Fluorescence-optical measurements of chloride movements in cells using the membrane-permeable dye diH-MEQ

Abstract

Fluorescence-optical measurements of the intracellular chloride concentration facilitate identification of chloride movements across the cell membrane of living cells. The two main dyes used for this purpose are 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) and 6-methoxy-quinolyl acetoethyl ester (MQAE). The use of both substances is impaired by their poor membrane permeability and therefore limited loading of the cells to be studied. Here we report the use of 6-methoxy-N-ethylquinolinium iodide (MEQ), a chloride-sensitive dye for which a membrane-permeable form is easily prepared. This makes the loading procedure as easy as with the acetoxymethyl (AM) forms of other dyes for sensing intracellular ions. In addition, the original method, which described absolute concentration measurements of chloride in the cytosol, was modified in so far as only relative measurements were made. This avoids the known limitations of single wavelength excitation and emission dyes with respect to exact concentration measurements. Moreover, to enhance the signal-to-noise ratio the driving force for chloride was considerably increased by changing the original direction of the anion flux in the cells under investigation. We verified the method by using fibroblasts and activating ICln, a putative chloride channel cloned from epithelial cells and of paramount importance in the regulatory volume decrease in these cells. In the presence of SCN- the MEQ quench measured in NIH 3T3 fibroblasts is dramatically enhanced in hypotonically challenged cells compared with cells under isotonic conditions. Antisense oligodeoxynucleotides sensing ICln considerably impeded the swelling-induced chloride current (ICl) in NIH 3T3 fibroblasts. Accordingly, the chloride movement measured by the SCN- quench of the MEQ signal was significantly reduced. Similar results can be obtained in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), two known blockers of chloride transport in the plasma membrane of a variety of cells. In conclusion, fluroscence-optical measurements using MEQ as the chloride-sensitive dye provide a reliable and easy-to-use method for measuring changes of the chloride flux across the cell membrane of living cells.

Cell Swelling Stimulates Cytosol to Membrane Transposition of ICln

“…Measurements of cellular anion entry were then performed by SCN Ϫ -quenching of the halide-sensitive fluorescent dye MEQ. In an earlier study it was demonstrated that the MEQ quench measured in NIH 3T3 fibroblasts is strongly enhanced in cells swollen by hypotonicity and that antisense oligodeoxynucleotides specifically knocking down ICln considerably impede both the MEQ quench and RVDC in NIH 3T3 fibroblasts (36). As shown in Fig.…”

“…diH-MEQ (Molecular Probes) was prepared as described earlier (36). Cells were loaded for 10 min with 5 M diH-MEQ, washed, and superfused with either a control solution (in mM: sodium gluconate 131, potassium gluconate 5.4, MgSO 4 0.8, calcium gluconate 1.2, NaH 2 PO 4 1, glucose 5.5, Tris 5, pH 7.4), or with a hypotonic solution (sodium gluconate reduced to 81 mM).…”

“…Cells were loaded for 10 min with 5 M diH-MEQ, washed, and superfused with either a control solution (in mM: sodium gluconate 131, potassium gluconate 5.4, MgSO 4 0.8, calcium gluconate 1.2, NaH 2 PO 4 1, glucose 5.5, Tris 5, pH 7.4), or with a hypotonic solution (sodium gluconate reduced to 81 mM). The solution was then exchanged rapidly for a 150 mM KSCN solution, and the decrease of the MEQ fluorescence due to cellular anion entry was taken as a measure for the anion permeability of the cell membrane (36). Measurements were performed on an Olympus IX70 microscope equipped with a monochromator (SpectraMaster, LSR) and a cooled charge-coupled device camera (Olympix FKI 300) controlled by the Merlin TM software (LSR).…”

Reciprocal Interactions Between Calcium and Chloride in Rod Photoreceptors

“…MEQ was loaded into cells after reducing it to DiH-MEQ by adding 30 M sodium borohydride (100 l) to MEQ (5 mg) under a continuous stream of nitrogen gas (Woll et al 1996). DiHMEQ enters cells during the incubation period (15 min), where it is oxidized and retained in the form of MEQ.…”

D2-like dopamine receptors promote interactions between calcium and chloride channels that diminish rod synaptic transfer in the salamander retina

“…MEQ was loaded into cells after reducing it to DiH-MEQ by adding 30 μM sodium borohydride (100 μl) to MEQ (5 mg) under a continuous stream of nitrogen gas (Wöll et al, 1996). DiH-MEQ that enters cells during the incubation period (15 min) is oxidized and retained in the form of MEQ.…”