The ability of the nicotinic acetylcholine receptor (nAChR) to undergo conformational transitions is exquisitely sensitive to its surrounding lipid environment. Previous work has highlighted a conformational selection mechanism, whereby different lipids stabilize different proportions of activatable resting versus nonactivatable conformations. In the absence of anionic lipids and cholesterol, the nAChR adopts an uncoupled conformation, which binds agonist with resting state-like affinity but does not usually undergo agonist-induced conformational transitions. Very slow (minutes to hours) transitions from uncoupled to coupled (resting, open and/or desensitized) conformations, however, can occur in membranes with relatively thick hydrophobic cores. Increasing membrane hydrophobic thickness 'awakens' uncoupled nAChRs by reducing the large activation energy barrier (or barriers) leading to coupled states, thus allowing conformational transitions to occur on an experimentally tractable timescale. Lipids shape activity by modulating the relative proportions of activatable versus nonactivatable conformations and by controlling the transitions between uncoupled and coupled conformations.
The 'capping' of loop-C at each transmitter binding site has been proposed to initiate the AChR channel-opening isomerization. We recorded single-channel currents from various loop-C constructs (adult mouse; both a-subunits, 188-198: VFYSCCPTTPY), both with and without ACh, to probe its role in binding vs. gating. One construct (11gly) had glycines at all positions, and others had n=9, 7, 5 or 3 glycines. The loop A mutation aA96H was added as a background to increase constitutive activity. 1) None of these constructs altered the unliganded gating activity.2) The open-probability of the 11gly-construct was not increased by 1mM ACh. 3) Reverting only two amino acids (bold) in the 11gly-construct to tyrosines generated AChRs that were activated by ACh, again without altering unliganded gating. These results suggest that loop-C is mainly involved in agonist binding. A sequential kinetic scheme has been used to describe brief and long unliganded openings in AChRs with background pore mutations (bdM2,9'-S). We compared this scheme with others using AChRs activated by low [ACh], in a construct that had only one functional binding site (aW149Fþbd9'SþεP121R). The interval durations were fitted across-concentrations using three different kinetic schemes, each having four states (C, O, AC and AO). The best fit was obtained by using the full cycle (MWC model). The estimated increase in the gating equilibrium constant with ACh (~740-fold) was similar to that obtained previously by saturating the binding site (~690-fold). These results show that a cyclic scheme describes brief/long unliganded openings at low [ACh] and can be used to estimate the association/dissociation rate constants to the highaffinity, open transmitter binding site.
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