Section 1

Section1 Page 1 Wednesday, July 18, 2001 1:18 PM

Somfor SAcetPhar

Ronald Cynthia

CONTENTS

1.1 Introduction1.2 Maintenance of Cel1.3 Creation and Maint

Expressing nAChR 1.3.1 Vector Selec1.3.2 Host Cell Ty

1.4 Isotopic Ion Flux AnAChR Ensembles 1.4.1 Efflux and In1.4.2 Techniques f

1.5 Radioligand Bindin1.5.1 Intact Cells

1.5.2 Membranes References

1.1 INTRODUCTION

Nicotinic acetylcholine rroles. They participate in neuronal circuits, and theihormones that in turn can

essential functional rolestherapy of brain and body

a diverse collection of su

1

2002 CRC Press LLCe Methodstudies of Nicotinic ylcholine Receptor macology

J. Lukas, John D. Fryer, J. Brek Eaton, and L. Gentry

l Lines Naturally Expressing nAChRenance of Cell Lines Stably and Heterologouslyof Defined Subunit Compositiontion, Transfection, and Subcloning pes Used and nAChR Subtypes Expressedssays for Rapid Characterization of Functional

flux Assaysor Cell Manipulation

g Assays Suspension and In Situand Detergent-Solubilized Preparations

eceptors (nAChRs) play many important physiologicalclassical excitatory neurotransmission to activate selectedr stimulation can modulate release of neurotransmitters orhave additional actions.17 Due to their wide dispersal and, nAChRs are ideal targets for the modulation by drug function in health and in disease.2-4, 810 nAChR exist asbtypes, each with a unique subunit composition.34, 7, 9, 11

Diversity of nAChR prodesigned to interact selectbehaviors or functions winteractions at other nACgenetics, understanding thunderstanding of the phymanipulated to combat di

Studies of nAChR phabinding. Another chapterphysiological studies of nAChR functional assaychapter by Marks refers toin synaptosomal preparafunction using intact celbinding analyses of nACoverview of the use of ceincluding a brief expositheterologously expressidescribed are those used iselectively cited in this outlined, but, with apologit been our intent to cite

1.2 MAINTENANCE EXPRESSING nA

Cell lines, naturally immoexpressing some of the sa(Table 1.1).

12

Limitations of their uses in such workduplicated here.

12

Howevbinding pharmacological system, taking advantage oand comparatively homogexperimental strategies. Cuseful and informative, assactivation or blockade affesurvival, intracellular meenzyme activity.

If a given cell line expcharacterization of that nis much simpler than phartissue or brain region in wcell lines, such as those coIMR-32), also may expre

2002 CRC Press LLCvides therapeutic opportunities, in that drugs might beively at an nAChR subtypes, thus contributing to specificithout causing adverse or undesired side effects due tohR subtypes. Even in an era dominated by molecular

e pharmacology of nAChR remains central to an improvedsiological roles of these receptors and how they can besease or, perhaps, to optimize human function. rmacology employ assays of nAChR function and ligand in this volume describes techniques used for electro-nAChR function, and Wonnacotts chapter elucidates

s based on measures of neurotransmitter release. The methods for isotopic ion flux assays of nAChR function

tions; in contrast, isotopic ion flux assays of nAChRls are described in this chapter. Techniques for ligandhR also are outlined here. The chapter begins with anll lines as models for studies of nAChR pharmacology,

ion of strategies for generation of cell lines stably andng functional and ligand binding nAChR. Methodsn our laboratory unless otherwise noted. Published workoverview provides additional examples of techniquesies to authors of such studies, it is not possible nor has

all such useful articles.

OF CELL LINES NATURALLY ChR

rtalized through carcinogenesis, have been useful factoriesme kinds of nAChR found in related, non-neoplastic cellsand attributes of cell lines in nAChR studies and examples have already been described elsewhere in detail not to beer, to summarize, cell lines allow functional and ligandprofiles to be derived and integrated in one experimentalf the potentially limitless quantities of easily manipulated

enous material to accommodate many kinds of assays andell lines also are suitable for somewhat indirect, but stillessments of nAChR pharmacology based on how receptorcts phenomena including gene expression, cell shape and

ssage levels, release of other chemical messengers, and

resses just a single nAChR subtype, then pharmacologicalAChR subtype is reasonably straightforward and usuallymacological characterization of the same subtype from ahich additional nAChR subtypes may be expressed. Somerresponding to autonomic neurons (e.g., PC12, SH-SY5Y,

ss multiple nAChR subtypes, requiring identification of

means for discriminating before pharmacological ch

Maintenance of cell lgraphs and instructional bspecific details that will nulation of pathogen- or inbiosafety cabinets (recircuair (HEPA) filters and venis central to handling the express nAChR. These tecalso protect cells from invewhere there are no assuranfree, additional precautiodifferent proportions of cataken. Some cell lines calaboratory areas through tillumination of the area, bsurpass the investment in

Conditions for cell innance of cells at 37

o

C, atthat would lead to concen

maintain neutral pH in bicate that nAChR assemblyat lower temperatures.

13,14

facilitate proliferative effecarcinoma cells.

15

Thus, cbefore adopting standard nAChR.

Cell culture medium cand across laboratories. Tstandard conditions of m(DMEM; high (4.5 mg/Lmg/ml (4 mM) L-glutami

TABLE 1.1Cell Lines Naturally Ex

nAChR Subtype

1

1

-nAChR

3

4*-nAChR

7-nAChR

2002 CRC Press LLCligand binding and functional properties of those nAChRaracterizations can be achieved confidently.

ines is both an art and a science. There are many mono-rochures pertaining to cell culture techniques that provideot be repeated here. Use of sterile technique and manip-fectious agent-free cells in Class II Type A, laminar flowlating all cabinet air through high efficiency particulate

ting 30% of the filtered air back into the laboratory room)types of cell lines mentioned in this chapter that naturallyhniques help to ensure the safety of the investigator andstigator- or laboratory-introduced contamination. In casesces that cell lines being developed or used are pathogen-

ns, such as use of Class II Type B cabinets exhaustingbinet air to the external (outside) environment, should be

n be safely manipulated under sterile conditions in openhe use of careful sterile technique and periodic ultravioletut costs of inevitable contaminations are likely to quicklya suitable biosafety cabinet.cubation are quite standard, typically involving mainte-

~95% relative humidity to slow evaporation from mediatration of salts and nutrients, and in 5% CO2 in air to helpcarbonate-buffered medium. However, some studies indi- and levels of expression are higher in cultures maintained In addition, it has been found that elevated CO2 levelscts of nAChR activation on pulmonary neuroendocrineareful consideration and pilot studies may be warrantedcell incubation conditions for studies of cells expressing

onstituents can vary for maintenance of different cell lineshe laboratory has adapted a number of clonal cell lines to

aintenance in Dulbeccos modified Eagles medium

pressing Specific nAChR Subtypes

Cell LinesTE671/RD human clonal line (rhabdomyosarcoma)RM0 rat muscle cell lineBC3-H-1 and C2 mouse muscle cell linesIMR-32 and SH-SY5Y human peripheral neuroblastoma linesPC12 rat pheochromocytoma cell lineIMR-32 and SH-SY5Y human peripheral neuroblastoma linesPC12 rat pheochromocytoma cell line) glucose, with 1 mM pyruvate, supplemented with 0.58ne), to which are added fetal calf serum (5% of DMEM

volume) and horse serumsupplemented, completebeen performed to ensuresame in this medium and relevant cell line. In somegrowth medium. For exainduces morphological dlines.

