Alteration of Mesopontine Cholinergic Function by the Lack of KCNQ4 Subunit.

The pedunculopontine nucleus (PPN), a structure known as a cholinergic member of the reticular activating system (RAS), is source and target of cholinergic neuromodulation and contributes to the regulation of the sleep-wakefulness cycle. The M-current is a voltage-gated potassium current modulated mainly by cholinergic signaling. KCNQ subunits ensemble into ion channels responsible for the M-current. In the central nervous system, KCNQ4 expression is restricted to certain brainstem structures such as the RAS nuclei. Here, we investigated the presence and functional significance of KCNQ4 in the PPN by behavioral studies and the gene and protein expressions and slice electrophysiology using a mouse model lacking KCNQ4 expression. We found that this mouse has alterations in the adaptation to changes in light-darkness cycles, representing the potential role of KCNQ4 in the regulation of the sleep-wakefulness cycle. As cholinergic neurons from the PPN participate in the regulation of this cycle, we investigated whether the cholinergic PPN might also possess functional KCNQ4 subunits. Although the M-current is an electrophysiological hallmark of cholinergic neurons, only a subpopulation of them had KCNQ4-dependent M-current. Interestingly, the absence of the KCNQ4 subunit altered the expression patterns of the other KCNQ subunits in the PPN. We also determined that, in wild-type animals, the cholinergic inputs of the PPN modulated the M-current, and these in turn can modulate the level of synchronization between neighboring PPN neurons. Taken together, the KCNQ4 subunit is present in a subpopulation of PPN cholinergic neurons, and it may contribute to the regulation of the sleep-wakefulness cycle.

The anthelmintic pyrantel acts as a low efficacious agonist and an open-channel blocker of mammalian acetylcholine receptors.

Pyrantel is an anthelmintic which acts as an agonist of nicotinic receptors (AChRs) of nematodes and exerts its therapeutic effects by depolarizing their muscle membranes. Here we explore at the single-channel level the action of pyrantel at mammalian muscle AChR. AChR currents are elicited by pyrantel. However, openings do not appear in clearly identifiable clusters over a range of pyrantel concentrations (1-300 microM). The mean open time decreases as a function of concentration, indicating an additional open-channel block. Single-channel recordings in the presence of high ACh concentrations and pyrantel demonstrate that the anthelmintic acts as a high-affinity open-channel blocker. When analyzed in terms of a sequential blocking scheme, the calculated forward rate constant for the blocking process is 8x10(7) M(-1) x s(-1), the apparent dissociation constant is 8 microM at a membrane potential of -70 mV and the process is voltage dependent. Pyrantel displaces alpha-bungarotoxin binding but the concentration dependence of equilibrium binding is shifted towards higher concentrations with respect to that of ACh binding. Thus, by acting at the binding site pyrantel activates mammalian AChRs with low efficacy, and by sterical blockade of the pore, the activated channels are then rapidly inhibited.

The noncompetitive inhibitor quinacrine modifies the desensitization kinetics of muscle acetylcholine receptors.

Quinacrine has been shown to act as a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAChR). However, its mechanism of action is still a matter of controversy. We analyzed in detail the action of quinacrine at both the single-channel and macroscopic current levels. The main effect of quinacrine is a profound concentration-dependent decrease in both the frequency of opening events and the duration of clusters elicited by high acetylcholine concentrations. Quinacrine also significantly increases (40-fold at 30 microM) the decay rate of macroscopic currents elicited by rapid perfusion of acetylcholine to outside-out patches. This decay is still well-described by a single exponential. Quinacrine has very little effect on the peak amplitude of the response, suggesting that it acts mainly on open channels. The recovery from desensitization after removal of acetylcholine is delayed in the presence of quinacrine. Results from both single-channel and macroscopic current recordings indicate that quinacrine increases the rate of nAChR desensitization and stabilizes the desensitized state. Interestingly, in equilibrium agonist-binding assays, quinacrine does not promote the typical high-affinity desensitized state. Thus, quinacrine seems to induce an intermediate state exhibiting the permeability but not the agonist binding properties of desensitization.

Amphetamine acts as a channel blocker of the acetylcholine receptor.

Non-competitive inhibitors (NCIs) of the nicotinic receptors (AChR) comprise a wide range of compounds. The chemical scaffold of amphetamine is similar to those of some NCIs. We investigated the effects of amphetamine (1-100 microM) on the muscle AChR by recording single-channel currents. The drug reduces the duration of the open state in a concentration-dependent manner and causes the appearance of brief closings, resembling the action of open-channel blockers. The forward rate constant for the blocking process is of the order of 10(7) M(-1) s(-1) and the blocking process is voltage dependent. The results are consistent with the steric block of the open channel as the primary action of amphetamine. At high drug concentrations the mechanism of inhibition deviates from that of classical open-channel blockers.