B

+ 10 (iM Progesterone ACh 1 |xM ACh 1 |iM ACh 1 jiM

Fig. 4. Progesterone inhibition of single-channels in an outside-out patch. (A) Schematic diagram of the experimental procedure used to examine ACh-evoked single channel activity. The media compositions are indicated in mM. (B) Application of brief ACh pulses (1 ¡M) evokes singlechannel activity in a membrane patch excised from a ciliary ganglion neuron. Left trace was obtained in control condition, middle trace during progesterone perfusion (10 ¡M, 55 s), and right trace during recovery (after 45 s of wash). "Open" or "closed" state of the nAChR are indicated by the label on the right. O , O , and O correspond to the current amplitude relative to the opening of 1, 2, or 3 channels, respectively.

Fig. 4. Progesterone inhibition of single-channels in an outside-out patch. (A) Schematic diagram of the experimental procedure used to examine ACh-evoked single channel activity. The media compositions are indicated in mM. (B) Application of brief ACh pulses (1 ¡M) evokes singlechannel activity in a membrane patch excised from a ciliary ganglion neuron. Left trace was obtained in control condition, middle trace during progesterone perfusion (10 ¡M, 55 s), and right trace during recovery (after 45 s of wash). "Open" or "closed" state of the nAChR are indicated by the label on the right. O , O , and O correspond to the current amplitude relative to the opening of 1, 2, or 3 channels, respectively.

measurements of the amplitude of discrete events activated by short pulses of ACh. Upon exposure to steroids, single-channel activity was profoundly decreased and a progressive recovery was readily observed when returning to the control conditions. Traces illustrated in Fig. 4 show that single-channel amplitudes remained unchanged by these experimental conditions, whereas the mean open-time of nAChRs is decreased.

Two important conclusions were made from these experiments: 1. steroid inhibition cannot be attributed to a reduction of the single channel conductance, and 2. steroid inhibition can take place even in the absence of the cytoplasmic machinery.

Steroids are lipophilic compounds that are known to diffuse through the cellular membrane and induce their action on gene expression by binding to receptors which are then translocated into the nucleus. Thus, addition of steroids in the extracellular medium in the low micromolar concentration range is expected to induce a partition of these molecules within the receptor-lipid environment in the cytoplasmic membrane. In addition, it has been a well-documented that cholesterol plays an important role in the stabilization of integral proteins such as LGCs and that a single muscular receptor can interact with as many as 5-10 cholesterol molecules (72). Thus, it could be expected that steroids act by perturbing the receptor environment. In favor if this hypothesis are recent studies using fluorescent probes and single-channel measurements, revealing that properties of both muscular and neuronal nAChRs depend on their lipid environment (73). Two factors demonstrate, however, that PROG inhibition of the neuronal nAChRs is not mediated by a membrane alteration. First, the inhibition is steroid-specific and addition of cholesterol, even at high doses, does not mimic the PROG inhibition (63,64). Second, PROG coupled with BSA (which is water-soluble and cannot diffuse into the membrane) induces similar or larger effects than free PROG (64,71). Thus, although membrane partitioning of the steroids should occur, it seems unlikely that these modifications could significantly alter the receptor properties, and it was previously concluded that steroids should act as negative allosteric modulators (63,64). A similar conclusion was more recently reached from biochemical and ion-flux assay experiments (71).

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