16,17

Cells are maintamm diameter dishes are u

flasks as a precaution agrecommended in climates

Antibacterial and antifto help combat microbiasupplements medium wit

amphotericin B (2

g/ml;

forming abilities in perfoincreases in basal

86

Rb

+

e

have been observed. ConnAChR and rates of recovadditives are to be presen

Each cell line has itexpedience, cells are typiLoosely adherent cells canto the plate surface, wheremild trypsinization. For thvolume (1.52 ml per 1balanced salt solution is apsolution is aspirated and ta period empirically deteanother). For especially aphosphate-buffered saline0.025% trypsin, respectivvested by addition of abodish; this medium is appsurface to dislodge cells. Hmedium, serially diluted (tcell clumping), and platedence within one week.

1.3 CREATION ANDSTABLY AND HEnAChR OF DEFI

Cell lines naturally expreyet been identified. In the

2002 CRC Press LLC (10% of DMEM volume; final osmolarity of serum- medium is ~330 milliosmolar). However, studies have nAChR expression by cells of the same passage is thein medium described in the initial report establishing the cases, induction of nAChR expression requires change inmple, serum deprivation and/or growth factor additionifferentiation and/or nAChR expression in some cellined on standard tissue culture plastic. Most often, 100-sed for cell maintenance, but growth in 25 cm2 or 75 cm2ainst microbial contamination is an option particularly or periods of high humidity.ungal agents are common additives to cell culture mediuml contamination. For example, the laboratory routinelyh penicillin (200 /ml), streptomycin (200 g/ml), and ~2M). Amphotericin B at ~120 M is used for its porerated patch electrophysiological recording, but modest

fflux from cells in the presence of 3 M amphotericin Bsequently, effects of cell culture additives on function ofery from any such effects should be documented if thoset during or shortly before studies of nAChR function. s own characteristic doubling rate. For the purpose ofcally passaged weekly when they approach confluence. be dislodged by streams of medium applied tangentiallyas more adherent cells can be dislodged in this way aftere latter approach, bulk medium is aspirated and a small

00-mm dish) of 0.25% trypsin in calcium-free Hanksplied to the dish for a few seconds. Typically, the enzyme

he dish is placed in the incubator for a few minutes (i.e.,rmined to yield rounded-up cells dislodged from onedherent cells or for economy, dishes can be rinsed with to remove serum proteins prior to digestion in 0.25% orely. Enzymatic digestion is terminated and cells are har-ut 110 ml of complete medium per 100-mm diameterlied from a pipette as a laminar stream across the disharvested cells are suspended to a specific volume in fresh

ypically no more than 1:10 in a given dilution to minimize at initial densities that should yield cultures near conflu-

MAINTENANCE OF CELL LINES TEROLOGOUSLY EXPRESSING

NED SUBUNIT COMPOSITION

ssing some important known nAChR subtypes have not

case of nAChR subtypes found in the brain, this may be

because mature, post-mittransformation, which machange with advances in nlines from transgenic animspecific promoter elementerologously expressing sptation in experimental resGeneration of heterologousubtypes can be characterusing many tissues or braexpress more than one nAgously expressing nAChRtransgenes as message, butsubunits capable of assemtional nAChR. Nevertheleof strategy and technique icells are outlined here.

1.3.1 V

ECTOR

S

ELECTIO

Initial success in stably an

to selection of host cell (Invitrogen) vector was chwhich gives high constituhuman host cell lines. pCEnuclear antigen (EBNA) ation of the vector in humaa requirement for insertiobecome silenced or subjechigh copy number for the tpressure via coordinate exsame plasmid.

Subsequently, there wvectors for the stable exbinding and/or functional vector in our studies.)

193

composed as binary (or h

nAChR, ternary

3

5

4-ntion markers is useful. (pcforms.) In one of the two ligand binding and functi

was used.

31

We also have hpromoters and have succethis purpose.

27,28

These appTet-on plasmids (contai

2002 CRC Press LLCotic neurons have lost their susceptibility to neoplasticy only occur in proliferating cells. This circumstance mayeuronal stem cell science and in creation of neuronal cellals in which oncogene expression is driven by neuron-

s. Nevertheless, development of cell lines stably and het-ecific nAChR subtypes has evolved to address this limi-ources. (Transient expression will not be discussed here.)s expression systems also offers advantages in that nAChRized pharmacologically in isolation, in contrast to studiesin regions or even some clonal cell lines that naturallyChR subtype. Creation of cell lines stably and heterolo- has been fraught with failures, not in ability to express in ability of messages to be translated into nAChR proteinbling into ligand binding and/or surface-expressed, func-ss, there have been some victories, and some of the detailsn stable, heterologous expression of nAChR in mammalian

N, TRANSFECTION, AND SUBCLONING

d heterologously expressing 7-nAChR18 can be attributed(see next section) and selection of vector. The pCEP4osen because it has the cytomegalovirus (CMV) promoter,tive expression of the downstream transgene of interest in

P4 also contains genes coding for the EpsteinBarr virusnd origin of replication (ORI) allowing episomal replica-n cells. That is, a vector was chosen that would eliminaten of the transgene into the host genome, where it mightt to variable regulation of expression. This helped to ensureransgene and its stable expression under constant selectivepression of the hygromycin resistance gene, all from the

ere succesful efforts in using integrating or episomalpression of several different nAChR subunits as ligandnAChR. (pcDNA3.1 (Invitrogen) is used as an integrating0 Particularly when the objective is to express nAChRigher order) complexes of subunits (e.g., binary 42-AChR), availability of vectors containing different selec-

DNA3.1 comes in zeocin, hygromycin, or G418 resistanceinitial reports of stable expression of neuronal nAChR asonal sites,31,32 a vector harboring an inducible promoterad reasons for expressing nAChR subunits from inducible

ssfully used the Tet-on and Tet-off systems (Clontech) forproaches require stable integration of both the pTet-off or

ning tetracycline-sensitive regulatory elements and also

conferring G418 resistancdownstream from a tetrac

To introduce nAChR sliterature as to whether elcially available lipofectionlatter two techniques give for transient transfection swithin a week of transfeinitial viability) that has tbalanced against cytotoxicinvestment rather than recgood and comparable succbut studies have not been recovery from transfectiogenes conferring antibiotiantibiotic for positive selethen assessed based on ndifferent times after selecoccurs by isolating a stablthen using it as host for icurves (plots of untransfeselection antibiotic) are acare done; kill curves are alof treatment with more th

Whether using episomisolated while surviving cfilter disc cloning, or amount of vaseline is appders, which are placed to cylinders is removed, andfrom the dish surface. Freferred to single wells in afrom the plate, and 5-mm but wiped free of excessflame-sterilized forceps. Aagainst the dish surface transferred to a well in a 2cells, and removed if desition to moistness of mediuof a pipette tip so that it wdipped into fresh mediumattached cells. Irrespectivare expanded for further radioligand binding.

