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A non-radioactive rubidium efflux assay for the identification of BKCa channel modulators
BKCa channels are fundamental in the control of cellular excitability and have been identified as potential therapeutic targets in many pathological conditions (Wu, 2003; Lawson and McKay 2005). Thus, BKCa channel modulators are a novel approach to regulate cell function and as such will be important as therapeutic agents and as experimental tools. The aim of this study was to optimise and validate a non-radioactive rubidium (Rb+) efflux assay (Terstappen, 1999) for the evaluation of novel compounds as BKCa modulators. Human Embryonic Kidney (HEK293) cells stably expressing the zero variant of the BKCa channel alpha subunit were grown to confluence in 96-well tissue culture plates. Cells were incubated for 4 hours in RbCl Buffer (mM: NaCl 150, CaCl2 2, MgCl2 1, NaH2PO4 0.8, glucose 5, HEPES 25 and RbCl 5.4). Cells were then washed four times with phosphate buffered saline (PBS) and exposed to KCl Buffer (mM: NaCl 150, CaCl2 2, MgCl2 1, NaH2PO4 0.8, glucose 5, HEPES 25 and KCl 5.4) containing different treatments. At different time points the Rb+ content of the extracellular (supernatant) and intracellular (cells lysed with 200μl 0.1% triton-X100) compartments was determined by atomic absorption spectrometry using a Thermo-Russell S-series Spectrometer with a Gilson 222XL auto-sampler. Rb+ efflux (%) was calculated and data expressed as mean values (± SEM). The intracellular content of Rb+ increased with incubation time reaching a maximum of 0.092 ± 0.008mM (n=24) at 4 hours. The RbCl buffer was effectively removed from the extracellular compartment with four PBS washes of the cells monolayer. In the presence of KCl buffer a Rb+ efflux of 27.5±0.6% (n=409) was observed that did not change significantly with time (2-30 min) and was not modified (31.6±1.2%, n=150) by the presence of 4-aminopyridine (1.0mM) and tetraethylammonium (10.0mM). NS1619 (3, 10, 30 and 100μM for 10 min), a BKCa channel opener (Olesen et al., 1994), evoked a significant (p<0.05) increase in the Rb+ efflux of 35.2±2.3%, 46.1±1.6%, 44.4±2.1% and 45.4±2.3% (n=32) respectively. In the presence of the selective BKCa channel blocker, iberiotoxin (0.10μM), NS-1619 (3-100μM for 10 min) failed to modify the Rb+ efflux relative to control level (21.8±2.7%, n=24). In conclusion an optimised assay that enables the characterisation of BKCa channels expressed in HEK293 cells was developed which provides a reliable framework for the screening of modulators of BKCa channels.
Lawson K & McKay NG (2005) Curr Pharm Design 12: 459-471. |
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