On the Mechanism Underlying the Irreversible Desensitization of Human Ganglionic Nicotinic Receptors

Abstract

alpha3-containing nicotinic acetylcholine receptors (AChRs) are expressed in autonomic ganglia and are thought to mediate fast synaptic transmission. These receptors have largely eluded rigorous kinetic characterization due to a combination of poor expression in heterologous systems and the total loss of receptor responsiveness during the course of excised-patch electrophysiology experiments. Recently, it has been demonstrated that a cysteine-to-alanine mutation at the −4’ position of the cytosolic M1-M2 linker can prevent this rundown in whole-cell patches of rodent superior cervical ganglion neurons (Campanucci et al., 2010). This work also suggested that this rundown, which plays a pivotal role in diabetic neuropathy, is due to oxidation of these cysteine residues. Here, we heterologously expressed human alpha3-subunit-containing AChRs in HEK-293 cells and found that the −4’ cysteine mutation cannot prevent the irreversible rundown of currents observed in outside-out patches of membrane. Furthermore, we found that the irreversible loss of receptor responsiveness does not occur in the whole-cell or cell-attached configurations, implying that patch excision leads to this phenomenon. We also investigated the effects of the reciprocal −4’ cysteine mutation in all five subunits of the human muscle AChRs, which do not natively have cysteines at this position. We found that introduction of the cysteines does not lead to an irreversible rundown, although it does slow down the rate of recovery from desensitization. Taken together, our results thus far suggest that the mechanism underlying the rundown of currents mediated by alpha3-containing AChRs in outside-out patches is fundamentally different from that mediated by cysteine oxidation in whole-cell recordings.