Mechanism of Tacrine Block at Adult Human Muscle Nicotinic Acetylcholine Receptors

Abstract

We used single-channel kinetic analysis to study the inhibitory effects of tacrine on human adult nicotinic receptors (nAChRs) transiently expressed in HEK 293 cells. Single channel recording from cell-attached patches revealed concentration- and voltage-dependent decreases in mean channel open probability produced by tacrine (IC50 4.6 μM at −70 mV, 1.6 μM at −150 mV). Two main effects of tacrine were apparent in the open- and closed-time distributions. First, the mean channel open time decreased with increasing tacrine concentration in a voltage-dependent manner, strongly suggesting that tacrine acts as an open-channel blocker. Second, tacrine produced a new class of closings whose duration increased with increasing tacrine concentration. Concentration dependence of closed-times is not predicted by sequential models of channel block, suggesting that tacrine blocks the nAChR by an unusual mechanism. To probe tacrine's mechanism of action we fitted a series of kinetic models to our data using maximum likelihood techniques. Models incorporating two tacrine binding sites in the open receptor channel gave dramatically improved fits to our data compared with the classic sequential model, which contains one site. Improved fits relative to the sequential model were also obtained with schemes incorporating a binding site in the closed channel, but only if it is assumed that the channel cannot gate with tacrine bound. Overall, the best description of our data was obtained with a model that combined two binding sites in the open channel with a single site in the closed state of the receptor.

Cholinergic drugs potentiate human nicotinic α4β2 acetylcholine receptors by a competitive mechanism

“…acetylcholine receptors expressed in M10 cells is 38 AM (Svensson and Nordberg, 1997) and for adult human muscle type nicotinic acetylcholine receptors expressed in HEK293 is 309 AM (Prince et al, 2002). With these values as reference, the present estimate of 14 AM for the apparent functional affinity of tacrine for human a4h2 nicotinic acetylcholine receptors does not appear unrealistic.…”

“…A more sophisticated mechanism is required to account for the anomalous concentration dependence of inhibition by galanthamine and rivastigmine. Various models for block of adult human muscle nicotinic acetylcholine receptors expressed in HEK293 cells by tacrine have been evaluated using single-channel data (Prince et al, 2002). The detailed kinetic analysis suggested that tacrine blocks the channel by binding to two sites in the open channel and to a site in the closed channel of the receptor.…”

“…Closed channel block is equivalent to binding to a specific nonallosteric site, which would prevent ion channel opening. Although there is an obvious difference in binding affinity of tacrine for neuronal and muscle type nicotinic acetylcholine receptors (Svensson and Nordberg, 1997;Prince et al, 2002), it is conceivable that the effects of the same drug on distinct nicotinic acetylcholine receptor subtypes are qualitatively similar. The two-site model applied to the present results of tacrine on human a4h2 nicotinic acetylcholine receptors predicts similar affinities for channel block (18 AM) and for the agonist binding site (14 AM).…”

Specific Inhibition of the Plasmodial Surface Anion Channel by Dantrolene

“…If this distant effect involves a 1:1 interaction with first-order rate constants, the nonluminal inhibitor will add a single exponentially decaying population of closings whose mean duration does not vary with inhibitor concentration. Any other combination of effects on open and closed channel durations would suggest a more complicated mechanism of inhibitor action, as commonly reported (46).…”

Regulation of Nicotinic Acetylcholine Receptor Channel Function by Acetylcholinesterase Inhibitors in Rat Hippocampal CA1 Interneurons

“…Many data suggest that the major subtypes of channels in rat hippocampal interneurons are composed of the ␣7 and ␣4␤2 subunits (Alkondon and Albuquerque, 1993;Jones and Yakel, 1997;Frazier et al, 1998;McQuiston and Madison, 1999). Tacrine, which was one of the first drugs used to treat AD (Terry and Buccafusco, 2003), was not a potentiator of nAChRs (Zwart et al, 2000;Samochocki et al, 2003) but rather inhibited the function of various nAChRs (Canti et al, 1998;Zwart et al, 2000;Prince et al, 2002). Huperzine A is isolated from the Chinese herb Huperzia serrata and enhances cognitive function (Zangara, 2003).…”