Exocytic Trafficking Is Required for Nicotine-induced Up-regulation of α4β2 Nicotinic Acetylcholine Receptors

The primary target for nicotine in the brain is the neuronal nicotinic acetylcholine receptor (nAChR). It has been well documented that nAChRs respond to chronic nicotine exposure by up-regulation of receptor numbers, which may underlie some aspects of nicotine addiction. In order to investigate the mechanism of nicotine-induced nAChR up-regulation, we have developed a cell culture system to assess membrane trafficking and nicotine-induced up-regulation of surface-expressed α4β2 nAChRs. Previous reports have implicated stabilization of the nAChRs at the plasma membrane as the potential mechanism of up-regulation. We have found that whereas nicotine exposure results in up-regulation of surface receptors in our system, it does not alter surface receptor internalization from the plasma membrane, postendocytic trafficking, or lysosomal degradation. Instead, we find that transport of nAChRs through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors. Therefore, nicotine appears to regulate surface receptor levels at a step prior to initial insertion in the plasma membrane rather than by altering their endocytic trafficking or degradation rates as had been previously suggested. The primary target for nicotine in the brain is the neuronal nicotinic acetylcholine receptor (nAChR). It has been well documented that nAChRs respond to chronic nicotine exposure by up-regulation of receptor numbers, which may underlie some aspects of nicotine addiction. In order to investigate the mechanism of nicotine-induced nAChR up-regulation, we have developed a cell culture system to assess membrane trafficking and nicotine-induced up-regulation of surface-expressed α4β2 nAChRs. Previous reports have implicated stabilization of the nAChRs at the plasma membrane as the potential mechanism of up-regulation. We have found that whereas nicotine exposure results in up-regulation of surface receptors in our system, it does not alter surface receptor internalization from the plasma membrane, postendocytic trafficking, or lysosomal degradation. Instead, we find that transport of nAChRs through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors. Therefore, nicotine appears to regulate surface receptor levels at a step prior to initial insertion in the plasma membrane rather than by altering their endocytic trafficking or degradation rates as had been previously suggested. Neuronal nicotinic acetylcholine receptors (nAChRs) 1The abbreviations used are: nAChR, nicotinic acetylcholine receptor; PBS, phosphate-buffered saline; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorting; BFA, brefeldin A; ER, endoplasmic reticulum; HA, hemagglutinin. in mammalian brain compose a family of proteins encoded by 11 genes (α2-7, α9-10, and β2-4) that assemble into pentameric ligand-gated ion channels (1Dani J.A. Biol. Psychiatry. 2001; 49: 166-174Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 2Elgoyhen A.B. Vetter D.E. Katz E. Rothlin C.V. Heinemann S.F. Boulter J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3501-3506Crossref PubMed Scopus (564) Google Scholar, 3Lindstrom J. North R.A. Handbook of Receptors and Channels. CRC Press, Inc., Boca Raton, FL1995: 153-175Google Scholar, 4McGehee D.S. Role L.W. Annu. Rev. Physiol. 1995; 57: 521-546Crossref PubMed Scopus (898) Google Scholar). Although all combinations of subunits that form functional nicotinic receptors can bind nicotine, α4- and β2-containing receptors form the high affinity nicotine binding site (5Picciotto M.R. Zoli M. Lena C. Bessis A. Lallemand Y. Le Novere N. Vincent P. Pich E.M. Brulet P. Changeux J.P. Nature. 1995; 374: 65-67Crossref PubMed Scopus (563) Google Scholar, 6Ross S.A. Wong J.Y. Clifford J.J. Kinsella A. Massalas J.S. Horne M.K. Scheffer I.E. Kola I. Waddington J.L. Berkovic S.F. Drago J. J. Neurosci. 2000; 20: 6431-6441Crossref PubMed Google Scholar, 7Whiting P.J. Liu R. Morley B.J. Lindstrom J.M. J. Neurosci. 1987; 7: 4005-4016Crossref PubMed Google Scholar, 8Flores C.M. Rogers S.W. Pabreza L.A. Wolfe B.B. Kellar K.J. Mol. Pharmacol. 1992; 41: 31-37PubMed Google Scholar, 9Marubio L.M. del Mar Arroyo-Jimenez M. Cordero-Erausquin M. Lena C. Le Novere N. de Kerchove d'Exaerde A. Huchet M. Damaj M.I. Changeux J.P. Nature. 