Steroid Mode Of Action

Inhibition by steroids of neuronal nAChRs can occur through several mechanisms illustrated in Fig. 3, which we shall review briefly. Namely, inhibition can be produced either by:

Fig. 3. Possible sites of steroid action. Each arrow symbolizes a possible site where steroids could act on the nAChR and correspond respectively to: 1. ACh-binding site with competitive inhibition, 2. the ionic pore with open channel blockade, 3. phosphorylation sites on the cytoplasmic loops, 4. lipid protein interaction, and 5. putative allosteric sites.

Fig. 3. Possible sites of steroid action. Each arrow symbolizes a possible site where steroids could act on the nAChR and correspond respectively to: 1. ACh-binding site with competitive inhibition, 2. the ionic pore with open channel blockade, 3. phosphorylation sites on the cytoplasmic loops, 4. lipid protein interaction, and 5. putative allosteric sites.

1. A competitive interaction with the natural ligand;

2. A block of the ionic pore (also referred to as open channel blocker: OCB);

3. activation of intracellular second messengers and protein phosphorylation/ dephosphorylation;

4. A partition of the steroid in the membrane lipid environment; and

5. Specific binding of the steroid on the LGC and induction of an allosteric interaction.

Studies designed to elucidate the steroid mode of action clearly demonstrate that active compounds such as PROG do not act as competitive inhibitors (63,64), nor as open channel blockers. In support of this conclusion, it was recently observed on cell lines expressing muscle or neuronal nAChRs that steroid incubation does not alter agonist or toxin binding (71). Furthermore, it was also found that increasing the agonist concentration failed to relieve PROG inhibition, which is another indication that steroids do not act by competition with the natural ligand (71).

Absence of channel blockade was illustrated by the voltage-independence of the inhibition and by determination of the single-channel events in small patches of membrane (63,64). Single-channel measurements can readily be obtained using the method of patch-clamp (22), and excision with a pipet of a small membrane area allows observation of opening and closing of one or a few nAChRs contained in this membrane patch. Typical recordings of single-channel activity, such as those illustrated in Fig. 4, made on membrane patches excised from dissociated quail ciliary ganglion neurons, permitted

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