2002 CRC Press LLCe) and the pTRE plasmid (containing the gene of interestycline response element).ubunit cDNA(s) into host cells, no consensus exists in theectroporation, any of a number of homemade or commer- aids, or calcium-phosphate precipitation is superior. The

higher initial survival of cells and are likely to be preferredtudies (involving study of cells and nAChR that they makection). Electroporation is a more harsh approach (lowero be custom-designed to optimize transfection efficiencyity for every type of host cell, but requires an equipmenturring costs for transfection reagents. In the laboratory,ess has been experienced using each of these approaches,done systematically. Typically, a 24- to 48-hour period ofn is allowed, thus permitting transgenes of interest andc resistance to be expressed before adding the appropriatection of stable transfectants. Efficiency of transfection isumbers of cells and/or numbers of colonies of cells attion. Higher success in expression of multiple subunits

e mono- or poly-clone expressing one nAChR subunit andntroduction of the second type of cDNA. Antibiotic killcted cell survival as a function of time in the presence ofquired for each prospective cell host before transfections

so required to assess any synergistic effects on cell survivalan one antibiotic.al or integrating vectors, clones surviving selection are

ell densities are still low, either by using ring cloning,a stab-and-grab technique. For ring cloning, a smalllied to the bottom edge of 5-mm diameter cloning cylin-isolate targeted cell colonies. Medium within the cloning 0.25% trypsin solution is applied until cells have liftedsh medium is added, and the suspended cells are trans-

24-well tray. For filter disc cloning, medium is aspirateddiameter filter discs previously soaked in trypsin solution solvent are laid over visually identified colonies using

fter 3 to 10 minutes, each filter disc is gently rubbedover the colony. Each disc with attached cells is then4-well tray filled with fresh medium, shaken to dislodge

red. The stab-and-grab technique simply involves aspira-m from a dish followed by positioning and manipulationipes up some cells from a colony. The pipette tip is then in a well in a 24-well tray and agitated to displace

e of the method for their physical isolation, cell clonessubcloning as needed and screening for function and/or

1.3.2 H

OST

C

ELL

T

YPES

Systematic studies are stilsuccess of nAChR subunistudies might progress to iof receptor expression, theassisted protein folding, aa fundamental understandcope with expression of nand/or ion permeability.

Cognizance of the unhost cell lines such as CHfection and heterologous ethat naturally make at leaexpress rat

7 subunits in

7- and

3

4*-nAChR.

18

sion of wild-type or mutan

attention was then turnedwas initially isolated fromearly control studies ind

observations that the SH-ogies and chemical pheno

epithelial cells have a shapolar morphologies (dendlar/lumenal dispositions). might share abilities withtransmembrane proteins. and heterologously expres

plus

4 subunits;

19, 2730

phomomeric, binary, and e

Success in heterologoleast some subclones of cells.

2022, 2426, 31

Data are

form) from the pCEP4 vneuroblastoma cells, PC1ronal cells. Stability of nsystematically evaluated. lines have the capacity to lines that lack such a capa

The issue of stabilityacross and within passagecell cultures, which presdefined as those passagedlines are commonly definegray area in nomenclature

2002 CRC Press LLC USED AND nAChR SUBTYPES EXPRESSED

l needed to determine roles that host cell types play in thet transfection and heterologous expression. Indeed, suchdentify critical molecular and cellular bases for regulationreby providing insight into phenomena such as chaperone-ssembly, and trafficking. These studies also may provideing of how and why some cells differ in their ability toAChR subtypes differing in kinetics of channel opening

published difficulties of nAChR expression in commonO and COS cells resulted in initial reasoning that trans-xpression of nAChR should be perfected first using cellsst some nAChR subtype. Hence, it was decided to over-the SH-SY5Y cell line known to naturally express human

,33 Bolstered by success with that approach and in expres-

t, human or chick 7 subunits in the same host cell line,19 to the SH-EP1 human epithelial cell line. This cell line

the same tumor that yielded the SH-SY5Y clone, buticated that it was native nAChR-null,33 consistent withEP1 and SH-SY5Y cell lines evolved divergent morphol-types as they were cloned.34 Nevertheless, neuronal and

red embryological lineage, and both types of cells exhibitrite-soma/axon compared to apical/basolateral or tissu-Consequently, reasoning was that SH-EP1 epithelial cells neuronal cells to process and properly express complexSH-EP1 cells have now been used successfully to stablys functional nAChR composed of 7, 4 plus 2, or 4reliminary data indicate successful expression of other

ven ternary complexes in these epithelial cells.us expression of nAChR has also been achieved using atHEK-293 human embryonic kidney and other fibroblastavailable showing expression of chick 7-nAChR (mutantector in HEK-293 cells as well as in IMR-32 human

2 rat pheochromocytoma cells, and CATH.a mouse neu-AChR expression in each of these cells has not been

Nevertheless, these studies clearly indicate that many cellexpress nAChR heterologously. How they differ from cellcity is yet to be determined. of transfection and uniformity in transgene expressions requires clarification and warrants discussion. Primaryumably have not become immortalized, are commonly ten times or less from initial seeding. Continuous celld as those cultures surviving over 24 passages (leaving a for cells carried through passages 11 to 23). Clonal cell

lines are those derived fromosomal makeup and numestablished. Continuous cmakeup stabilizes growthoccur to give cells a compor gene expression of inte6 months or about 30 papassages and may be as Even established cell linenAChR when carried for lines are considered to be passages without evidencenot ensure that all passagsame stock in different lawill do so through 20 to same amounts of nAChRligand binding sites may other gene critical to synthquality assurance is requinecessarily catastrophic iflong as frozen stocks of lfresh passage of cells.

While not systematicvariations in levels of nACand on the same dish. Heteven after cells identified is not clear whether variabin the cell cycle, cellcell

Colleagues practicingences in membranes of cegenes but derived from thchoice of a single clone fr

that two clones isolated frfection, selection, subcloretaining capacity to expr

1.4 ISOTOPIC ION CHARACTERIZA

Isotopic ion flux assays acharacterize function of vo

Ion flux assays complemeelectrophysiological analstopped-flow studies like parable to that for the fas

2002 CRC Press LLCm a single cell. Flow cytometric studies show that chro-bers can vary wildly as a tumor-derived cell line becomesell lines usually arise when a particular chromosomal and cell phenotype. However, recombinations still canetitive growth advantage but could result in loss of genesrest. The policy is not to carry any cell line for more thanssages (realizing that cells in stocks are no less than 5far as 20 passages removed from the true first passage).s, such as PC12 or SH-SY5Y, sporadically show loss ofmore than 20 passages from the stocks. Engineered cellstably transfected if they have been carried for at least 24 of loss of transfected gene expression. However, this doeses from the same frozen stock, or cells carried from theboratories, will express the same quantities of nAChR or30 passages every time. Even if a cell line expresses the subunit message, expression of nAChR as functional orstill differ because of differences in expression of someesis, assembly, and/or maturation of nAChR. Continuousred to monitor transgene expression. However, it is not a cell line loses expression while being maintained, soow passage cells are maintained in abundance to begin a

ally evaluated, experience indicates that there may behR expression even across cells within the same passage

erogeneity in levels of nAChR expression returns quickly,for their high level of expression have been subcloned. Itility in nAChR expression reflects dependence on position contacts, or other cellular features. electrophysiology have remarked about physical differ-lls expressing different nAChR subtypes from transfectede same host cell stocks. It should not be surprising, givenom dozens arising from a transfection of 105 to 106 cells,om a master stock might evolve differently during trans-ning, and passage to exhibit different properties whileess nAChR.

