Mark G. McNamee

Desensitization of the nicotinic acetylcholine receptor: molecular mechanisms and effect of modulators.

Summary1.Loss of response after prolonged or repeated application of stimulus is generally termed desensitization. A wide variety of phenomena occurring in living organisms falls under this general definition of desensitization. There are two main types of desensitization processes: specific and non-specific.2.Desensitization of the nicotinic acetylcholine receptor is triggered by prolonged or repeated exposure to agonists and results in inactivation of its ion channel. It is a case of specific desensitization and is an intrinsic molecular property of the receptor.3.Desensitization of the nicotinic acetylcholine receptor at the neuromuscular junction was first reported by Katz and Thesleff in 1957. Desensitization of the receptor has been demonstrated by rapid kinetic techniques and also by the characteristic “burst kinetics” obtained from single-channel recordings of receptor activity in native as well as in reconstituted membranes. In spite of a number of studies, the detailed molecular mechanism of the nicotinic acetylcholine receptor desensitization is not known with certainty. The progress of desensitization is accompanied by an increase in affinity of the receptor for its agonist. This change in affinity is attributed to a conformational change of the receptor, as detected by spectroscopic and kinetic studies. A four-state general model is consistent with the major experimental observations.4.Desensitization of the nicotinic acetylcholine receptor can be potentially modulated by exogenous and endogenous substances and by covalent modifications of the receptor structure. Modulators include the noncompetitive blockers, calcium, the thymic hormone peptides (thymopoietin and thymopentin), substanceP, the calcitonin gene-related peptide, and receptor phosphorylation. Phosphorylation is an important posttranslational covalent modification that is correlated with the regulation and desensitization of the receptor through various protein kinases.5.Although the physiological significance of desensitization of the nicotinic receptor is not yet fully understood, desensitization of receptors probably plays a significant role in the operation of the neuronal networks associated in memory and learning processes. Desensitization of the nicotinic receptor could also possibly be related to the neuromuscular disease, myasthenia gravis.

Purification of membrane proteins

As stated at the beginning of this chapter, it is not possible to present a single step-by-step protocol for the purification of all membrane proteins. We have discussed many of the individual techniques employed to purify membrane proteins and the problems associated with their application. A successful purification protocol will require the use of a combination of these techniques. It should be clear from the examples given, however, that affinity chromatography is by far the most useful technique available. The greatest single obstacle to performing a successful purification is the ability to maintain solubilized proteins in fully dispersed monomeric micelles without inactivating the protein. The best combination of techniques and the optimal conditions for their use can be determined only by trial and error. This process should become easier as a greater number of examples become available and as greater use is made of HPLC and FPLC techniques.

Reconstitution of acetylcholine receptor function in lipid vesicles of defined composition.

The effect of specific lipids on the functional properties of the acetylcholine receptor were examined in reconstituted membranes prepared from purified Torpedo californica acetylcholine receptor and various defined lipids. Cholesterol and negatively charged lipids greatly enhanced the ion influx response of the vesicles as measured by the effect of a receptor agonist on cation translocation across the vesicles. Part of the lipid-dependent effects could be attributed to alterations in the average size of the vesicles. All lipid mixtures used permitted complete incorporation of receptor and retention of ligand binding properties. Quantitative differences in ion flux properties suggest a modulating role for specific lipids in acetylcholine receptor function.

Lipid modulation of nicotinic acetylcholine receptor function : the role of membrane lipid composition and fluidity

The effects of membrane lipid composition and fluidity on AChR ion channel function were studied after reconstituting the receptor with sphingomyelin, phosphatidylcholines with different degrees of unsaturation, or different neutral lipids. AChR ion flux activity was shown to be retained in some membranes of both high and low fluidity, as measured by the steady-state anisotropy of the membrane probes diphenylhexatriene and trimethylammonium diphenylhexatriene. The results suggest that lipid composition is more important than bulk membrane fluidity in determining AChR ion channel function.

Mutations in the M4 domain of Torpedo californica acetylcholine receptor dramatically alter ion channel function

Site-directed mutagenesis was used to mutate alpha Cys418 and beta Cys447 in the M4 domain of Torpedo californica acetylcholine receptor expressed in Xenopus laevis oocytes. The M4 region is a transmembrane domain thought to be located at the lipid-protein interface. By whole-cell voltage clamp analysis, mutation of both alpha subunits to alpha Trp418 increased maximal channel activity approximately threefold, increased the desensitization rate compared with wild-type receptor, and shifted the EC50 for acetylcholine from 32 microM to 13 microM. Patch measurements of single-channel currents revealed that the alpha Trp418 increased channel open times approximately 28-fold at 13 degrees C with no effect on channel conductance. All of our measured functional changes in the alpha Trp418 mutant are consistent with a simple kinetic model of the acetylcholine receptor in which only the channel closing rate is altered by the mutation. Our results show that changes in protein structure at the putative lipid-protein interface can dramatically affect receptor function.

DESENSITIZATION OF CENTRAL CHOLINERGIC MECHANISMS AND NEUROADAPTATION TO NICOTINE

This review focuses on neuroadaptation to nicotine. The first part of the paper delineates some possible general mechanisms subserving neuroadaptation to commonly abused drugs. The postulated role of the mesocorticolimbic neuroanatomical pathway and drug-receptor desensitization mechanisms in the establishment of tolerance to, dependence on, and withdrawal from psychoactive drugs are discussed.The second part of the review deals with the pharmacological effects of nicotine at both pre- and postsynaptic locations within the central nervous system, and the still-perplexing upregulation of brain nicotine-binding sites seen after chronic nicotine administration. A special emphasis has been put on desensitization of presynaptic cholinergic mechanisms, and postsynaptic neuronal nicotinic-receptor function and its modulation by endogenous substances. A comparison with the inactivation process occuring at peripheral nicotinic receptors is also included.Finally, a hypothesis on the possible connections between desensitization of central cholinergic mechanisms and neuroadaptation to nicotine is advanced. A brief comment on the necessity of fully understanding the effects of nicotine on the developing nervous system closes this work.

