ROLES OF NICOTINIC ACETYLCHOLINE RECEPTOR β SUBUNIT CYTOPLASMIC LOOPS IN ACUTE DESENSITIZATION AND SINGLE-CHANNEL FEATURES

To evaluate physiological roles of the large, second cytoplasmic loops (C2) situated between the M3 and M4 transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits. We have constructed chimeric beta 2 (beta 2 chi) and beta 4 (beta 4 chi) subunits in which the "nested" C2 domains (but not the "proximal" sequences of similar to 14 residues immediately adjacent to the M3 or M4 domains) of these beta subunits were replaced by the corresponding sequence from the serotonin 5-HT3A receptor subunit. We previously reported that heterologously expressed nAChR containing alpha 4 and beta 2 chi subunits displayed a faster whole-cell current decay in its agonist response compared to responses of all-wild-type alpha 4 beta 2-nAChR. This suggests an unexpected, functional role for the C2 domain of the beta 2 subunit in alpha 4 beta 2-nAChR acute desensitization. Here we report that there also is faster desensitization of alpha 4 beta 4 chi-nAChR relative to alpha 4 beta 4-nAChR stably and heterologously expressed in the human SH-EP1 cell-line. In addition, cell-attached, single-channel recording shows that both acetylcholine-activated alpha 4 beta 2 chi- and alpha 4 beta 4 chi-nAChR have a significantly lower mean open probability, shorter mean open-time, and a longer mean closed-time than their fully wild-type counterparts while not having different conductance amplitudes. These findings reveal microscopic bases for the faster desensitization of alpha 4*-nAChR containing chimeric instead of wild-type beta subunits. Our findings also remain consistent with novel and unexpected roles of beta subunit-nested C2 domains in modulation of alpha 4*-nAChR function. (C). 2014 IBRO. Published by Elsevier Ltd. All rights reserved.