Scanning curves and kinetics of the acetylcholine / acetylcholine receptor hysteresis

Abstract

The high affinity binding of the neurotransmitter acetylcholine (AcCh) to the nicotinic acetylcholine receptor (nAcChR) of the electric organ of Torpedo californica exhibits a pronounced hysteresis and scanning loops. Dialysis conditions lead to an equilibrium binding curve which is coincident with the upper hysteresis branch; K = 5 × 10 −9 M, 4° C; and one AcCh molecule binds to the receptor (R) monomer of M r ≈ 290 000. Kinetic analysis of the changes in free and bound AcCh during the open-system conditions of dialysis, which releases the metastability, shows that AcCh (A) binding proceeds along an induced-fit pathway according to A + R h ⇌ AR h ⇌ AR vh, describing the ligand-induced transition from the high affinity state (R h; K h ≈ 10 −7 M) to the very high affinity state (R vh; K vh ⪡ K ). The rate constant of the step AR h → AR vh is k 2 = 6 × 10 −3 s −1 and that of the reverse step is k −2 = 3 × 10 −4 s −1. Direct binding of A to free R vh can be neglected. Therefore, the state R vh does not pre-exist; it is induced and only stable, as AR vh, by bound AcCh. The metastability can be described in terms of long-lived AR vh · R 1 hybrid dimers. Physiologically, the steric hindrance in the metastable hybrid may be viewed as a saving device; the functionally important, receptor channel-active R 1 conformer is, at low AcCh concentrations ([A] < 1 μ M), prevented from converting into the desensitized states R h and AR vh.