The effect of cholesterol on agonist-induced flux in reconstituted acetylcholine receptor vesicles

The AChR has been successfully incorporated into functional vesicles and planar bilayer membranes by several procedures [l-9], using soybean lipids [ 1,35,7], total lipids isolated from electroplax membranes [6], or mixtures of lipids resembling the composition of electroplax membranes [2]. In [9] a neutral lipid fraction from asolectin was required to obtain a maximum agonist-induced ion flux, and a-tocopherol, phylloquinone or coenzyme Qlo were able to replace the neutral lipid component. Here, we describe the reconstitution of the AChR from Torpedo culifomica electroplax tissue in defined lipid mixtures. In agreement with [9], phospholipids alone do not result in effective reconstitution of the permeability control properties of the receptor. However, if cholesterol is included with the phospholipids at concentrations comparable to those found in native membranes, ion permeability control can be recovered.

Lipid modulation of nicotinic acetylcholine receptor function: the role of neutral and negatively charged lipids

The effects of negatively charged and neutral lipids on the function of the reconstituted nicotinic acetylcholine receptor from Torpedo californica were determined with two assays using acetylcholine receptor-containing vesicles: the ion flux response and the affinity-state transition. The receptor was reconstituted into three different lipid environments, with and without neutral lipids: (1) phosphatidylcholine/phosphatidylserine; (2) phosphatidylcholine/phosphatidic acid; and (3) phosphatidylcholine/cardiolipin. Analysis of the ion flux responses showed that: (1) all three negatively charged lipid environments gave fully functional acetylcholine receptor ion channels, provided neutral lipids were added; (2) in each lipid environment, the neutral lipids tested were functionally equivalent to cholesterol; and (3) the rate of receptor desensitization depends upon the type of neutral lipid and negatively charged phospholipid reconstituted with the receptor. The functional effects of neutral and negatively charged lipids on the acetylcholine receptor are discussed in terms of protein-lipid interactions and stabilization of protein structure by lipids.

Effects of phospholipase A2 on the binding and ion permeability control properties of the acetylcholine receptor.

Abstract An acidic phospholipase A2 (EC 3.1.1.4) isolated from Naja naja siamensis venom blocks acetylcholine receptor function in excitable post synaptic membrane vesicles from Torpedo californica electroplax. Specifically, the phospholipase acts catalytically to prevent the large increase in sodium efflux induced by carbamylcholine. The efflux inhibition can be correlated with specific hydrolysis of phospholipids in the membrane. During the time course of inhibition, the binding affinity of the receptor for carbamylcholine increases 10-fold, a phenomenon associated with receptor desensitization. Prolonged treatment of the membranes with phospholipase A2 causes nonspecific lysis of the vesicles. Incorporation of unsaturated fatty acids or lysophosphatidylcholine into Torpedo membranes also blocks carbamylcholine-induced sodium efflux. The fatty acids have no effect on the binding affinity of the receptor, and lysophosphatidylcholine causes a small decrease in receptor affinity for carbamylcholine. Lysophosphatidylethanolamine and most saturated fatty acids have no direct effect on sodium efflux, but the lysophosphatides cause vesicle lysis. All of the inhibitory effects of the phospholipase and the fatty acids can be reversed and/or prevented by treatment of the vesicles with bovine serum albumin.

Differential desensitization properties of rat neuronal nicotinic acetylcholine receptor subunit combinations expressed inXenopus laevis oocytes

Summary1. Chronic administration of nicotine up-regulates mammalian neuronal nicotinic acetylcholine receptors (nAChRs). A key hypothesis that explains up-regulation assumes that nicotine induces desensitization of receptor function. This is correlated with behaviorally expressed tolerance to the drug.2. The present experiments were conducted to: (a) obtain information on the nicotine-induced desensitization of neuronal nAChR function, a less understood phenomenon as compared to that of the muscle and electric fish receptor counterparts; (b) test the hypothesis that different receptor subunit combinations exhibit distinct desensitization patterns.3.Xenopus laevis oocytes were injected with mRNAs encoding rat receptor subunitsα2,α3, orα4 in pairwise combination with theβ2 subunit. The responses to various concentrations of acetylcholine (ACh) or nicotine were analyzed by the two electrode voltage clamp technique.4. Concentration-effect curves showed that nicotine was more potent than ACh for all the receptor subunit combinations tested. Only theα4β2 combination exhibited a depression of the maximum effect at concentrations higher than 20µM nicotine.5. After a single nicotine pulse, receptor desensitization (calculated as a single exponential decay) was significantly slower forα4β2 than for eitherα3β2 orα2β2.6. Concentrations of nicotine that attained a near maximum effect were applied, washed, and re-applied in four minute cycles. The responses were calculated as percentages of the current evoked by the initial application. Following 16 minutes of this protocol, theα4β2 combination showed a greater reduction of the original response as compared to theα2β2 andα3β2 subunit combinations. Taking points 5 and 6 together, these experiments suggest that theα4β2 receptor subtype desensitizes at a slower rate and remains longer in the desensitized state.7. Becauseα4β2 is the main receptor subunit combination within the brain and is up-regulated by nicotine, our data may be important for understanding the molecular basis of tolerance to this drug.