The additional ACh binding site at the α4(+)/α4(−) interface of the (α4β2)₂α4 nicotinic ACh receptor contributes to desensitization

Abstract

BACKGROUND AND PURPOSENicotinic ACh (α4β2)2α4 receptors are highly prone to desensitization by prolonged exposure to low concentrations of agonist. Here, we report on the sensitivity of the three agonist sites of the (α4β2)2α4 to desensitization induced by prolonged exposure to ACh. We present electrophysiological data that show that the agonist sites of the (α4β2)2α4 receptor have different sensitivity to desensitization and that full receptor occupation decreases sensitivity to desensitization. EXPERIMENTAL APPROACHTwo-electrode voltage-clamp electrophysiology was used to study the desensitization of concatenated (α4β2)2α4 receptors expressed heterologously in Xenopus oocytes. Desensitization was assessed by measuring the degree of functional inhibition caused by prolonged exposure to ACh, as measured under equilibrium conditions. We used the single-point mutation α4W182A to measure the contribution of individual agonist sites to desensitization. KEY RESULTS(α4β2)2α4 receptors are less sensitive to activation and desensitization by ACh than (α4β2)2β2 receptors. Incorporation of α4W182A into any of the agonist sites of concatenated (α4β2)2α4 receptors decreased sensitivity to activation and desensitization but the effects were more pronounced when the mutation was introduced into the α4(+)/α4(−) interface. CONCLUSIONS AND IMPLICATIONSThe findings suggest that the agonist sites in (α4β2)2α4 receptors are not functionally equivalent. The agonist site at the α4(+)/α4(−) interface defines the sensitivity of (α4β2)2α4 receptors to agonist-induced activation and desensitization. Functional differences between (α4β2)2α4 and (α4β2)2β2 receptors might shape the physiological and behavioural responses to nicotinic ligands when the receptors are exposed to nicotinic ligands for prolonged periods of times.

Targeting α4β2 Nicotinic Acetylcholine Receptors in Central Nervous System Disorders: Perspectives on Positive Allosteric Modulation as a Therapeutic Approach

“…Assuming a translation of the in vitro window current to an in vivo setting, such a persistent cholinergic activity could likely be a contributing phenomenon in volume transmission. Here, the wider window current at lower ACh concentrations observed with HS receptors [22,23] especially supports a role for this stoichiometry in this transmission mode.…”

“…It is thus believed to relate to the residual nAChR activity in a scenario of tonic exposure to an agonist, such as in volume transmission as described below, or upon administration of an exogenous nicotinic agonist. Here, it has been observed that the HS stoichiometry displays window current at lower and presumably more physiological agonist levels than the LS receptor [22,23]. Analogously, the window currents for various a4b2* agonists differ between the two stoichiometries [24].…”

“…Intriguingly, the high-and low-sensitive ACh binding sites in the LS receptor display differential responses to various nAChR ligands [19][20][21]. Additionally, the HS stoichiometry is more sensitive to nicotine-induced up-regulation and more sensitive to desensitization induced by low concentration of agonists [13,22], whereas the LS stoichiometry has a higher permeability to Ca 2+ [16] and more rapid desensitization kinetics [13] than the HS receptor.…”

“…The ACh window current (grey area) of the a4b2 nAChR is defined by the overlap of the receptor activation curve (solid line) and desensitization curve (dashed line) and indicates an agonist concentration range in which the receptor is expected to be tonically active. This has been addressed for a4b2 nAChR in multiple studies [14,[22][23][24].…”

“…Furthermore, nAChR agonists are notoriously known for their bell-shaped pharmacology (e.g. [67]), which may be due to increased desensitization at higher doses [14,[22][23][24], thus making dosing of these drugs somewhat of a challenge.…”