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.

[1]  N. Marrion,et al.  Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons , 1998, Nature.

[2]  P. Strong.  Potassium channel toxins. , 1990, Pharmacology & therapeutics.

[3]  Neil V Marrion,et al.  Calcium-activated potassium channels , 1998, Current Opinion in Neurobiology.

[4]  M. Lazdunski,et al.  Properties of the apamin-sensitive Ca2+-activated K+ channel in PC12 pheochromocytoma cells which hyper-produce the apamin receptor. , 1986, The Journal of biological chemistry.

[5]  V. Gribkoff,et al.  Effects of channel modulators on cloned large-conductance calcium-activated potassium channels. , 1996, Molecular pharmacology.

[6]  D. Julius,et al.  New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor , 1994, Nature.

[7]  R. Tsien,et al.  Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. , 1989, The Journal of biological chemistry.

[8]  R. Tsien,et al.  Fluorescence ratio imaging: a new window into intracellular ionic signaling , 1986 .

[9]  A. Weston,et al.  The effects of BRL 34915 and nicorandil on electrical and mechanical activity and on 86Rb efflux in rat blood vessels , 1986, British journal of pharmacology.

[10]  D. Jenkinson,et al.  Bis-quinolinium cyclophanes: 6,10-diaza-3(1,3),8(1,4)-dibenzena-1,5(1,4)- diquinolinacyclodecaphane (UCL 1684), the first nanomolar, non-peptidic blocker of the apamin-sensitive Ca(2+)-activated K+ channel. , 1998, Journal of medicinal chemistry.

[11]  B. Hille.  Ionic channels of excitable membranes , 2001 .

[12]  W. Stühmer,et al.  Cloned Ligand-gated Channels Activated by Extracellular ATP (P2X Receptors) , 1997, The Journal of Membrane Biology.

[13]  O. McManus,et al.  Purification and reconstitution of the high-conductance, calcium-activated potassium channel from tracheal smooth muscle. , 1994, The Journal of biological chemistry.

[14]  I. Watanabe,et al.  Differential Effects of Thiopental on Neuronal Nicotinic Acetylcholine Receptors and P2X Purinergic Receptors in PC12 Cells , 1997, Anesthesiology.

[15]  W. Catterall.  Structure and function of voltage-gated ion channels. , 1995, Annual review of biochemistry.

[16]  Brian Cox,et al.  HTS approaches to voltage-gated ion channel drug discovery , 1998 .

[17]  R. North,et al.  Ligand- and voltage-gated ion channels , 1995 .