Multiple conductance classes of mouse nicotinic acetylcholine receptors expressed in Xenopus oocytes.

Abstract

Acetylcholine receptor (AcChoR) subunit mRNAs transcribed from mouse BC3H-1 cDNAs were in'ected into Xenopus oocytes and the expressed AcChoR channels were examined by single channel recording. Injection of a-, A8-, r-, and 8subunit mRNAs produced two predominant channel classes with conductances of =50 and -412 pS, while infrequent openings of -25-pS channels were also observed. Injection of a-, (3-, and y-subunit mRNAs produced a single class of ==12-pS AcChoR channels, which resembled the smallest conductance channels present in apy& inected oocytes. Assembly of -less channels may thus explain the lowest conductance AcChoR channels in aPyv-i iected oocytes and might also account for similar channels that have been observed in vertebrate skeletal muscle.The nicotinic acetylcholine receptor (AcChoR) of vertebrate skeletal muscle is a channel-forming pentamer thought to be composed of either a2,fy8 or aJ8e subunits. The Xenopus oocyte expression system (1-3) has been used in recent years to explore the roles of different subunits in determining receptor properties. By this means it has been shown that two principal conductance classes (40 and 60 pS) of mammalian AcChoRs differ in subunit composition, the larger conductance channel having an E subunit in place of y (4). It has also been reported that functional channels can be assembled when one of the subunits is missing. In the case of Torpedo AcChoRs, weak responses to AcCho were observed in some oocytes that were lacking y or 8 subunits (5, 6). The single channel properties of receptors lacking y or 8 subunits have not been described. Although both kinds of receptors assemble less efficiently and the 8-less receptors have reduced agonist affinity (5), the weak responses to AcCho might also reflect a reduced conductance or a briefer open time. We were interested in examining the properties ofthese receptors because they might offer an explanation for the small conductance channels (10-25 pS) observed in developing amphibian muscle (7,8). We injected mRNAs encoding the mouse a, 3, y, and 8 subunits into Xenopus oocytes and found that multiple conductance classes of channels were expressed on the oocyte membrane, and one of these, the smallest, is due to deletion of the 8 subunit. MATERIALS AND METHODSmRNA Preparation and Inection. The cDNA clones encoding mouse AcChoR subunits were generously provided by Jim Boulter (Salk Institute). mRNAs for a, (3, y, and 8 subunits were individually transcribed with SP6 polymerase. The plasmids were linearized with HindIII (clones BMA 407, BMB 49, and BMD 451 encoding a, A, and 8 subunits, respectively) or EcoRI (clone BMG 419, encoding fy subunit).The transcription reactions were carried out under standard conditions (Promega) and typically contained 2-3 ug of linearized cDNA template. The transcripts were resuspended in nuclease-free water at a concentration of 200 ng/pAl (asubunit transcript) or 100 ng/,Al (J3-, y-, and 8-subunit tran-

Potentiation and Inhibition of Neuronal Nicotinic Receptors by Atropine: Competitive and Noncompetitive Effects

“…Ion currents and indirect effects mediated by endogenous mAChRs were not observed in the present experiments. The absence of endogenous mAChRs, which has also been reported in other oocyte studies (13,(27)(28)(29)(30), is possibly due to the collagenase treatment and defolliculation of the oocytes (20).…”

Acetylcholine Receptors Direct Rapsyn Clusters to the Neuromuscular Synapse in Zebrafish

“…In frog the ␦ subunit is absolutely required for functional expression of both embryonic and adult-type AChRs (Paradiso and Brehm, 1998), and our findings from sop indicate that a similar dependence exists for fish. However, unlike fish and frogs, the ␦ subunit is apparently not essential for surface expression of embryonic-type AChRs in mammals (Kullberg and Brehm, 1990;Liu and Brehm, 1993). This species difference raises the interesting possibility that mutations resulting in omission of the ␦ subunit may be viable in the human population.…”

“…In contrast, the ␤ subunit is essential for assembly and function of muscle nicotinic receptors in all species (Liu and Brehm, 1993;Paradiso and Brehm, 1998). The ␦ subunit is not required for assembly and function in mammals (Kullberg et al, 1990), but it is required for expression of frog AChRs (Paradiso and Brehm, 1998). Therefore, the genes coding for the ␤ and ␦ subunits were the primary candidates we set out to test.…”

Slow-Channel Transgenic Mice: A Model of Postsynaptic Organellar Degeneration at the Neuromuscular Junction

“…This indicates that there is abundant ␦-subunit protein in the transgenic endplates. Also, the channel properties of ␦-less channels have been studied and have much lower conductances and shorter open times than the channels measured in the transgenic muscle fibers (Kullberg et al, 1990). Finally, we cannot rule out a role of other transgene actions that would not be present in the SCCMS, such as a dominant negative effect of overexpressed ⑀ subunit, that alters AChR D) were stained for cholinesterase (brown stain, hematoxylin and eosin counterstain) to localize motor endplates (A, C), glyoxal bis (2-hydroxyanil) (GBHA, red stain, methylene blue counterstain) to localize accumulation of ionized calcium (B), and for acid phosphatase (red stain, hematoxlin counterstain) to localize lysosomal activity (D).…”