Nicotinic acetylcholine receptors (nAChRs) are important targets in drug discovery due to their high physiological importance and their implications in the pathogenesis and poisoning.Accordingly, robust high thoughput screening technologies are necessary to test a large number of potential therapeutically active substances in combination with high expressing nAChR cell lines. We have, therefore, established an eight channel automatic patch clamp technique with the pituitary tumor cell line (GH₄C₁), stably transfected with the human a7 nAChR, to investigate the ligand induced receptor current.

Method: 

Rat pituitary tumor cell line (GH₄C₁) stably transfected with the human a7 nAChR (Genionics, Schlieren, Switzerland) were maintained in Dulbecco's modified Eagle's medium (DMEM). Cells were grown at 37 °C and 5% CO₂ / humidified air up to 70–80% confluence. The cells were harvested with an enzyme-free buffer. Patch clamp experiments were done in a external solution with 140 mM NaCl and a potassium free internal solution. Whole-cell patch clamping under voltage-clamping conditions (-70 mV) was performed with planar electrodes in an 8-channel patchliner system (Nanion Technologies GmbH, Munich, Germany).

Result: 

Cholinergic induced inward sodium currents with 20 to 400 pA could be assessed. The receptor activity could be triggered concentration depended by epibatidine (1–500 nM) and nicotine (1–100 mM). The ligand induced inward currents stopped within a few ms, because of receptor desensitisation. This desensitisation of receptors could be prevented by adding of 100 nM PNU 120596. Under such conditions, ligand induced current increased even to 2 nA.

Conclusion: 

The GH₄C₁ cell line is appropriate to investigate functional properties of the a7 nAChR with a chip based automated patch clamp system. Thus, different substances tested for their effect on the functionality of the a7 nicotinic receptor.