Functional Properties of Human Nicotinic AChRs Expressed by IMR-32 Neuroblastoma Cells Resemble Those of (cid:2) 3 (cid:3) 4 AChRs Expressed in Permanently Transfected HEK Cells

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 (cid:2) 3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express (cid:2) 3, (cid:2) 5, (cid:2) 7, (cid:3) 2, and (cid:3) 4 AChR subunits. From these subunits, several types of (cid:2) 3 AChRs as well as homomeric (cid:2) 7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect (cid:2) 3 (cid:3) 4 AChRs. (cid:2) 7 AChR function was not detected, yet we estimate that there are 70% as many surface (cid:2) 7 AChRs in IMR-32 when compared with (cid:2) 3 AChRs. Agonist potencies (EC 50 values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16 (cid:4) 1 (cid:5) M) (cid:6) nicotine (Nic; 48 (cid:4) 7 (cid:5) M) (cid:7) cytisine (Cyt; 57 (cid:4) 3 (cid:5) M) (cid:8) acetylcholine (ACh; 59 (cid:4) 6 (cid:5) M). 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 (cid:9) 1 (300 (cid:5) M ACh; (cid:9) 60 mV). Assays that used mAbs confirmed the predominance of (cid:2) 3- and (cid:3) 4-containing AChRs in IMR-32 cells. Although 18% of total (cid:2) 3 AChRs contained (cid:3) 2 subunits, no (cid:3) 2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface (cid:2) 3 (cid:3) 2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in (cid:2) 3 (cid:3) 2 transfected HEK cells. Characterization of various (cid:2) 3 AChRs expressed in HEK cell lines revealed that the functional properties of the (cid:2) 3 (cid:3) 4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC 50 values) for this line was DMPP (14 (cid:4) 1 (cid:5) M) (cid:8) Cyt (18 (cid:4) 1 (cid:5) M) (cid:6) Nic (56 (cid:4) 15 (cid:5) M (cid:6) ACh (79 (cid:4) 8 (cid:5) M). The efficacies of both Cyt and DMPP were (cid:2) 80% when compared with ACh and the desensitization rate was 2 s (cid:9) 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 (cid:2) 3 (cid:3) 4 AChRs.

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