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Original Article |
3ß4 AChRs Expressed in Permanently Transfected HEK Cells
Correspondence to: Jon Lindstrom, 217 Stemmler Hall, University of Pennsylvania Medical School, Philadelphia, PA 19104-6074. Fax:(215) 573-2015 E-mail:jslkk{at}mail.med.upenn.edu.
We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human 3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express 3, 5, 7, ß2, and ß4 AChR subunits. From these subunits, several types of 3 AChRs as well as homomeric 7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect 3ß4 AChRs. 7 AChR function was not detected, yet we estimate that there are 70% as many surface 7 AChRs in IMR-32 when compared with 3 AChRs. Agonist potencies (EC50 values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16±1 µM) > nicotine (Nic; 48 ± 7 µM) 
cytisine (Cyt; 57 ± 3 µM) = acetylcholine (ACh; 59 ± 6 µM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s-1 (300 µM ACh; -60 mV). Assays that used mAbs confirmed the predominance of 3- and ß4-containing AChRs in IMR-32 cells. Although 18% of total 3 AChRs contained ß2 subunits, no ß2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface 3ß2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in 3ß2 transfected HEK cells. Characterization of various 3 AChRs expressed in HEK cell lines revealed that the functional properties of the 3ß4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC50 values) for this line was DMPP (14 ± 1 µM) = Cyt (18 ± 1 µM) > Nic (56 ± 15 µM > ACh (79 ± 8 µM). The efficacies of both Cyt and DMPP were 
80% when compared with ACh and the desensitization rate was 2 s-1. These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to 3ß4 AChRs.
Key Words: nicotinic receptor, autonomic, patch clamp
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