FLUX ASSAYS FOR RAPID TION OF FUNCTIONAL nAChR ENSEMBLES

re a proven and classical (over 25 years of use) means toltage- or ligand-gated ion channels in clonal cell lines.35,36nt, and in many ways offer advantages over, more tediousyses of channel function. Ion flux assays adapted for

those used in enzymology give temporal resolution com-test electrophysiological studies and are fully suitable for

intricate study of nAChR

nAChR or other channelstopped-flow studies, butstudies. Nevertheless, ionthroughput analyses of nAinstrumentation. Ion flux from the entire populatiosummed across >10

8

recep

with ~10

3

surface receptors

using ion flux assays witypically remain constant oion flux at time zero. Whenit has reflected time-depensible radiotracer.

Agonist dose-responsecells plated at equal densdifferent dose of the test lsamples exposed to a maxion flux is determined as aboth agonist and fully-blopositive control or test samSpecific ion flux is plottedare analyzed using nonline

where F is the measured sto yield the parameters F

tration giving one-half of n (> 0) as the Hill coefficierelative to maximal respotest ligands may producethan the maximum responfor these ligands plateau,be estimated by the concedose-response curves are inhibition of functional rhigher concentrations, buthalf of its maximal effect

test ligand giving one-half>100% of standard agonidose-response curves shouten times higher than the a

not be more than double r

Antagonist dose-respoof samples treated with difagonist at a constant conceof concentration of test lig

/ (1 + (IC

50

/[L])

n

), where

2002 CRC Press LLC functional kinetics.37 Membrane vesicles very rich ins of interest and resistant to fluid pressures attained in not to whole cells, are preparations of choice for such flux assays using intact cells are ideally suited for high-ChR function using very simple techniques and commonassays integrate responses for the ensemble of nAChRn of cells in a cell culture dish or microwell, typicallytors (>105 cells per 15.5-mm diameter well (24-well tray) per cell).38,39 Cellular nAChR responses can be determinedth a temporal resolution of seconds, and ion flux ratesver a period of 45 to 60 seconds extrapolated through zero bi- or multiphasic kinetics of ion flux have been observed,

dent inactivation of nAChR rather than exhaustion of acces-

profiles for test ligands can be obtained from studies ofity in wells in a multiwell array each incubated with aigand. Positive control (total) responses are determined inimally efficacious dose of a standard agonist. Nonspecific negative control in samples lacking agonist or containingcking antagonist. Nonspecific ion flux is subtracted fromple responses to yield specific ion flux for those samples.

as a function of concentration of test ligand, and the dataar regression fits to the formula F = Fmax / (1 + (EC50/[L])n),pecific ion flux and [L] is the molar ligand concentration,

max as the maximum ion flux, EC50 as the ligand concen-the maximal ion flux response to the standard agonist, andnt for the process. Efficacy of test ligand can be determinednse to standard agonist in positive control samples. Some maximal responses at high concentrations that are lessse to the standard agonist. If agonist dose-response curves then they act as partial agonists and their potencies canntration giving one-half of their maximal effect. If agonistbell-shaped, then the test ligand may be exhibiting self-esponses and/or inducing desensitization of nAChR at its potency also can be estimated as the dose giving one-. EC50 values also will differ from the concentration of a of its maximal effect for the super efficacious drug havingst efficacy. To provide valid measures of agonist activity,ld include measurements at agonist concentrations at leastpparent EC50, and responses at those concentrations shouldesponses obtained at the apparent EC50.nse curves for test ligands can be obtained from studiesferent doses of the test ligand in the presence of a standardntration. Specific ion flux results are plotted as a function

and, and the data are analyzed using the formula F = FmaxF is the measured specific ion flux and [L] is the molar

ligand concentration, to y

absence of antagonist, IC

5

bition of ion flux response0 for an antagonist in this can be expressed as IC

50

va

to negative control levelsexpressed as the concentrblock.

Competitive or noncotinguished based on studantagonist concentrations.

values, thus showing that fconcentrations increase, apparent efficacy in the facobserved EC

50

values as nlarly, competitive antagonincreases in apparent IC

50

efficacious dose range incrwill not shift appreciablymaximally efficacious ran

to K

i

values (measures of which there is half-maximtion used (although agonis

value for practical reason

antagonists requires additi

estimated as the concentrEC

50

value for an agonist

value obtained from such onist K

i

values can be deteof agonist dose-response by agonist and competitiv

Ion flux assays can alsof blockade can be evalutreatment with agonist. Inbe gained by studies of iocontaining different conceexperiments (e.g., N-meth

give insights into ion selechannels can be assessed to flux in samples treated

On balance, ion flux asto that obtained from whorecording. Single channel cell current recording hasin seconds or less) and inare not open long enoug

2002 CRC Press LLCield the parameters Fmax as the maximum ion flux in the0 as the ligand concentration giving a half-maximal inhi-, and n as the Hill coefficient for the process, which is 90% complete

ended that some cell samples be used to determine thed the amount of 86Rb+ remaining in the extracellular fluid.

86Rb+ can be calculated from the latter value and from volume and the concentration of extracellular K+ and

alculated specific activity of 86Rb+, the determined amount and an assumed or determined intracellular concentrationcally ~120 mM), intracellular volume accessible to 86Rb+

b+, plated cells are removed from the incubator, and sub-nducted behind a Lucite shield at room temperature (with in part to slow functional desensitization. Loading isf medium into a shielded collection flask and application

M NaCl, 5.4 mM KCl, 2 mM CaCl2, 5 mM glucose, 50

0 milliosmolar; typically 3, 2, or 1 ml, respectively, per

well for 12-, 24-, or 48-wused as the efflux bufferantagonist, and serum comcontaining nicotinic ligandso it is better to avoid usiand cell rinsing are repeaminutes. Rinse buffer is thnicotinic ligands of choicand stored at 4oC until usstudies show that frozen ethanol or dimethylsulfoxoption is to create high coin small quantities or of cwater, 10 mM d-tubocuramethyllycaconitine in ethaAfter incubation for a presefflux medium is collecte0.1% sodium dodecyl sulenkov counting of aqueoefficiency (glass/plastic v~95% efficiency.

