Incorporation of the β3 Subunit Has a Dominant-Negative Effect on the Function of Recombinant Central-Type Neuronal Nicotinic Receptors

Abstract

The ␤3 neuronal nicotinic subunit is localized in dopaminergic areas of the central nervous system, in which many other neuronal nicotinic subunits are expressed. So far, ␤3 has only been shown to form functional receptors when expressed together with the ␣3 and ␤4 subunits. We have systematically tested in Xenopus laevis oocytes the effects of coexpressing human ␤3 with every pairwise functional combination of neuronal nicotinic subunits likely to be relevant to the central nervous system. Expression of ␣7 homomers or ␣/␤ pairs (␣2, ␣3, ␣4, or ␣6 together with ␤2 or ␤4) produced robust nicotinic currents for all combinations, save ␣6␤2 and ␣6␤4. Coexpression of wild-type ␤3 led to a nearly complete loss of function (measured as maximum current response to acetylcholine) for ␣7 and for all functional ␣/␤ pairs except for ␣3␤4. This effect was also seen in hippocampal neurons in culture, which lost their robust ␣7-like responses when transfected with ␤3. The level of surface expression of nicotinic binding sites (␣3␤4, ␣4␤2, and ␣7) in tsA201 cells was only marginally affected by ␤3 expression. Furthermore, the dominant-negative effect of ␤3 was abolished by a valine-serine mutation in the 9Ј position of the second transmembrane domain of ␤3, a mutation believed to facilitate channel gating. Our results show that incorporation of ␤3 into neuronal nicotinic receptors other than ␣3␤4 has a powerful dominant-negative effect, probably due to impairment in gating. This raises the possibility of a novel regulatory role for the ␤3 subunit on neuronal nicotinic signaling in the central nervous system.

Elucidation of Molecular Impediments in the α6 Subunit for in Vitro Expression of Functional α6β4* Nicotinic Acetylcholine Receptors

“…First, co-expression of chimeric ␣6/␣3 or ␣6/␣4 subunits (␣6-NTD fused with ␣3-or ␣4-TMD/ICL) with ␤2, ␤2␤3, and ␤4 subunits results in formation of functional receptors in both mammalian cells and oocytes (22)(23)(24)(25)(26)(27). Second, the minute responses observed for ␣6␤2␤3 and ␣6␤4␤3 nAChRs in oocytes have been found to be dramatically enhanced by the introduction of a ␤3 V273S mutant in the receptors (28,29). Finally, expression of functional ␣6␤2␤3 and ␣6␣4␤2␤3 nAChRs in oocytes has recently been accomplished by linking subunits in pentameric constructs; this concatemerization somehow makes up for the absence of whatever cellular factors that enables the formation of functional wild type (WT) receptors in neurons (26).…”

Identification of N-terminal Extracellular Domain Determinants in Nicotinic Acetylcholine Receptor (nAChR) α6 Subunits That Influence Effects of Wild-type or Mutant β3 Subunits on Function of α6β2*- or α6β4*-nAChR

“…Recently (12), it was observed that coexpression with a large excess of human nAChR wild-type ␤3 subunits has a dominantnegative effect on the function of human ␣6*-nAChR, whereas coexpression with a large excess of human mutant ␤3 subunits (valine 273 to serine at position 9Ј in the putative second transmembrane domain; ␤3 V273S ϭ ␤3 V9ЈS ) potentiates the function of human ␣6␤2*-and ␣6␤4*-nAChR. This observation is surprising, because knock-out studies strongly suggest that the naturally expressed murine ␣6*-nAChR containing ␤2 and ␤3 subunits are functional and that ␣6 and ␤3 subunits are needed to show sensitivity of these receptors to certain ␣-conotoxins (4 -7).…”

Structural and functional diversity of native brain neuronal nicotinic receptors

“…It has been shown that the β3 subunit co-assembles with several nAChR subunit combinations but, in all cases other than α3β3β4, it appears to have a dominant negative effect that leads to the absence of the functional expression of the assembled β3* receptor complex [41,42]. However our ex-vivo studies [31] indicate the great propensity of β3 to assemble with α6 subunit, and α6* receptor expression in β3 knock-out mice is decreased in the cell bodies and nerve terminals of dopaminergic neurons.…”