β3 subunit is present in different nicotinic receptor subtypes in chick retina

https://doi.org/10.1016/S0014-2999(00)00067-4Get rights and content

Abstract

Although the neuronal nicotinic β3 subunit was cloned several years ago, it has only recently been shown to form heteromeric channels when associated with other nicotinic subunits, and very little information is available concerning its assembly in the native nicotinic receptors of the nervous system. Using subunit-specific antibodies and immunoprecipitation experiments, we have identified the retina as being the chick central nervous system (CNS) area that expresses the highest level of the β3 subunit. Sequential immunopurification experiments showed that there are at least two populations of β3-containing receptors in chick retina: in one, the β3 subunit is associated with the α6 and β4 subunits; in the other more heterogeneous population, the β3 subunit is associated with the α2, α3, α4, β2 and β4 subunits. Both of these receptor populations bind [3H]epibatidine and a number of nicotinic receptor agonists with high affinity (nM) and nicotinic receptor antagonists with a lower affinity (μM). The greatest pharmacological difference between the two populations is the affinity for the α-conotoxin MII, which inhibits binding to α6-containing receptors and not that to β3-containing receptors. We also searched for the presence of the β3 subunit associated with the α-bungarotoxin binding subunits α7 and/or α8 in retina and chick brain. Immunoprecipitation studies using anti-β3 antibodies did not detect any specific α-bungarotoxin labeled receptors, thus, indicating that the β3 subunit is not present in the α-bungarotoxin receptors of these areas.

Introduction

Neuronal nicotinic acetylcholine receptors are widely expressed in the vertebrate nervous system, where they function as postsynaptic receptors to excite neurons or presynaptic receptors to modulate neurotransmitter release Role and Berg, 1996, Wonnacott, 1997. These receptors are a family of acetylcholine-gated cation channels consisting of different subtypes, each of which has a specific anatomical distribution in the central (CNS) and peripheral nervous system Sargent, 1993, Gotti et al., 1997a, Lindstrom, 2000.

Eight vertebrate α-subunits (α2–α9) and three β subunits (β2–β4) have been cloned. When individually expressed in heterologous systems, the α7, α8, and α9 subunits form functional homomeric channels that are blocked by the snake toxin α-bungarotoxin, whereas the α2, α3, α4 and α6 subunits form channels only when coexpressed in combination with the β2 or β4 subunits (heteromeric channels), and these channels are not blocked by α-bungarotoxin McGehee and Role, 1995, Role and Berg, 1996, Lindstrom, 2000.

Neither the α5 nor the β3 subunits can form functional channels when coexpressed heterologously together with another α or β subunit, which is why they were referred to as orphan subunits for a long time Sargent, 1993, Role and Berg, 1996. The participation of the α5 subunit in the formation of acetylcholine-activated channels has recently been demonstrated. It forms functional channels in oocytes only when coexpressed with the α4 and β2 Ramirez-Latorre et al., 1996, Fucile et al., 1997, or with the α3 and β2 or α3 and β4 subunits, but not when expressed alone (Wang et al., 1996).

β3 subunit mRNA is expressed in several areas of the mammalian CNS, where it extensively colocalizes with the α6 subunit (Le Novère et al., 1996). It has recently been reported that a mutated form of the human β3 subunit (β3V273T) can be coexpressed in oocytes with the human α3 and β4 subunits to form functional channels whose pharmacological and biophysical properties are different from those of the α3β4 combination (Groot-Kormelink et al., 1998). It has also been reported that the β3 subunit can co-assemble in oocytes with a mutated form of the α7 subunit (L247Tα7), an assembly that changes the functional and pharmacological profile of L247Tα7 receptors (Palma et al., 1999).

Identifying the subunit composition of native neuronal nicotinic acetylcholine receptor subtypes is a difficult but worthwhile task, since recent results have shown that the functional and pharmacological properties of heterologous subtypes may be influenced by the types of cells in which they are expressed (Lewis et al., 1997). This means that it is difficult to deduce the pharmacological properties of native receptors from those obtained in heterologous systems. The only biochemical data on the presence of the β3 subunit in neurons have been obtained for the rat cerebellum, in which it has been found that the β3 subunit is present in oligomeric receptors together with the α4, β2, and β4 subunits (Forsayeth and Kobrin, 1997).

In order to investigate the role of the β3 subunit in native chick receptors, we immunopurified β3-containing receptors using subunit-specific antibodies. We decided to use chick retina as a source of β3 subunits because previous work by Hernandez et al. (1995) has shown that β3 mRNAs are undetectable in most chick brain compartments but relatively abundant in the developing retina and trigeminal ganglia.

Section snippets

Subunit-specific antibodies

Polyclonal antibodies against the α2, α3, α4, α5, α6, α7, α8, β2, β3, β4 peptides were raised as previously described (Vailati et al., 1999). Two different peptides were chosen for all of the subunits: one located in the cytoplasmic loop between M3 and M4 (CYT), and the other located at the COOH terminal (COOH). The antibodies raised against the peptides were purified on an affinity column made by coupling the corresponding peptide to CNBr-activated Sepharose 4B (Pharmacia) according to the

Characterization of the anti-β3 antibodies

The antibodies raised against peptides of the β3 subunit were tested on a fusion protein of the chick β3 subunit expressed in bacteria containing the sequence 308–372 of the intracellular loop of the subunit itself.

Fig. 1 shows the Western blot analysis of the bacteria homogenate obtained before (lanes 1 and 3) and after (lanes 2 and 4) induction with isopropyl-β-d-thiogalactoside. Only the antibodies directed against the intracellular peptide (KGHVDRYSFSDTEEKETTLKSKLPG) were able to recognize

Discussion

There is little information concerning the distribution, role, and function of the β3-containing nicotinic receptors. It has only recently been shown that a mutated form of the β3 subunit is able to form functional channels. The only previously available data concerned its cloning Deneris et al., 1989, Hernandez et al., 1995, its mRNA distribution in chick and rat brain Hernandez et al., 1995, Le Novère et al., 1996 and its presence with the α4, β2, and β4 subunits in oligomeric receptors

Acknowledgements

We would like to thank Mr. Kevin Smart and Ms. Ida Ruffoni for their aid with the manuscript. This work was supported in part by grants from Fabriques de Tabac Réunies, Neuchâtel, Switzerland, the Italian Ministry of University and Scientific and Technological Research, and the European Programme “Training and Mobility of Researchers”, Contract No. ERB4061PL97-0790.

References (24)

  • S. Fucile et al.

    α5 Subunit forms functional α3β4α5 nAcetylcholineRs in transfected human cells

    Neuroreport

    (1997)
  • V. Gerzanich et al.

    Comparative pharmacology of epibatidinebatidine: a potent agonist for neuronal nicotinic acetylcholine receptors

    Mol. Pharmacol.

    (1995)
  • Cited by (0)

    View full text