22Na+ influx assays of in detail elsewhere and was variations on methods etial use in studies of nAChare used as for 86Rb+ effluxgrowth medium by rinsingmedium. For cell rinses aor serum-free cell culture were preincubated for longcharacterization, HEPES-1.8 mM CaCl2, 1.2 mM Madjusted to pH 7.4 with Nibration. For ion flux assapart with sucrose, e.g., 0.215 mM HEPES, pH 7.4, ~3for influx assay reduces influx of radiotracer and mactivation. Just before starof incubation in Na+-freeinhibit action of the Na+-Kremoved by aspiration, anmM ouabain (to continue 5 Ci/ml), and nicotinic lito 2 min for studies in thrinsed 3 to 4 times over ~0

2002 CRC Press LLCell trays). In some early studies, cell culture medium was. However, phenol red can have activity as an nAChRponents can include esterases that can cleave ester bond-s like acetylcholine, suberyldicholine, or succinylcholine,ng medium and serum in efflux assays. Buffer aspirationted two more times for a total time of 20 seconds to 2en replaced with fresh ion efflux buffer usually containinge. Whereas stock solutions of efflux buffer are made upe, drugs are made fresh daily from powder unless controlaqueous stocks or stocks dissolved in other media (e.g.,ide) give the same results as freshly prepared ligand. Anncentration stock solutions of more expensive ligands soldommonly used ligands. Stocks of 1 M carbamylcholine inrine in efflux buffer, 1 M nicotine in water, and 100 mMnol can be stored at 20C without loss of drug activity.

cribed period (1 to 5 minutes for studies in the laboratory),d for counting, and cells are dissolved in 0.01 N NaOH,fate for further analysis. 86Rb+ can be quantified by Cer-us samples in liquid scintillation counters at ~25%/45%ials) or by scintillation counting in scintillation fluid at

nAChR (and other channel) function have been describedill not be repeated here.35,36 86Rb+ influx assays, designedstablished for 22Na+ influx assays, also have seen substan-R.4244 Cells are plated and processed and solution volumes assays. Sample processing is initiated by removal of cell

cells and incubation for up to 30 min in rinse/equilibrationnd equilibration, some published methods used completemedium (~330 milliosmolar),41,44 sometimes because cells times with nicotinic ligands. For simpler pharmacologicalbuffered salt solutions (e.g., 150 mM NaCl, 5 mM KCl,gCl2, 0.8 mM NaH2PO4, 10 mM glucose, 15 mM HEPES,aOH, ~310 milliosmolar)42 are used for rinses and equil-ys, Na+-free influx buffer (replacing NaCl in whole or in5 M sucrose, 5 mM KCl, 1.8 mM CaCl2, 10 mM glucose,25 milliosmolar)44 is substituted. Use of Na+-free medium

extracellular monovalent cations that could compete forinimizes changes in membrane potential during nAChR

ting the influx assay, all samples are subjected to 1 minute influx buffer supplemented with 1 to 2 mM ouabain to

+-ATPase. To initiate influx, this medium is then simplyd cells are bathed in influx buffer supplemented with 1

inhibition of 86Rb+ uptake via Na-K-ATPase), 86Rb+ (aboutgands of choice. After a prescribed period of time (20 sec

e laboratory), the influx period is terminated as cells are.5 to 3 minutes to remove extracellular 86Rb+. 86Rb+ uptake

is quantified by Cerenkovsolved in 0.01 N NaOH, 0

86Rb+ or 22Na+ influx asto that obtained through 86types of studies. For examment of cells with nicotinare used to monitor nAChpotentially complicated ifand loss of loaded 86Rb+ finitiate nAChR functionalization of 86Rb+ efflux datinitiation, even when usedligands, adequately accousuperior to either kind osensitivity (signal when urial), which translates intenergetic than those from86Rb+ can be detected usinfluid, again making them ato purchase and dispose othe clear choice for ion fl

1.4.2 TECHNIQUES FOR

Sample handling in isotoptional or (for some steps or aspiration. In typical 86Rwell tray is 12 seconds, aFor a typical efflux periodplate. Plates are tilted durimore gently via laminar aspiration and applicationbottom edge of the well, anof some cells or a spot of

A recently developedion flux assays provides hiin experimental design, sureproducibility than sequlysine-coated wells and loon Falcon or Corning mulplates are currently manuflevel relative to the lips aseal between well lips whunder uniform finger pressplates as usual into a shand aligned top to top ove

2002 CRC Press LLC or liquid scintillation counting of cellular samples dis-.1% sodium dodecyl sulfate.says can and do provide essentially equivalent informationRb+ efflux assays. Influx assays have advantages for someple, studies of nAChR desensitization involving pretreat-

ic ligands are simpler to interpret initially if influx assaysR function. Use of 86Rb+ efflux assays for such studies is ligand pretreatment causes transient activation of nAChRrom cells before challenge doses of ligand are applied to analysis. However, it has been demonstrated that normal-a to the amount present in cells at the time of efflux assay in studies involving pretreatment of cells with nicotinicnts for any loss of loaded 86Rb+. 86Rb+ efflux assays aref influx assay in terms of resolution (signal:noise) andsing the same amount of isotope and/or biological mate-o lower cost. Moreover, emissions from 86Rb+ are less 22Na+, which translates into improved safety. In addition,g Cerenkov counting without use of scintillation vials ordvantageous economically and in terms of safety (no needf organic scintillants). On balance, 86Rb+ efflux assays areux analyses of nAChR function.

CELL MANIPULATION

ic ion flux assays most commonly involves using conven-in the method) repeating pipettes for solution application

b+ efflux assays, the sample processing interval for a 24-nd sample wells are processed sequentially one at a time. of 5 minutes, 10 minutes pass while processing a singleng solution exchange so that fluids are removed or appliedflow to the cell plating surface. Pipette tips for solution also are positioned consistently at one point inside thed care must be taken to prevent or minimize displacement

cells to minimize data scatter. novel approach (the flip-plate method) for conduct ofgher throughput, gentler sample handling, more flexibilityperior temporal accuracy, and improved sample-to-sampleential pipetting methods. Cells are seeded into poly-D-aded with 86Rb+ (in this example for efflux assays) as usualtiwell plates. Only Falcon or Corning brands of multiwellactured so that the lips at the top of each well are elevated/t the edge of the plate. This allows formation of a tighten two plates are opposed top to top and pressed togetherure. 86Rb+ loading medium is aspirated from wells of cell

ielded collection flask, but then the cell plate is invertedr the first of two rinse plates set up in advance to contain

fresh efflux buffer (2 ml pfirmly, and the ensemble bathes the cells. After a fethe rinse solution to fall binverted over the second can be washed and reusedaspiration step.) There is ain the cell plate (typicallycellular 86Rb+ and rinses. Tual rinse solution is aspira10 seconds total time per efflux plate set up in aefflux buffer. Efflux is initiflipping them, allowing thin wells of the cell plate. held firmly together (somand gently flipped, allowinto the efflux plate. Althwells of the cell plate, thisnormalization. The wells solution as was used in thdodecyl sulfate to dissolveefflux plate and the cell premaining intracellular isolation vials for Cerenkov oconvenient, more economiCerenkov counting using beta plate-reading liquid schas been used routinely inis, however, not recommetolerance in well dimensiomethod has also been usedVariants on the flip-plate ausing cells or substrate ad

1.5 RADIOLIGAND

Pharmacological characteing of radiolabeled nicotifor specific radioligand binradioligand acting with reother nAChR subtypes) orat a given nAChR subtypelabeled epibatidine, nicoti(e.g., 125I-labeled -bungainteracting at overlapping

2002 CRC Press LLCer 15.5-mm diameter well). The plates are held togetheris gently and slowly flipped (rotated) so that rinse bufferw seconds, the plates are then gently flipped back to allowack into the first rinse plate. The cell plate is lifted off,

rinse plate, and rinsed in the same fashion. (Rinse plates; the bulk of isotope is safely removed during the initial small, consistent, residual volume of buffer that remains 30 to 40 L for a 24-well plate) after removal of extra-herefore, to maintain exact test concentrations, the resid-ted from each well as quickly as possible (usually aboutplate). The cell plate is then inverted and aligned over andvance to contain 2 ml of nicotinic ligands of choice inated by again holding the plates firmly together and gentlye test concentrations of drug to fall into and bathe cells