1999; 398: 805-810Crossref PubMed Scopus (511) Google Scholar) and are therefore thought to be the primary nAChR subtype affected by the relatively low nicotine concentrations (100-500 nm) found in the blood of smokers (10Benowitz N.L. Kuyt F. Jacob P.I. Clin. Pharmacol. Ther. 1982; 32: 758-764Crossref PubMed Scopus (256) Google Scholar). Chronic exposure of α4β2 receptors to nicotine, as occurs in smokers, results initially in receptor desensitization, followed by subsequent up-regulation of high affinity nicotine binding sites (11Schwartz R.D. Kellar K.J. Science. 1983; 220: 214-216Crossref PubMed Scopus (420) Google Scholar). Upon nicotine withdrawal, the increased number of nAChRs recover from desensitization, resulting in excess activity in the nAChR system. It has been proposed that this cycle of nicotine-induced receptor up-regulation may contribute to the negative symptoms associated with nicotine withdrawal, resulting in continued tobacco consumption and, eventually, nicotine dependence (12Dani J.A. Heinemann S. Neuron. 1996; 16: 905-908Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). The mechanisms and cellular machinery required for nicotine-induced up-regulation remain unknown. However, a large body of research supports a consensus on several relevant points. First, up-regulation is observed in the brains of human smokers (13Breese C.R. Adams C. Logel J. Drebing C. Rollins Y. Barnhart M. Sullivan B. Demasters B.K. Freedman R. Leonard S. J. Comp. Neurol. 1997; 387: 385-398Crossref PubMed Scopus (228) Google Scholar) as well as in chronically nicotine-treated animal models (8Flores C.M. Rogers S.W. Pabreza L.A. Wolfe B.B. Kellar K.J. Mol. Pharmacol. 1992; 41: 31-37PubMed Google Scholar, 14Marks M.J. Pauly J.R. Gross S.D. Deneris E.S. Hermans-Borgmeyer I. Heinemann S.F. Collins A.C. J. Neurosci. 1992; 12: PubMed Google Scholar) and in nAChRs R. P.J. Lindstrom J. Mol. Pharmacol. Google Scholar, M. Sullivan J.P. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar, P. S. Mol. Pharmacol. Google Scholar, M. J. Pharmacol. Ther. 1995; Google that up-regulation cellular It has been that increased nicotine binding in receptor number rather than receptor affinity R. P.J. Lindstrom J. Mol. Pharmacol. Google Scholar, R.D. Kellar K.J. J. PubMed Scopus Google Scholar, M.J. J.A. Collins A.C. J. Pharmacol. Ther. Google Scholar, E.M. S. PubMed Scopus Google that up-regulation receptor rather than in the receptors. chronic nicotine does not nAChR levels M.J. Pauly J.R. Gross S.D. Deneris E.S. Hermans-Borgmeyer I. Heinemann S.F. Collins A.C. J. Neurosci. 1992; 12: PubMed Google The relevant of nAChRs in neuronal at the plasma membrane, exposure to membrane In cell surface receptor activity is by insertion and of receptors at the plasma membrane Annu. Rev. 1997; PubMed Scopus Google Scholar). it is that of neuronal is at in by the and of receptors R.A. Science. 1999; PubMed Scopus Google Scholar, C. Y. R.A. M. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). receptor has been to be a of the potential of J.L. P. M. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, mechanisms the number of surface receptors at the plasma membrane may be in nicotine-induced up-regulation. this we the up-regulation and trafficking of surface α4β2 nAChRs a cell culture that functional α4β2 receptors. We that in this system, chronic nicotine the up-regulation of surface α4β2 nAChRs in a that is to nAChR up-regulation observed in the brains of human smokers and in animal models chronically to of nAChR trafficking that up-regulation is not of endocytic trafficking at a step prior to insertion of α4β2 nAChRs into the plasma and at in and in with and and of into by C. Mol. Biol. 1987; 7: PubMed Scopus Google Scholar). the and nAChR subunits a from of and a from of and of by the with and at a at from from the to a to the of with a of with on a from and on a to a of with a of and with of acetylcholine the for to a in the at low at and on the of a and from with and genes or in and for in binding from culture in binding and by at for in and by through a by a step at for The from the by at in a and a of in binding in for binding The number of nicotinic binding sites by binding of to previously M.J. Collins A.C. J. Pharmacol. Ther. Google Scholar). by of in the binding as and binding a of followed by a and of on on and with and to for to membrane with primary or at the or nAChR and for at with phosphate-buffered to and with for and with for in and and in and for the the with at in for and in on for The observed on surface receptors as with primary the with PBS, to and at for the to internalization of surface receptors. the and with and by as of α4β2 of surface receptors as in the to lysosomal in order to in the the and in for The for in primary or at in and a in for the for with or at in and or and and for as with receptor and and and the receptor as