Addicted to Nicotine
A National Research Forum

Section IV: Biology of Nicotine Addiction
Neil E. Grunberg, Ph.D., Chair

MOLECULAR BIOLOGY AND KNOCKOUTS OF NICOTINIC RECEPTORS

Marina Picciotto, Ph.D.
Department of Psychiatry
Yale University School of Medicine

Introduction

Nicotine in tobacco exerts its actions on physiology and behavior by binding to nicotinic receptors in the brain. These receptors are large proteins spanning nerve cell membranes that normally translate the external signal of the neurotransmitter acetylcholine into an electrical signal that affects processes inside the nerve cell. These "nicotinic acetylcholine receptors" (nAChRs) can affect nerve cell function because they act as a gate for the passage of positively charged sodium, potassium, and calcium ions across the cell membrane. This ion flow can then increase the excitability of nerve cells and, most notably, can result in increased release of neurotransmitters that neurons use to communicate with other nerve cells. Outside of the brain, nicotinic receptors are also found in muscle and in nerve cells of the autonomic (fight or flight) nervous system. These receptors are also activated by nicotine and contribute to physiological responses to tobacco.

Each functional receptor is made up of five components, or subunits, which are similar to each other in sequence and structure. These subunits combine together like a wagon wheel to form the ion pore that is opened by binding of nicotine or acetylcholine. Nicotinic subunits are divided into several families. Those found in muscle form one family; the two other families are both found in nerve cells but have different structures. One family can make functional receptors only when at least one alpha-type subunit combines with at least one beta-type subunit to make up part of the five-component receptor (alpha2-alpha6 combined with beta2-beta4). The second family are able to make functional receptors even when all five components of the receptor are identical (alpha7-alpha9).

What We Know

Despite the many different subunits expressed in the brain, experiments indicate that nAChRs in the autonomic ganglia are primarily composed of alpha3 and beta4 subunits, whereas brain receptors are primarily composed of alpha4 and beta2 subunits, with the other subunits combining with these receptors in subsets of nerve cells. alpha7 subunits are present in many brain areas, including the hippocampus and the cortex, and appear to be able to make functional receptors on their own. One important question for ongoing research is which of the effects of nicotine on the central nervous system are mediated through alpha7 subunit-containing receptors and which are mediated through nAChRs containing the beta2 subunit. It is known, for example, that the alpha7 subunit is present at high levels in the hippocampus, an area of the brain involved in learning and memory. In addition, it is known that many nicotinic subunits, including alpha3, alpha4, alpha5, alpha6, beta2, beta3, and to some extent alpha7, are present in the mesolimbic dopamine system, an area of the brain thought to be involved in rewarding properties of drugs of abuse. These are therefore receptors that might contribute to cognitive and rewarding aspects of nicotine.

Given this wide diversity of receptors sensitive to nicotine, it is of interest to determine which subtypes of the nAChR mediate the effects responsible for tobacco consumption. A particularly useful tool in this search are transgenic animals that lack specific subunits or subtypes of the nAChR. These types of animals, termed "knockout" mice, can be generated using modern genetic engineering techniques and have been extremely useful in determining the functional role of many proteins that have been identified through molecular cloning. Mice lacking the beta2 subunit of the nAChR have been developed, as have mice lacking the alpha7 subunit. The beta2 subunit mutant mice have been used to examine the reinforcing properties of nicotine in an animal model.

The nucleus accumbens (NAc) and the ventral tegmental area (VTA) are brain areas thought to be responsible for the reinforcing effects of nicotine. These areas form the mesolimbic dopaminergic system and are critical brain reward regions that mediate the reinforcing actions of many drugs of addiction. Nicotine, like ethanol, cocaine, and amphetamine, can increase levels of dopamine in the NAc, and lesions of the mesolimbic dopamine neurons attenuate nicotine self-administration in rats. Nicotine can stimulate dopamine release in the brains of wild-type mice, but beta2 subunit mutant mice show no increase in extracellular dopamine levels following nicotine treatment. Using the self-administration paradigm, it has been possible to examine directly the reinforcing properties of nicotine in these beta2 mutant animals. Wild-type mice will self-administer low-dose nicotine after they have been trained to self-administer cocaine. In contrast, mutant mice extinguish self-administration behavior when nicotine is substituted for cocaine.

These experiments demonstrate that the beta2 subunit is a necessary component of the receptor mediating nicotine reinforcement and suggests a method to determine other components of this receptor.

What We Need To Know More About

There are still many questions about nicotinic receptor function. Future experiments using mice deficient in various a subunits of the nAChR will identify the partners of beta2 in mediating the addictive properties of nicotine and could contribute to rationale drug design of a treatment for nicotine addiction. Much of what we know about the structure of neuronal nicotinic receptors is based on studies of the muscle receptors, and a great deal of structural information still remains to be gathered. In addition, more data are needed on the relative ion permeability of different subunit combinations in the brain. Electrophysiological studies are beginning to show how nicotinic receptors are able to exert their actions on nerve cells, but much more research is needed to characterize which subunits are responsible for the effects of nicotine on different neurotransmitter systems. Finally, the links among the molecular biology of nicotine receptors, their physiology, and the ultimate role of individual receptor subtypes in complex behaviors are just beginning to be established. A multidisciplinary approach to nicotinic receptor function could unite a large body of work on the behavioral pharmacology of nicotine with the newer body of knowledge on the molecular biology of these receptors.

Recommended Reading

Corrigall, W.A.; Franklin, K.B.; Coen, K.M.; and Clarke, P.B. The mesolimbic dopaminergic system is implicated in the reinforcing effects of nicotine. Psychopharmacology (Berl) 107(2-3):285-289, 1992.

Dani, J.A., and Heinemann, S. Molecular and cellular aspects of nicotine abuse. Neuron 16(5):905-908, 1996.

Koob, G.F. Neural mechanisms of drug reinforcement. Ann N Y Acad Sci 654:171-91, 1992.

Le Novere, N., and Changeux, J.P. Molecular evolution of the nicotinic acetylcholine receptor: An example of multigene family in excitable cells. J Mol Evol 40:155-172, 1995.

McGehee, D.S.; Heath, M.J.; Gelber, S.; Devay, P.; and Role, L.W. Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors. Science 269(5231):1692-1696, 1995.

Orr-Utreger, A.; Goldner, F.M.; Saeki, M.; Lorenzo, I.; Golberg, L.; De Biasi, M.; Dani, J.A.; Patrick, J.W.; and Beaudet, A.L. Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-bungarotoxin binding sites and hippocampal fast nicotinic currents. J Neurosci 17(23):9165-9171, 1997.

Picciotto, M.R.; Zoli, M.; Lena, C.; Bessis, A.; Lallemand, Y.; Le Novere, N.; Vincent, P.; Pich, E.M.; Brulet, P.; and Changeux, J.-P. Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain. Nature 374:65-67, 1995.

Picciotto, M.R.; Zoli, M.; Rimondini, R.; Lena, C.; Marubio, L.M.; Pich, E.M.; Fuxe, K.; and Changeux, J.-P. Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine. Nature 391(6663):173-177, 1998.

Wonnacott, S.; Drasdo, A.; Sanderson, E.; and Rowell, P. Presynaptic nicotinic receptors and the modulation of transmission release. In: Block, G., and Marsh, J., eds. The Biology of Nicotine Dependence. Chichester: Wiley, 1990. pp. 87-105.