Human α3β4 Neuronal Nicotinic Receptors Show Different Stoichiometry if They Are Expressed in Xenopus Oocytes or Mammalian HEK293 Cells

Abstract

BackgroundThe neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the α3 and β4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the α5 and/or β2 subunits. We compared the properties of human α3β4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and α∶β subunit ratio.Methodology/Principal FindingsTwo distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the α subunit, as reported for α4β2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9′ of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike α4β2 channels, for α3β4 receptors the putative two-α form is the predominant one in oocytes (at 1∶1 α∶β cRNA ratio). This two-α form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-α form is the predominant one in HEK cells transfected with a 1∶1 α∶β DNA ratio or in oocytes at 9∶1 α∶β RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-α form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-α form gave rise to short bursts (14 ms) of high conductance (39 pS).Conclusions/SignificanceLike other neuronal nicotinic receptors, the α3β4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected.

Autoantibody-induced internalization of nicotinic acetylcholine receptor α3 subunit exogenously expressed in human embryonic kidney cells

“…For this assay, we established HEK293 cells stably co-expressing α3 and β4 subunits (HEK293-α3β4 cells). In accordance with previous reports that α3 subunits could form functional receptors when expressed in combination with β4 subunits on the HEK293 cell surface (Krashia et al, 2010), we successfully co-expressed the α3 and β4…”

Unraveling the High- and Low-Sensitivity Agonist Responses of Nicotinic Acetylcholine Receptors

“…Single-channel measurements have implied that the (␣4) 3 (␤2) 2 receptor displays larger conductance than the (␣4) 2 (␤2) 3 receptor , and the same has been reported for (␣3) 3 (␤4) 2 versus (␣3) 2 (␤4) 3 (Krashia et al, 2010). Combined with results obtained on homomeric receptors (Rayes et al, 2009), it seems to be a general mechanistic feature of the nAChRs that agonist binding to three sites results in maximal activation.…”

“…Although mixtures of different stoichiometries can explain biphasic concentration-response curves, the lack of a detailed explanation for the different agonist sensitivities has made interpretation of certain experimental data difficult and has hampered rational drug design. Hence, a thorough understanding of the underlying mechanism is highly desirable and may be relevant to other Cys-loop receptors, including the major ganglionic nAChR ␣3␤4 subtype, which also assemble in two different stoichiometries (Krashia et al, 2010).…”

Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

“…One could therefore use site-specific mutagenesis and biophysical characterization of activation mechanisms in recombinant receptors to find the types of subunits that line the agonist-binding pockets. By working with recombinant receptors, however, one cannot exclude the possibility that the ratio of subunit cDNA transfected, the type of the expressing cell, or a mutation might influence the receptor's subunit composition (e.g., 45,57,58). We nevertheless argue that the specific mutations we introduced are less likely to change the subunit stoichiometry of the recombinant receptors studied here (SI Text, section 3).…”