The Effects of β3 Subunit Incorporation on the Pharmacology and Single Channel Properties of Oocyte-expressed Human α3β4 Neuronal Nicotinic Receptors

Abstract

We compared the main properties of human recombinant ␣3␤4␤3 neuronal nicotinic receptors with those of ␣3␤4 receptors, expressed in Xenopus oocytes. ␤3 incorporation decreased the channel mean open time (from 5.61 to 1.14 ms, after approximate correction for missed gaps) and burst length. There was also an increase in single channel slope conductance from 28.8 picosiemens (␣3␤4) to 46.7 picosiemens (␣3␤4␤3; in low divalent external solution). On the other hand, the calcium permeability (determined by a reversal potential method in chloride-depleted oocytes) and the pharmacological properties of ␤3-containing receptors differed little from those of ␣3␤4. The main pharmacological difference in ␣3␤4␤3 "triplet" receptors was a 3-fold decrease in the potency of lobeline relative to acetylcholine. Nevertheless, there was no change in the rank order of potency for agonists (epibatidine > > lobeline > cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, nicotine > acetylcholine > carbachol for both receptors; measured at low agonist concentrations). Sensitivity to the competitive antagonists trimetaphan (0.2-1 M) and dihydro-␤-erythroidine (30 M) was similar for the two combinations, with a Schild K B for trimetaphan of 76 and 66 nM on ␣3␤4 and ␣3␤4␤3, respectively. The change in single channel conductance confirms that ␤3 replaces a ␤4 subunit in the pentamer. The absence of pronounced differences in the pharmacological profile of the triplet receptor argues against a role for the ␤3 subunit in the formation of agonist binding sites, whereas the changes in channel kinetics suggest an important effect on receptor gating. The shortening of the burst length of ␤3-containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics.Among neuronal nicotinic subunits, ␤3 was long considered an "orphan" as it does not form functional recombinant receptors if expressed as a classical heteromeric combination, i.e.together with either an ␣ or a ␤ subunit. Because of the sequence similarity between ␤3 and ␣5 we tested the hypothesis that, just like ␣5, ␤3 would form functional receptors only if co-expressed together with both an ␣ and a ␤ subunit. By a reporter mutation strategy, we showed that ␤3 is indeed incorporated into functional recombinant ␣3␤4␤3 receptors (1). In ␣3␤4␤3 receptors, ␤3 is present as a single copy, which replaces one of the ␤ subunits (versus two copies each for the ␣3 and ␤4 subunits (2)); note that ␤3 coassembles with other ␤ subunits in rat native cerebellar nAChRs 1 (3). The question now arises of whether the ␤3 subunit can change the properties of neuronal nicotinic receptors. There are several reasons for investigating this problem. First of all it is important to establish whether any such change introduced by the presence of ␤3 affects the role that these receptors may have in physiological processes in the central nervous system or in the pharmacology of tobacco addiction. Furthermore, if ␤3-containing receptors have distinctive biophysical or pharmacol...

Functional Characterization of the α5(Asn398) Variant Associated with Risk for Nicotine Dependence in the α3β4α5 Nicotinic Receptor

“…Rat ␣3␤4 receptors expressed in BOSC 23 cells predominantly shows a 34-pS conductance class (Ragozzino et al, 1997). Overall, our findings are similar to the previously published single-channel data on wild-type ␣3␤4 and ␣3␤4␣5 receptors (e.g., Nelson and Lindstrom, 1999;Boorman et al, 2003). Our single-channel data indicate that the ␣5(Asp398) and ␣5(Asn398) variants behave similarly in single-channel recordings.…”

Subcellular Trafficking, Pentameric Assembly, and Subunit Stoichiometry of Neuronal Nicotinic Acetylcholine Receptors Containing Fluorescently Labeled α6 and β3 Subunits

“…␤3 subunits do not drastically alter the EC 50 for ACh or nicotine when incorporated into nAChRs (Boorman et al, 2003), but they do profoundly alter single-channel kinetics (Boorman et al, 2003). Channel burst duration was significantly shortened for nAChRs containing ␤3 versus those without it (Boorman et al, 2003), suggesting that ␤3 reduces the probability of channel opening, P open .…”

“…␤3 also increases ␣6-specific binding activity in HEK293 cells (Tumkosit et al, 2006). Uncertainty exists, however, because others have reported that ␤3 incorporation into nAChRs acts as a dominant negative (Boorman et al, 2003;Broadbent et al, 2006), suppressing ACh-evoked responses by an incompletely understood gating mechanism. This effect occurred apparently without significantly altering the surface expression of nAChRs.…”

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

“…V273T subunits participate in formation of ␣3␤4*-nAChR (23,24), help to define nAChR subtypes capable of containing ␤3 subunits, thus providing insights into roles of ␤3*-nAChR in nicotinic signaling. However, there remain puzzles about the makeup of functional, all-human or all-mouse ␣6*-nAChR, especially ␣6␤3*-nAChR.…”