Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha 3 beta 4 AChRs expressed in permanently transfected HEK cells

We characterized the functional and molecular properties of nicotinic acety lcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfect ed human embryonic kidney (HER) cells. IMR-32 cells, like neurons of autono mic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the pr operties of functional AChRs in these cells overwhelmingly reflect alpha3 b eta4 AChRs. alpha7 AChR function was not detected, yet we estimate that the re are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC50 values) followed the rank order of 1,1-dime thyl-4-phenylpiperazinium (DMPP; 16 +/- 1 muM) > nicotine (Nic; 48 +/- 7 mu M) greater than or equal to cytisine (Cyt; 57 +/- 3 muM) = acetylcholine (A Ch; 59 +/- 6 muM). All agonists exhibited efficacies of at least 80% relati ve to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 muM ACh; -60 mV). Assays that used mAbs confirme d the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. A lthough 18% of total alpha3 ACbRs contained beta2 subunits, no beta2 subuni t was detected on the cell surface. Chronic Nic incubation increased the am ount of total, but not surface alpha3 beta2 AChRs in IMR-32 cells. Nic incu bation and reduced culture temperature increased total and surface AChRs in alpha3 beta2 transfected HEK cells. Characterization of various alpha3 ACh Rs expressed in HEK cell lines revealed that the functional properties of t he alpha3 beta4 cell line best matched those found for IMR-32 cells. The ra nk order of agonist potencies (EC50 values) for this line was DMPP (14 +/- 1 muM) = Cyt (18 +/- 1 muM) > Nic (56 +/- 15 muM > ACh (79 +/- 8 muM). The efficacies of both Cyt and DMPP were similar to 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with th e potential to express several human nicotinic AChR subtypes, the functiona l properties of AChRs expressed by IMR-32 are completely attributable to al pha3 beta4 AChRs.