increase that develops immediately after the application of the hyposmotic challenge. Since N2A neurons have been used as an expression system of exogenous channels, the presence of osmolarity-regulated channels in these cells is an important aspect that deserves the attention of researchers who may wish to express and study the properties of transport proteins in this cell line.
In this paper, we report, for the first time, experimental evidence of multiphoton photolysis of a caged proton compound, 2-nitrobenzaldehyde (o-NBA), using a new sensor system that utilizes fluorescent-labeled nanocapsules, i.e., a fluorescent nanostructured shell of micrometric size and nanometric thickness. The photolabile compound undergoes one-photon absorption in the UV range (200−380 nm), and the mechanism that leads to proton release is based on the well-known 2-nitrobenzyl photochemistry, which has been used for many photoactivatable-caged compounds. Because the use of UV excitation can cause biological damage, we changed our focus to multiphoton absorption−uncaging processes. The induced pH decrease was monitored by imaging changes in the pH-dependent emission of fluorescein isothiocyanate that was embedded in a nanostructured system (so-called “nanocapsules”). The nanocapsules with covalently bound dyes allow improved stability in fluorescence monitoring. Moreover, an original image processing method is introduced to quantify the uncaging. Using a femtosecond Ti:sapphire laser that was operating at 720 nm, with a pulse width of ∼200 fs at the sample, delivered through an adapted confocal laser scanning head and a 1-min exposure time with high power (45−50 mW), we obtained appreciable photolysis of 2-nitrobenzaldehyde. So far, we demonstrated that fluorescent-labeled nanocapsules are a suitable system as fluorescence sensors.
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