A physical model of nicotinic ACh receptor kinetics

If only one molecule is bound to the receptor, it opens for a short time; two bound molecules result in a long opening of the receptor. It was however noted, that an excess of brief openings, that appeared in some recordings at high agonist concentrations, is not consistent with this interpretation (Colquchoun et al., 1985, Sine et al., 1987, Hallermann et al., 2005). Thus, the question of how the binding sites contribute to channel gating is still open.

The GAKM assumes that the ACh-AChR binding is formed as a result of the interaction between the πelectron systems originating from the agonist-bindingsites-aromatic-amino-acids, and the cholinum nitrogen, native to the quaternary ammonium ligands (reviewed by Arias (2000)).What is usually not discussed is the fact that close to the ACh binding site there is an electrically noneutral amino acid, the negatively charged aspartic acid, which may have some influence on the agonist binding or tracking. To check the role of this amino acid for the ACh receptor’s kinetics we performed experiments on the αD200Q receptor (Mukhtasimova et al., 2005) with the negatively charged aspartic acid removed and replaced by a polar amino acid (glutamine). Previous studies suggested that the amino acid αD200 played a role in the process of receptor activation, i.e. in the process that starts after a binding of the agonist (O’ Leary et al., 1992, Akk et al., 1996). In our present studies we go a bit further: we examine the role of this amino acid in the process of the receptor block.

We studied the receptor’s kinetics, in both wild and mutated versions, to better understand the limits of the validity of the GAKM discussed above. This model has very severe consequences for our understanding of the structure of the receptor. Applications of various blockers, such as the open-channel blockers and the use of GAKM predicts, for example, the amino acid content of an ion channel (Leonard et al., 1988). Validity of the models describing how the receptor is built is thus very strongly dependent on the model of the receptor’s kinetics.

The classical open-channel blockers’ theory is based on the observation that the voltage dependence in the blockers’ action occurs only when the blockers are electrically charged (Neher et al., 1978, Akk et al., 2003). It turns out, however, that there exist electrically neutral molecules (such as HC (Bouzat et al., 1996, Nurowska et al., 2002) and physostigmine (Wachtel, 1993)), which block the ACh receptor with a very strong voltage dependence. They prove that the voltage dependency is quite a complex phenomenon, which is not only a consequence of the molecule charge. Another doubt about the GAKM comes from the fact that the open-channel blockers action is very often dependent on the agonist concentration. The occurrence of this dependence is so common that some authors consider it as the main feature of the open-channel blockers (Buisson et al., 1998). This is in direct contradiction with the claim of the open-channel blockers’ theory that the blocker enters into the ionic channel: once the blocker is in the channel, the agonist concentration should not influence the block.

In this paper we introduce a model of the receptor blocking which resolves the problems discussed above. Our model explains how an electrically neutral blocker (HC) induces a voltage dependent effect. It also explains the dependence of the blocker action on the agonist concentration. This model naturally and universally explains receptor desensitization. Molecular rearrange-ments causing receptor desensitization were, up to now, poorly understood. Our model of the receptor block requires a new kinetic model of the AChR gating. The formulation of this new kinetic model is also given and its consequences for the kinetic theory are discussed.

The human kidney cell line HEK 293 was routinely cultured in DMEM, supplemented with heatinactivated foetal calf serum (10%), L-glutamine (4mM), penicillin (100 units/ml -1 ) and streptomycin (100 µg/ ml -1 ). Cells were incubated in an atmosphere containing 5% CO 2 at 37 • C. One day before transfection, cells were seeded into culture dishes.

Transfection. Mouse nAChR subunit (α, β, δ, γ) cD-NAs in the expression vector pRBG4 (Sine, 1994) and the mito-GFP construct were transiently expressed in HEK 293 cells using LipofectAmine Plus TM Reagent (Life Technologies, Inc). The WT subunits and the mutated α-subunit with the D200 (aspartic acid) to Q (glutamine) substitution were the generous gift of Prof. Steven Sine (Receptor Biology Lab., Mayo Foundation, Rochester). Mito-GFP was a kind gift from Prof. Adam Szewczyk (Nencki Institute, Warsaw). A total of 1 µg of cDNA per 35 mm culture dish in the ratio 2:1:1:1:1 (α:β:δ:γ:GFP) was use

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