and with primary at receptor Inc., and or and observed with a channels not in of α4β2 receptors to and with surface proteins with for in and by in and with and in The into with or nicotine at for the the in and in The for on and by at for at with for at to in and in from in by to and by with of and with the to the at and for the prior to the of the at at all and all at α4β2 receptors to and by in The and in and at with for and and in surface α4β2 receptors at in for in and in and at the in and in to at and for at the the in and in to and The of for a in the is the from at of and as for surface with primary the and in and with at for the to internalization of surface receptors. The in and to for to membrane the of the the with and by fluorescence-activated cell as in the is the from at α4β2 nAChRs in of nicotine-induced nAChR up-regulation have used to assess the number of cellular receptor binding sites M. Sullivan J.P. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar, P. S. Mol. Pharmacol. Google Scholar, M. J. Pharmacol. Ther. 1995; Google Scholar). have in at cell culture that a large of binding sites are P. S. Mol. Pharmacol. Google Scholar). the relevant receptors that contribute to the and of neuronal at the cell we to on this of receptors. to this it has been to the of nicotine on the surface-expressed α4β2 nAChR in in to the in surface in J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In it to of receptor in neuronal cell models to the of the nAChR the of to and the of for surface in Therefore, to find and system to up-regulation and trafficking of surface α4β2 we surface of and α4β2 nAChRs in cell assess surface we in with and with the of the to surface-expressed receptors. is of into functional receptors M. P. S. C.R. E. Neuron. Full Text PDF PubMed Scopus Google surface observed or with or subunits not However, and subunits receptors that at the surface of by with the the surface-expressed α4β2 nAChRs by to functional Although we not in or not the and subunits in a to of acetylcholine at concentrations from to from at membrane from to we observed at membrane not with has been previously for the α4β2 nAChR subtype B. M. Sullivan J.P. J. Neurosci. 1996; 16: PubMed Google Scholar). α4β2 receptors in we to nicotine up-regulation in this cell system. and nAChRs Chronic nicotine-induced up-regulation of we the number of binding sites of the nicotinic in the and of chronic nicotine with and genes for and or to nicotine for The of nicotine it is in the of nicotine levels found in the blood of smokers (10Benowitz N.L. Kuyt F. Jacob P.I. Clin. Pharmacol. Ther. 1982; 32: 758-764Crossref PubMed Scopus (256) Google Scholar) and therefore is a relevant from and nicotine and binding binding activity as with or or of nicotine binding sites than that nicotine is of up-regulation of binding sites in this cell system. Although binding sites we in the up-regulation of the surface-expressed receptors. assess the of nicotine to up-regulation of the surface of we cell surface of α4β2 receptors a to several associated with of surface levels in First, the in as by to that the of surface in on and to of and not of the of which of the and the levels than of this we the and dependence of surface nAChR up-regulation. to nicotine for at concentrations from to for The and with the at The and at with and with The with and of by previously with receptor binding of with nicotine a increased cell surface of α4β2 receptors in a with up-regulation of and to a for surface receptor up-regulation. to nicotine for from to prior to and nicotine up-regulation of surface α4β2 receptors of than observed of nicotine exposure Therefore, up-regulation of α4β2 surface nAChRs is and and the of up-regulation is nicotine is we to exposure to the surface α4β2 nAChRs surface receptor up-regulation. as we observed up-regulation of the receptors with nicotine, the up-regulation of surface α4β2 nAChRs. Therefore, nicotine on binding sites is not required for the of up-regulation. ion required for up-regulation, we several that α4β2 nAChR activity by First, exposure to a of up-regulation in the of nicotine However, that as not up-regulation of surface α4β2 nAChRs that of the binding not ion are required for surface receptor up-regulation. with this the of and nicotine up-regulation to the as nicotine Therefore, nicotine is of up-regulation of surface α4β2 receptors in the of activity the of surface nAChR up-regulation that we observed in are to up-regulation of nAChR observed in human smokers, animal and cell (11Schwartz R.D. Kellar K.J. Science. 