Functional analysis of native and recombinant ion channels using a high-capacity nonradioactive rubidium efflux assay.
A nonradioactive cell-based rubidium (Rb(+)) efflux assay for functional analysis of native and recombinant ion channels has been developed. Cells are first loaded with rubidium, a tracer for potassium, and after channel activation, rubidium distribution between intracellular and extracellular space is determined by atomic absorption spectroscopy. The relative amount of rubidium in the cell supernatant is a direct measure of channel activity. The broad utility of the method is demonstrated by analysis of a range of different ion channels. Ligand-gated ion channels like nicotinic acetylcholine receptors and purinergic P2X receptors were studied in native PC-12 cells. Calcium-activated potassium channels were analyzed in native (small-conductance calcium-activated potassium channel, SK(Ca)) as well as recombinant cell lines (large-conductance calcium-activated potassium channel, BK(Ca)). Also recombinant voltage-gated potassium channels (Kv1.1, Kv1.4) were amenable to this functional analysis. The method is particularly useful for identification of ion channel modulators in drug discovery since it allows functional analysis with high capacity.
Terstappen
G C
GC
Bayer AG, Pharma Research Centre, Wuppertal, D-42096, Germany. gct66554@glaxowellcome.co.uk
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D016716:PC12 Cells; D010455:Peptides / Q000494:pharmacology; D015221:Potassium Channels / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011978:Receptors, Nicotinic / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012413:Rubidium / Q000032:analysis / Q000378:metabolism*; D012604:Scorpion Venoms / Q000494:pharmacology; D013054:Spectrophotometry, Atomic / Q000379:methods; D013498:Suramin / Q000494:pharmacology; D014162:Transfection
D010455:Peptides; D015221:Potassium Channels; D011978:Receptors, Nicotinic; D018048:Receptors, Purinergic P2; D011994:Recombinant Proteins; D012604:Scorpion Venoms; D013498:Suramin; C064719:iberiotoxin; D012413:Rubidium