Elsevier

Neuropharmacology

Volume 44, Issue 6, May 2003, Pages 765-771
Neuropharmacology

Properties of neuronal α7 mutant nicotinic acetylcholine receptors gated by bicuculline

https://doi.org/10.1016/S0028-3908(03)00054-6Get rights and content

Abstract

We have shown previously that mutating to threonine the leucine residue in the M2 domain of the α7 nicotinic acetylcholine receptor (human L248T, L248T; chick L247T, L247T) converts bicuculline (BIC) from an antagonist into an agonist. In this work we studied the functional properties of the BIC-activated channels and report that, in Xenopus oocytes injected with L248T subunit cDNA, BIC activates single-channel currents that have similar conductances, but shorter mean burst duration, than the channels activated by ACh. In contrast, both the conductance and kinetics of the channels activated by either ACh or BIC are substantially the same in oocytes expressing L247T receptors. We have also shown previously that if Cys 189 and 190, which are thought to be at or near the transmitter binding site, are additionally mutated to Ser, the new mutant receptor (L247TC189SC190S) has a reduced affinity for ACh. We now find that the EC50 in the BIC dose-current response relation, as well the characteristics of the channels activated by BIC, are similar in oocytes expressing either L247T or L247T–C189S–C190S receptors. On the other hand, ACh activation of L247T–C189S–C190S receptors gates channels whose mean open time and burst duration are much shorter than those of ACh-gated L247T-channels. Therefore, the gating kinetics of both L248T and L247R–C189S–C190S receptor-channels change when BIC is replaced by ACh; and we conclude that both ACh and BIC activate mutant α7 receptors with different patterns of activation.

Introduction

The neuronal α7 nicotinic acetylcholine receptor (α7-AChR) is widely expressed throughout the central nervous system and is believed to play an important role in synaptic transmission because it is located at both pre- and post-synaptic sites (Radcliffe and Dani, 1998; Fabian-Fine et al., 2001), and because it is highly permeable to Ca2+ (Seguela et al., 1993; Fucile et al., 2000).

Using site-directed mutagenesis, it has been shown that the α7-AChR mutated in the leucine ring in the M2 channel-lining region exhibits special functional properties, namely: (i) slow desensitization; (ii) high affinity for the transmitter; (iii) lack of inward rectification; and (iv) conversion of many α7-AChR antagonists into agonists (Bertrand et al., 1992; Palma et al., 1996). Furthermore, while α7 subunit-deficient mice show unaltered mechanisms of learning and memory formation (Orr-Urtreger et al., 1997), mice that have α7-AChRs carrying the leucine-to-threonine (L250T) mutation in the M2 channel domain die 2–3 d post partum showing abnormal neuronal apoptosis (Orr-Urtreger et al., 2000), and transgenic L247Tα7-mutant mice (i.e. with a chick mutation equivalent to L250T in the mouse and to L248T in the human) exhibit altered emotional and memory mechanisms (Ragozzino et al., 2000). Thus, those findings indicate that mutations for threonine in the leucine ring of the M2 domain may provide useful tools to study neurological disorders including Alzheimer’s disease, schizophrenia and epilepsy, in which the α7-AChRs dysfunction is presumably involved (Changeux and Edelstein, 2001; Chini et al., 1994; Lena and Changeux, 1997; Weiland et al., 2000).

In a previous work we showed that bicuculline (BIC), the classical GABAA receptor antagonist, inhibits the human α7-AChR, but activates the L248T–AChR heterologously expressed in Xenopus oocytes (Demuro et al., 2001). For this work, we decided to determine the properties of the mutant α7-AChR channels gated by BIC, and compare them with those of the channels gated by ACh. We find that the channel properties differ, presumably because ACh and BIC act on overlapping but not identical biding sites of α7-nAChRs.

Section snippets

Oocyte cDNA injection

Recombinant DNA plasmids encoding the human L248T α7 (L248T), chick L247T α7 (L247T) and chick L247T/C189–C190 α7 (L247TC189SC190S) subunits in pcDNA3 vector were intranuclearly injected into stage VI oocytes (2 ng cDNA in 10 nl buffer). The protein sequence alignment of human and chick α7 subunits indicating the residues mutated to generate L248T, L247T and L247T–C189S–C190S nAChRs is illustrated in Fig. 1. Preparation of oocytes and injection procedures were as detailed elsewhere (Palma et

Spontaneous channel activity

Recordings were performed in outside-out patches obtained from ACh- and BIC-responsive oocytes that had been injected with L248T subunit cDNA. In accordance with previous findings (Fucile et al., 2002a, Fucile et al., 2002b), all the 16 excised patches examined (14 oocytes from seven donors) had a basal channel activity that was blocked by MLA (not shown) and which displayed a quite variable frequency (5–50 Hz). These ‘spontaneous’ channels were ‘silent’ in divalent ion-free medium (Fig. 2A,

Discussion

Bicuculline is routinely used as a potent antagonist of GABAA receptors, and we have recently shown that BIC also blocks competitively the homomeric neuronal α7 AChRs; but when these are mutated in the leucine ring (L248T) they are gated by BIC (Demuro et al., 2001). Here we studied further the actions of BIC on neuronal α7 receptors, which play important roles in many nervous system functions and disorders (McGehee et al., 1995; Pettit et al., 2001; Lodge, 2001).

We report that both BIC and ACh

Acknowledgements

We thank Dr. Marc Ballivet for providing the cDNAs encoding chick wt. α7 and L247Tα7 nAChR subunits, and Dr. Anna M. Mileo for making the hmut1 and Dr. Benedetta Barabino for the making cmut2 mutant subunits. The cDNA encoding the human wt. α7 subunit was a kind gift from Janssen (Belgium). This paper was supported by MIUR (Co-finanziamento and Neuroscienze to F.E.).

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