1983; 220: 214-216Crossref PubMed Scopus (420) Google Scholar, C.R. Adams C. Logel J. Drebing C. Rollins Y. Barnhart M. Sullivan B. Demasters B.K. Freedman R. Leonard S. J. Comp. Neurol. 1997; 387: 385-398Crossref PubMed Scopus (228) Google Scholar, R. P.J. Lindstrom J. Mol. Pharmacol. Google Scholar, M. Sullivan J.P. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar, P. S. Mol. Pharmacol. Google Scholar, M.J. J.A. Collins A.C. J. Pharmacol. Ther. Google Scholar, M.J. Collins A.C. J. Pharmacol. Ther. 1983; Google Scholar). are for cell and and of the surface-expressed of are a system to the of receptor trafficking to nicotine-induced up-regulation. the of has been for receptors that with and their J.L. P. M. Neuron. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In a in of nAChR in stabilization of receptor and up-regulation. We therefore to of the α4β2 nAChRs from the plasma membrane affected by the trafficking of α4β2 nAChRs have not been we internalization of surface nAChRs by receptors in at for with the of the The and at or at in for and with and for at the receptors at the surface However, to for the surface receptors in a and to with internalization through the endocytic pathway that the we observed in to of the surface we internalization in the of which is to and found that the nAChRs at the surface the not In we internalization with from not receptors and therefore not to surface receptors. we observed of surface receptors as with the results that the internalization we observed to be the of not we that our endocytic we the of nicotine on in the as in the internalization that nicotine to the the as in the nAChRs into the in the of nicotine that exposure to nicotine does not the initial internalization of receptors from the plasma high concentrations of nicotine used in to that be as and as However, at high and low not in internalization be the results by we a to the of internalization and chronic nicotine The surface α4β2 nAChRs with primary the and to at for the points. The to and surface nAChRs with and receptors that by the primary and at the cell surface the to at the of a of receptors from the surface be the of this we internalization to surface receptors. The surface receptors from the plasma membrane the with of receptors at the surface the we internalization rates or to the surface receptors and observed that the at or internalization the of the not Therefore, this a and internalization We the of nicotine on internalization of surface α4β2 nAChRs this in with the nicotine to the internalization of surface receptors the and the of from the plasma membrane not from that of Although nicotine not to the internalization of the it that chronic with nicotine to up-regulation may in in the of this we internalization of nAChRs a with the nicotine-treated a in surface as with However, surface receptor the of receptor not chronically with nicotine and results concentrations of nicotine to not Therefore, α4β2 receptor is at the surface of the chronically nicotine-treated the at which the is from the plasma membrane is to that of Therefore, chronic exposure to nicotine the of nAChR from the cell of of up-regulation that nicotine may by the of nAChR α4β2 nAChR and resulting in up-regulation. this we to trafficking of the α4β2 nAChRs from the plasma membrane to the affected by First, to the that the surface receptors are to from the plasma membrane, we with α4β2 receptors and a of lysosomal α4β2 nAChRs at the plasma membrane, from the However, of the α4β2 receptors with at this we of nAChRs with receptor or not of from which proteins are to the plasma Therefore, it appears that surface α4β2 receptors are and to lysosomal nicotine the of transport to the we to in the degradation of the surface receptors. Therefore, we the of the surface-expressed of receptors a surface degradation in the or of with a that surface The of the proteins by and subsequent with the nAChR In the of nicotine, the surface nAChR at that receptor is not to the plasma membrane is to the and results are with our and and that the internalization that we to surface receptors is not to be to the of nicotine the not the surface nAChRs as with the that exposure to nicotine does not the degradation of surface α4β2 nAChRs in the of nAChRs of from the we not in internalization or degradation of surface α4β2 nAChRs in the of nicotine, the that in postendocytic trafficking of may contribute to up-regulation. to assess the of endocytic trafficking to up-regulation, we a negative of associated previously to be required for of plasma membrane proteins from F. A. N. Y. A. I. M. M. Y. Mol. Biol. 2000; PubMed Scopus Google Scholar). the