After a prescribed incubation period, the plates are againetimes gently swirled once to displace pericellular 86Rb+)ing assay solution containing effluxed 86Rb+ to fall backough a small amount of residual volume remains in the quantity is uniform and can be determined to allow dataof the cell plate are then filled with the same volume ofe efflux plate but containing 0.01 N NaOH, 0.1% sodium the cells and their contents. Samples from each well of thelate can then be used to determine levels of effluxed andtope, respectively, for each sample after transfer to scintil-r liquid scintillation counting. It has been found to be morecal, and less labor intensive simply to prepare the plates foran E&G Wallac (now Perkin-Elmer) Tri-Lux 1450 Micro-intillation and luminescence detector. The flip-plate method 6-, 12-, 24- and 48-well formats. Use of a 96-well formatnded, as surface tension and the requirement for higherns and orientation make assays problematic. The flip-plate successfully after appropriate adaptation for influx assays.pproach have potential applicability in virtually any assayherent to or immobilized on a test plate.

BINDING ASSAYS

rization of nAChR can be assessed based on specific bind-nic ligands and on competition by unlabeled compoundsding. Central to this approach is identification of a suitableasonable selectivity (binding with much lower affinity to specificity (showing no binding to other nAChR subtypes)(s). Most radioligands for nAChR are agonists (e.g., 3H-ne, acetylcholine, or cytisine) or competitive antagonists

rotoxin or -cobratoxin; 3H-labeled methyllycaconitine) sites on the extracellular face of nAChR. However, high

affinity radioligands targeor phencyclidine) have bnAChR to characterize bRadioligand binding studconfirm, sites of action oacting as competitive antinhibit binding of radiolabcomparable affinities. By functional assays might aligands binding in the ioinhibitors of radiolabeled

Essential to radioliganumbers of sites and affinration curves are determinfragments incubated with(total) binding is determinbinding is determined as to an at least 100-fold esubtracted from total bindspecific binding as a funclinear regression fits commsimplest analyses assumeuse the formula B = Bmabinding and [L] is the ranumber of specific bindinaffinity of nAChR for thebinding (> 0). Analyses cclass of specific binding siin a particular preparation

Many monographs, rein further detail transformof appropriate experimentpoints will be summarizeunder conditions where fithe KD for the radioliganThe highest amount of bo10% of the free radioligastudies using 3H-epibatidisubtypes of 10 pM or leincreased reaction volumetaining enough receptor toradioligand concentrationto help ensure approach toin concert with saturatioradioligand binding are tymin, but slower rates of

2002 CRC Press LLCting the channel domain (e.g., 3H-labeled histrionicotoxineen used in studies of preparations highly enriched ininding sites for noncompetitive functional antagonists.ies can be used to make predictions about, or to helpf functionally potent compounds. For example, ligandsagonists or as agonists in functional assays should alsoeled agonists or competitive antagonists to nAChR withcontrast, ligands acting as noncompetitive antagonists inct with equal potency as competitive inhibitors for radio-n channel, but should display no or lower affinity asagonist or competitive antagonist binding. nd-based characterization of nAChR is determination ofities for specific radioligands. Radioligand binding satu-ed for fixed amounts of intact cells or cellular membrane different concentrations of radioligand. Positive controled in samples exposed to radioligand alone. Nonspecific

a negative control in samples exposed to radioligand andxcess of nonradiolabeled probe. Nonspecific binding ising to yield specific binding for those samples. Plots of

tion of radioligand concentration are analyzed using non-only found now in data analysis software packages. The

achievement of equilibrium conditions (see below) andx / (1 + (KD/[L])n), where B is the observed amount ofdioligand concentration to yield parameters Bmax for theg sites, KD for the dissociation constant (a measure of

radioligand), and n as the Hill coefficient for radioligandan be extended to assess whether or not more than onetes displaying different affinities for the radioligand existsand how many receptors exist in each class of binding sites.view articles, or data analysis software packages explains for analysis of radioligand binding assays and features

al design and interpretation. Only some of the more salientd here. Binding saturation curves should be conductedxed receptor concentration in assay mixtures is less thand under use and less than the radioligand concentration.und radioligand in a saturation assay should be less thannd in that sample. This is particularly critical in bindingne, which can have KD values for binding to some nAChRss. Such a high affinity for 3H-epibatidine means thats are required to lower receptor concentration while main- give significant levels of radioligand binding. The highest in a saturation curve should be ten times higher than KD Bmax values. Kinetics of ligand binding should be assessedn binding analyses. Association rate constants (kon) for

pically diffusion controlled yielding values of ~108/mol association are observed for some of the more bulky

radioligands, such as 125I-stants (konobs) are related tis the dissociation rate coThere are several ways toHowever, KD values for rabe consistent with kineticMoreover, if koff values armeans of sample processiresult. If koff is less than 0be ~12 hours or more, anthat saturation analyses arachieved unless samples ahalf time for radioligand lowered in reaction mixtu

Radioligand binding sradiolabeled ligand interacan be obtained from stuwith different doses of testude) and a constant concative control (nonspecificsingle concentration of rapositive control or test samor not equilibrium conditprocess have the general samount of binding, [L] isspecific binding sites in ththe ligand concentration gn as the Hill coefficient fobe expressed tentatively inof the binding in the pooccupy all nAChR interac50% of radioligand bindiare then based on the test of inhibition of radioligancentration of ligand givingthe Cheng-Prusoff converof radioligand and KD is saturation analysis.

In principle, competitdistinguished based on stcompetitor concentrationsdifferent concentrations oHowever, these types of sinduce conformational chare not always totally andteric models of nAChR h

2002 CRC Press LLClabeled -bungarotoxin. Observed association rates con-o true kon by the formula konobs = koff + kon [L], where koffnstant and [L] is the concentration of radioligand used. determine koff that will not be discussed in detail here.dioligand binding obtained from saturation curves should determinations because of the relationship KD = koff/kon.e greater than 1/min, then dissociation is too fast for mostng to give reliable data. KD values >10 nM are likely to.001/min, then half times for dissociation (0.693/koff) willd KD values

states of nAChR,46 perhapsand giving experimental competitive mechanisms o

For 125I-labeled -buradiolabeled toxin stocks and reaction mixtures coprecaution helps to prevenslows irradiation decomposolution at 20oC. Nonspunlabeled -bungarotoxinsuggested by the manufacbeen able to define nonspin Ringers buffers that do

1.5.1 INTACT CELLS

Radioligand binding assayassayed either while in suassays using plated cellstypically prepared on muequilibrated in ice-cold Ri1.3 mM MgCl2, 33 mM Taspirated and replaced wior without unlabeled hompriate single concentrationto the size of the cell cultumixture is removed by aspcold, fresh Ringers bufferand rinse buffer addition oare dissolved in 0.01 N Nathen transferred to approbound or to scintillation vtitation of 3H-labeled radi

Cells for radioligand bmedium from large platerinsing cells with ice-coldbuffer to each dish. Cellsdish bottom with an anglcells dislodged by pipettsuspension. Cells are genwithdrawn, the supernataRingers buffer. The sampland the pelleted cells are for the intended assay. Samm polycarbonate) are su

2002 CRC Press LLC making those states refractory to binding of other ligands,results indistinguishable from predictions based on non-f radioligand binding block. ngarotoxin binding assays conducted in the laboratory,

are supplemented with 1 mg/ml of bovine serum albumin,ntain at least 0.1 mg/ml of bovine serum albumin. Thist adsorption of radiotoxin to stock or reaction tubes andsition of probe. Stock samples are maintained in aqueousecific binding is defined in samples containing 2 M

. 3H-labeled agonists are maintained at 20oC in solventsturer. For 3H-labeled epibatidine binding assays, we haveecific binding using samples containing 100 M nicotine not require supplementation with bovine serum albumin.

