α4‐β2 and other nicotinic acetylcholine receptor subtypes as targets of psychoactive and addictive drugs

Abstract

1 Xenopus oocytes were injected with various muscle and neuronal nicotinic acetylcholine receptor (ACh receptor, cholinoceptor) subunit RNA combinations and their pharmacological properties studied using two-electrode voltage clamp. The functional expression of one of these combinations, rat a4-2fi2, has not been previously described. The a4-2 mRNA is a splicing variant transcribed from the a4 gene. In the experiments reported here, the a4-2#2 subtype was functionally indistinguishable from the a4-1#2 subtype.2 For each subtype, the relative potency of nicotine compared with acetylcholine was obtained by estimating the relative concentration of nicotine which would elicit the same current response as 0.1JIM ACh. The ratios of these concentrations (nicotine: ACh) for the mouse muscle ACh receptor-(alily6) was 96.1:1. In contrast, the ratios for the rat neuronal subtypes were: a2fi2, 1.01:1; a3fi2, 2.01:1; a4f2, 0.76:1 and a4-2#2, 0.76:1. The much greater relative nicotine sensitivity of the neuronal subtypes as compared with muscle receptors illustrates their potential to mediate the psychoactive and addictive effects of nicotine. However, it does not appear that the differences in relative nicotinic sensitivity among the neuronal receptors themselves can be used as a simple discriminative tool in neuronal tissue. 3 The slopes of the log dose-log response curves at low ACh concentrations were all greater than 1 but less than 2, suggesting that at least two agonist binding sites mediate the functional response of each hetero-oligomer. 4 The response of all the neuronal subtypes to ACh could be inhibited by the psychoactive drugs mecamylamine, amitriptyline, phencyclidine, trifluoperazine and promethazine. With the exception of the very potent antagonist, mecamylamine, the degree of block of the peak current to ACh produced by 10pM concentrations of these drugs was remarkably similar (around 50%). 5 The degree of inhibition produced when the antipsychotic drug, trifluoperazine, was co-applied with ACh increased as the duration of application increased. Such an effect was not observed with promethazine, a related phenothiazine derivative which does not have antipsychotic actions.

Potentiation of Human α4β2 Neuronal Nicotinic Acetylcholine Receptor by Estradiol

“…The ␣4-1 and ␣4-2 splice variant sequences are identical except for the last three amino acids changing a WLAAC C-terminal tail to WLAGMI. Rat ␣4 -1␤2 nAChRs have previously been shown to be inhibited by E (Paradiso et al, 2000), yet our observations would suggest that the relatively minor splice variation found in ␣4-2␤2 nAChRs (widely expressed in rat brain and described previously as pharmacologically indistinguishable from ␣4-1␤2; Connolly et al, 1992) could confer the capability of E potentiation to these receptors.…”

“…Alternative splicing of the rat ␣4 subunit (␣4-1 and ␣4-2) results in two variants of the rat ␣4␤2 nAChR (Connolly et al, 1992). The ␣4-1 and ␣4-2 splice variant sequences are identical except for the last three amino acids changing a WLAAC C-terminal tail to WLAGMI.…”

Unconventional ligands and modulators of nicotinic receptors

“…Even though ion flux studies carried out in neuronally differentiated PC12 cells and in SH-SY5Y cells have indicated that PCP inhibits noncompetitively the activation of ganglionic nAChRs with an IC50 of approximately 10 M (Connolly et al, 1992;Fryer and Lukas, 1999), the differential sensitivity of different neuronal nAChRs to PCP remains to be determined. Nevertheless, considering that PCP concentrations in the CSF can be as high as 6 M after high-dose intoxication (Donaldson and Baselt, 1979), it is likely that the psychotic effects induced by this highly addictive drug are at least in part the result of its actions on nAChRs in the brain.…”

“…Functional blockade of neuronal nAChRs has also been observed with PCP (Connolly et al, 1992;Fryer and Lukas, 1999). Even though ion flux studies carried out in neuronally differentiated PC12 cells and in SH-SY5Y cells have indicated that PCP inhibits noncompetitively the activation of ganglionic nAChRs with an IC50 of approximately 10 M (Connolly et al, 1992;Fryer and Lukas, 1999), the differential sensitivity of different neuronal nAChRs to PCP remains to be determined.…”

The α7 Nicotinic Acetylcholine Receptor Subunit Exists in Two Isoforms that Contribute to Functional Ligand-Gated Ion Channels

“…This lack of sequence variants of nicotinic receptors is surprising, given that most genes encoding AChR subunits contain between 5 and 10 exons, and thus numerous splice possibilities exist. Reports indicate that the ␣4 subunit exists in two isoforms, but when these respective isoforms are incorporated into functional channels containing the ␤2 subunit, they are indistinguishable on the basis of their pharmacology and biophysics (Connolly et al, 1992). The ␣1 subunit has also been shown to exist in multiple sequence variants, but only one of these isoforms assembles functional ACh receptors (Newland et al, 1995).…”