of is and the However, a negative in the of results in the of endocytic as well as the in large are to of resulting in of surface proteins M. Y. A. S. M. J. Sci. PubMed Scopus Google Scholar). in the postendocytic trafficking of nAChR proteins required for up-regulation, of this pathway with up-regulation. the in the cell we the of a in with of or negative In receptor the cell in as However, in the receptor with in large that the in the cell as has been previously in cell M. Y. A. S. M. J. Sci. PubMed Scopus Google Scholar). with α4β2 and α4β2 receptors in that α4β2 nAChR trafficking to the the the of α4β2 observed in α4β2 and Therefore, the is in postendocytic trafficking of α4β2 nAChRs to the We surface of α4β2 receptors in α4β2 with or the negative is observed in all α4β2 or that that we observed from in α4β2 or α4β2 with we observed nicotine-induced up-regulation a with nicotine in α4β2 and trafficking of nAChRs is we observed up-regulation of α4β2 nAChRs results are with the of nicotine on internalization and degradation rates of surface receptors and that up-regulation is of through the from the for nAChR our that internalization of the α4β2 nAChRs from the plasma membrane trafficking in the postendocytic pathway is in nicotine-induced up-regulation of surface α4β2 nAChRs. We therefore to the of receptor insertion of receptors into the plasma membrane to up-regulation. It has been well documented that nicotine-induced up-regulation of high affinity nicotine binding sites does not from in which that up-regulation is not it has been that up-regulation of nicotine binding sites can in the of R. P.J. Lindstrom J. Mol. Pharmacol. Google that de is not required for up-regulation. In our system as we have found that the and not up-regulation of surface nAChRs not membrane proteins are through the secretory pathway to the plasma trafficking of through the secretory pathway required for up-regulation, of secretory pathway nicotine-induced up-regulation. is a that transport of proteins from the endoplasmic to J. J.S. R.D. Full Text PDF PubMed Scopus Google Scholar, R.A. J. R.D. Science. PubMed Scopus Google Scholar). assess the of secretory to the plasma membrane in up-regulation, we surface in the of to or with nicotine for prior to for surface exposure to nicotine the up-regulation of surface nAChRs with to a in surface which is of the of in secretory pathway not However, with nicotine, we not up-regulation of surface Instead, nAChR surface not than with that secretory pathway is for nicotine-induced up-regulation. to surface up-regulation or up-regulation occurs in the of BFA, the transport of of receptors to the plasma membrane, we binding to assess nicotine-induced up-regulation of the of nAChRs in the of exposure of to nicotine for up-regulation of binding sites to the of the nicotine exposure not the up-regulation of binding sites that the mechanism up-regulation is in that the transport of receptors to the plasma membrane is that the site of up-regulation is at or prior to the ER, the site of in the up-regulation is at the ER, of nAChR subunits required for up-regulation in the in which α4β2 is we with a with and subunits and for prior to and with and to the of the of in a with In the with that the of the is in the ER, of nAChR subunits that can be to nAChRs by nicotine The of nicotine-induced nAChR up-regulation is to models of exposure to results in surface receptor to However, in the of chronic exposure to results in up-regulation, which has been proposed to be a mechanism to receptors at the cell surface (12Dani J.A. Heinemann S. Neuron. 1996; 16: 905-908Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Therefore, nicotine-induced up-regulation to be to the activity of cell surface receptors. We have a culture cell system and a surface to the up-regulation and trafficking of surface-expressed α4β2 nAChRs in We have to on the surface-expressed which in are the relevant receptors required for membrane In our cell system, we and surface of functional α4β2 nAChRs and nicotine-induced up-regulation of and surface α4β2 nAChRs and The up-regulation that we is to previously high affinity nicotine binding site up-regulation in and animal models R. P.J. Lindstrom J. Mol. Pharmacol. Google Scholar, M. Sullivan J.P. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar, P. S. Mol. Pharmacol. Google Scholar, M.J. Collins A.C. J. Pharmacol. Ther. 1983; Google Scholar). that up-regulation can be cellular machinery that is in Therefore, the for up-regulation of nAChRs may be not with to nicotine for the of cell surface receptors as In mechanisms and that can be this system may be in of receptors in neuronal and, can be in the of neuronal or and of α4β2 nAChR endocytic trafficking has that surface-expressed nAChRs are and from the plasma membrane and into a lysosomal degradation pathway and Therefore, surface nAChRs are by receptors from the secretory Chronic exposure of surface nAChRs to nicotinic is for of surface receptor up-regulation does not alter the endocytic trafficking of receptors mechanism that has been proposed for up-regulation is that nicotine of the receptors from a pathway to their surface and R. P.J. Lindstrom J. Mol. Pharmacol. Google Scholar). However, our that chronic exposure to nicotine has on the of the surface nAChRs from the plasma membrane or their degradation in the and postendocytic with a negative does not up-regulation of nAChRs that of nAChR surface is not in endocytic trafficking or the lysosomal degradation of the surface a that has been previously as a mechanism for up-regulation R. P.J. Lindstrom J. Mol. Pharmacol. Google Scholar). In to our degradation and internalization by of nAChRs in on and In our the of surface receptors and therefore not the of in the degradation of nAChRs through a It is that the that observed by R. P.J. Lindstrom J. Mol. Pharmacol. Google Scholar) be by of degradation of receptor as a of However, our results a of a of nicotine on the lysosomal degradation Although postendocytic trafficking does not contribute to up-regulation, the insertion of nAChRs into the plasma membrane through the secretory pathway appears to be required for nicotine-induced up-regulation of surface receptors. of secretory pathway with results in of nicotine-induced surface receptor up-regulation de does not to be required for up-regulation, not up-regulation R. P.J. Lindstrom J. Mol. Pharmacol. Google Scholar). have been of the of on up-regulation R. P.J. Lindstrom J. Mol. Pharmacol. Google Scholar, M. Sullivan J.P. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar). However, the in of of binding sites in the of and nicotine with that exposure may in in that nicotine-induced up-regulation we have in our cell system and found of or that at in the on up-regulation. and S. F. that a of is for nicotine-induced up-regulation. In with binding in the of that surface receptor up-regulation is by of secretory pathway to the plasma membrane, the of receptors is by nicotine that the up-regulation is at or prior to the of receptors and their subsequent transport through the secretory for our that of nAChRs relevant to up-regulation may in the endoplasmic First, we find that nicotine-induced up-regulation of surface nAChRs not the up-regulation of binding the of with is with of nAChR, it has been that the step of surface appears to be at the D.S. J. Biol. PubMed Scopus Google Scholar, J.M. Y. N. E. Neurosci. PubMed Scopus Google Scholar). the ER, are several potential in receptor that be by nicotine to and into functional receptors is a for into secretory and from the M. A. Science. 1999; PubMed Scopus Google Scholar). In as a to of nAChRs have that nicotine and which may be of increased into functional receptors R. S. M. C. J. Neurosci. PubMed Google Scholar). to in order to proteins to degradation and of A. Rev. Mol. Biol. PubMed Scopus Google Scholar). nAChR appears to be by in the system J. PubMed Scopus Google and this step be by nicotine to up-regulation. receptor subunits that to in the is at which the are and into transport can and neuronal nicotinic receptor family have been to with and J.M. Y. N. E. Neurosci. PubMed Scopus Google Scholar, Lindstrom J. P. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Lindstrom J. P. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). nicotine is and to we and have that with surface receptors is to up-regulation, exposure to up-regulation P. S. Mol. Pharmacol. Google Scholar). Therefore, it is to a by the surface receptors in to nicotine, to a that at a site to up-regulation. pathway is to be nAChR chronic of with or the of nicotine and of which the ion activity of surface is to up-regulation of R. P.J. Lindstrom J. Mol. Pharmacol. Google Scholar, M. Sullivan J.P. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar). a for nicotine-induced up-regulation, the of nAChR with at the cell surface a system. results in increased number of nAChRs from the to the the observed up-regulation of surface nicotinic receptors. The be to the in receptor at the and, are in to into neuronal and animal models and their in nicotinic receptor trafficking and nicotine-induced We and for of the and We for nicotinic acetylcholine receptor and and for