SUSPENSION AND IN SITU

s of nAChR on the cell surface require use of intact cellsspension or seeded on assay plates.41, 47 To initiate binding in situ, cell culture growth medium is aspirated. Cellsltiwell trays, such as for ion flux assays, are rinsed andngers buffer (150 mM NaCl, 5 mM KCl, 1.8 mM CaCl2,ris, pH 7.4, ~310 milliosmolar). The final rinse buffer is

th assay (Ringers) buffer containing the radioligand withologous ligand or test ligands of choice and at the appro- or range of concentrations. Reaction volumes are tailoredre plate or well. After a prescribed period, the incubationiration. Samples are then rinsed two to three times in ice- over a period of minutes (either by sequential aspirationr by application of the flip-plate technique). Rinsed cellsOH, 0.1% sodium dodecyl sulfate. Dissolved samples arepriate vessels for -counting of 125I-labeled radioligandials to which liquid scintillation fluid is added for quan-oligands.inding assays in suspension are prepared first by aspiratings (100-mm diameter in this example) seeded with cells, Ringers buffer, and adding ~1 ml of ice-cold Ringers are then gently harvested mechanically by scraping theed rubber or polypropylene policeman and collecting thee-delivered streams of tangentially applied buffer as atly centrifuged for 3 to 5 min at 500 g, excess buffer isnt is discarded, and the cell pellet is suspended again ine is centrifuged, the supernatant withdrawn and discarded,resuspended in fresh Ringers buffer to a density suitablemple aliquots placed in centrifuge tubes (16 mm 100

pplemented with radioligand and/or unlabeled ligands of

choice and incubated for for 125I-labeled -bungaroassays. To end the reactiobuffer, and centrifuged onnatants are aspirated andsuspended again in 3 to 4a third cycle of dilute suspin 0.01 N NaOH, 0.1% sobound radioligand as desprocess samples using thestudies using radioligandscells in situ. However, thereaction volumes and qua

125I-labeled bungaror 7-nAChR, can be usnAChR subtypes on the cwith intact cells to quantepibatidine, including thonAChR (typical KD of ~1equivalent samples contanicotine are used to defineplus intracellular pools of3H-epibatidine binding toto membrane fractions fepibatidine and nicotine tpositively charged and recarbamylcholine should nsamples containing 3H-eptidine binding to intraceepibatidine binding to cemended that full carbamyusing both intact cell andetermine whether there itions into cells. If biphascells, then this indicates treceptors is blocked by caThe second phase of such is entering the cell at hireceptors, which would apAt a minimum, these studiadequate to block only su

Assays using intact internalization of surfaceshould be chosen to ensuperiods are appropriate to

2002 CRC Press LLCa prescribed period. Sample volumes are typically 200 ltoxin binding assays and 600 l for 3H-epibatidine bindingn, samples are diluted in 3 to 4 ml of ice-cold Ringersly for the time it takes to accelerate to 5000 rpm. Super- discarded, and cell pellets are rapidly but gently re- ml of buffer and centrifuged. The process is repeated forension and centrifugation, and the cell pellets are dissolveddium dodecyl sulfate before being processed to quantitatecribed previously. Because longer times are needed to cell suspension protocol, it is recommended that binding with fast dissociation rates follow the protocol for intact cell suspension protocol is more economical in terms ofntities of reagents used.otoxin, which has KD values of ~1 nM for 1*-nAChRed in assays with intact cells directly to quantify thoseell surface. In principle, it is also possible to use assaysify surface and intracellular binding sites for 3H-labeledse on 42-nAChR (typical KD of ~10 pM), 34*-

00 pM) or 7-nAChR (typical KD of ~1 nM). Otherwiseining 3H-epibatidine only or 3H-epibatidine plus 10 M total and nonspecific binding, respectively, to cell surface nAChR. Our studies have shown that levels of specific intact cells are indistinguishable from levels of bindingrom the same number of cells, showing access of 3H-o all nAChR pools, even in unbroken cells. Theoretically,latively membrane-impermeant nicotinic ligands such asot enter the cell and block intracellular nAChR. Therefore,ibatidine plus carbamylcholine should display 3H-epiba-llular and nonspecific sites only, allowing specific 3H-ll surface sites to be calculated. However, it is recom-lcholine competition dose-response profiles be obtainedd membrane preparations (see below). This will helps any movement of carbamylcholine at higher concentra-ic competition profiles are obtained when using intact

hat the fraction of binding sites corresponding to surfacerbamylcholine with high affinity at lower concentrations.a competition profile would indicate that carbamylcholinegher concentrations and gaining access to intracellularpear to be blocked by carbamylcholine with lower affinity.es would help identify a concentration of carbamylcholinerface receptors.

cells should involve incubation at 0 to 4oC to prevent nAChR complexed with radioligand; assay conditionsre that ligand and receptor concentrations and incubation

achieve binding equilibrium at these lower temperatures.

1.5.2 MEMBRANES AND

Preparation of membranemedium removal, rinsing, Cell suspensions are gentlis withdrawn and discardhypo-osmotic 5 mM Tris, of membranes from smalcold Ringers buffer. Sampa Polytron at setting 65, minimize foaming of the tubes (16 mm 100 mm pThe supernatant is withdrain fresh Ringers buffer susuitable for the intended athe sample is centrifugedtime. Brief sonication casuspension of membranessupplemented with 0.4 msealed tubes maintained bungarotoxin or 3H-epibastored at 80oC as capreparations with preservradioligand binding sites preparations. Membrane fTris, pH 7.4) have been uexperience suggests that nconditions compared to assalt solutions.

For centrifugation-basof choice, and radioliganpolycarbonate). Reaction addition, and reaction tubfor the prescribed period.ice-cold Ringers buffer strifuged at ~40,000 g for 1membrane pellets are respension is most efficient sample to be blended to a of the buffer is added to dcycle of dilute suspensionin 0.01 N NaOH, 0.1% sobound radioligand as descgands that have slower dbut they would underestimating radioligands. Centr

2002 CRC Press LLCDETERGENT-SOLUBILIZED PREPARATIONS

fragments for radioligand binding assays begins withand mechanical harvesting of cells as described previously.y centrifuged for 3 to 5 min at 500 g, and the supernatanted. The cell pellet is suspended again either in ice-cold,pH 7.4 (to help ensure swelling of cells and maximal yieldl diameter cells such as PC12 and SH-SY5Y) or in ice-les are subjected to homogenization for 45 seconds using

using a probe tip, suspension volume, and vessel size tosuspension. The homogenate is transferred to centrifugeolycarbonate) and sedimented at ~40,000 g for 10 minutes.wn and discarded, and the membrane pellet is suspendedpplemented with 0.4 mg/ml of sodium azide to a densityssay. If obtained by homogenization in hypotonic buffer,

and resuspended in fresh Ringers buffer one additionaln be used at this point to aid in obtaining a uniform. It has been found that resuspension in Ringers bufferg/ml sodium azide allows preservation of membranes inat 4oC for many months without loss of 125I-labeled -tidine binding capacity. Cell pellets can be frozen andn tissues (brain, muscle) and still yield membraneed nAChR radioligand binding sites. However, nAChRare not well preserved by frozen storage of membraneractions suspended in hypo-osmotic buffers (e.g., 25 mMsed by others in nAChR radioligand binding assays, butonspecific binding is increased substantially under thesesays done in Ringers or other physiological, extracellular

ed assays, membrane sample aliquots, unlabeled ligandsd are placed in centrifuge tubes (16 mm 100 mmmixtures are gently flicked to ensure mixing after eaches are placed on an orbital shaker throughout incubation To end the reaction, samples are diluted in 3 to 4 ml ofupplemented with 0.1% bovine serum albumin and cen-0 minutes. Supernatants are aspirated and discarded, and

uspended in 3 to 4 ml of buffer and centrifuged. Resus-if ~250 l of buffer is added to the tube, allowing thefine paste during vigorous vortex mixing, before the bulkilute the suspension. The process is repeated for a third

and centrifugation, and the final cell pellets are dissolveddium dodecyl sulfate before being processed to quantitateribed previously. These assays can be used with radioli-

issociation constants (e.g., 125I-labeled -bungarotoxin),ate numbers of nAChR if assayed with quickly dissoci-ifugation assays of 125I-labeled -bungarotoxin binding

give lower nonspecific binassays (but, see below).

For filtration-based asbased assays but in boroWhatman GF/C filters arbeing rinsed with 3 ml of RAfter a prescribed period200 to 600 l volume arsamples of 6 ml volume cato a polyethyleneimine-cunbound radioligand throuagain to each reaction tuvacuum, preferably before25-mm diameter filter diskcapture comparable quant125I-labeled radioligand bcounting immediately. Tegeometry and sample placdetection of isotope. If dbinding sites, then they shomedium, and left on a shinitiating liquid scintillatioacross samples should bestandard. This is the methoGF/C filtration-based assawith greater ease and recomparable resolution if albumin is used to rinse theapplication, as well as in

If levels of purity of capture anionic nAChR-radisc assay developed by mixtures to the center odiameter, round-bottom wsolution (50 mM NaCl, 5wash solution is removed disc over in the well. Thisdetermination of bound ra

Specific 3H-epibatidinused in immunoprecipitatigradients) is quantified unAChR and bound 3H-epibto a final concentration of

125I-labeled -bungarovolume containing 100 lfinal concentration. React

2002 CRC Press LLCding levels (hence, more resolution) than most filtration

says, sample aliquots are prepared as for centrifugation-silicate glass tubes (typically 12 mm 75 mm). Also,e presoaked in ~0.1 mg/ml of polyethyleneimine beforeingers buffer just prior to application of reaction samples.

of incubation with orbital shaking, reaction samples ofe diluted in 3 ml of ice-cold Ringers buffer. Reactionn be processed directly. Each diluted suspension is appliedoated filter. Vacuum is then applied to draw buffer andgh the filter. Three to four ml of Ringers buffer is addedbe, and the contents are transferred to the filter under the filter has dried. The rinse process is repeated twice;s or filter pads used in semiautomated sample processors

ities of membrane sites. If discs have been used to captureinding sites, they can be inserted into test tubes for sts should be conducted to ensure that counting wellement in the counting tube are compatible with maximaliscs have been used to capture 3H-labeled radioliganduld be dried, placed in vials containing liquid scintillation

aker overnight to ensure suspension of radiolabel beforen counting. Uniformity in efficiency of isotope detection

ascertained using a liquid scintillation counting internald of choice for 3H-labeled nAChR agonist binding assays.ys for 125I-labeled -bungarotoxin binding can be done

producibility than centrifugation-based assays and withRingers buffer supplemented with 0.1% bovine serum polyethyleneimine-coated filters prior to and after sample

reaction mixtures.nAChR allow, ion exchange techniques can be used todioligand complexes. The version of the Whatman DE81Schmidt and Raftery48 involves application of reaction

f dry, 25-mm diameter DE81 discs placed in ~40-mmells of a campers egg carton. Three to four ml of wash

mM NaPO4, 0.1% Triton X-100, pH 7.4) are added, andafter 2 to 3 min by aspiration, taking care to flip the filter process is repeated twice before discs are processed fordioligand as for vacuum-filtered samples.e binding to nonionic detergent-solubilized nAChR (e.g.,on assays or to quantitate nAChR fractionated on densitysing GF/C filtration to resolve free 3H-epibatidine fromatidine after the latter have been reprecipitated by addition 20% polyethylene glycol-8000.toxin binding assays are typically done in a 200 l reaction

of membranes and 50 l of radiotoxin at four times theion times are typically one hour at room temperature for

competition assays, to set uanalyses. 3H-epibatidine busing 600 l reaction voluepibatidine at six times thdone in 6 ml reaction volfor assays in the presence times are typically 1 hour equilibrium. For most stuligand to define nonspecistudies if needed, membrato assess unlabeled ligandthen membrane suspensioligand, it should be addedvolume to ensure that theligand stock solution.

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2002 CRC Press LLCp dissociation studies, and for pre-equilibration saturationinding competition and kinetics assays are typically donemes containing 100 l of membranes and 100 l of 3H-

e final concentration. 3H-epibatidine saturation assays areumes to ensure adequately low concentration of nAChRof 1100 pM radioligand. 3H-epibatidine binding reactionat room temperature, which is usually adequate to achievedies, the sequence of reagent addition is dilution buffer,fic binding if needed, unlabeled ligand for competitionne suspension, and then radioligand. If studies are designed competition toward initial rates of radioligand binding,

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NICOTINIC RECEPTORS IN THE NERVOUS SYSTEMTable of ContentsSECTION IChapter 1: Some Methods for Studies of Nicotinic Acetylcholine Receptor PharmacologyCONTENTS1.1 INTRODUCTION1.2 MAINTENANCE OF CELL LINES NATURALLY EXPRESSING nAChR1.3 CREATION AND MAINTENANCE OF CELL LINES STABLY AND HETEROLOGOUSLY EXPRESSING nAChR OF DEFINED ...1.3.1 Vector Selection, Transfection, and Subcloning1.3.2 Host Cell Types Used and nAChR Subtypes Expressed

1.4 ISOTOPIC ION FLUX ASSAYS FOR RAPID CHARACTERIZATION OF FUNCTIONAL nAChR ENSEMBLES1.4.1 Efflux and Influx Assays1.4.2 Techniques for Cell Manipulation

1.5 RADIOLIGAND BINDING ASSAYS1.5.1 Intact Cells Suspension and In Situ1.5.2 Membranes and Detergent-Solubilized Preparations

REFERENCES