Distribucion de Los Canales de Calcio Tipo N

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Neuron, Vol. 9, 1099-1115, December, 1992, Copyright 0 1992 by Cell Press

Biochemical Properties and SubcellularDistribution of an N-typeCalcium Channel al SubunitRuth E. Westenbroek,* Johannes W. Hell,*Conception Warner,* Stefan J. Dubel,+ Terry P. Snutch,+and William A. Catterall**Department of PharmacologyUniversity of WashingtonSeattle, Washington 98195+Biotechnology Laboratoryand Departments of Zoology and NeuroscienceUniversity of British ColumbiaVancouver, British ColumbiaCanada V6T 123

SummaryA site-directed anti-peptide antibody, CNB-1, that recog-nizes the al subunit of rat brain class B calcium channels(rbB) immunoprecipitated 43% of the N-type calciumchannels labeled by [lz51]o-conotoxin. CNB-1 recog-nized prot eins of 240 and 210 kd, suggesting the pres-ence of two size forms of this al subunit. Calcium chan-nels recognized by CNB-1 were localized predominantlyin dendrites; both dendriti c shafts and punctate synapticstructures upon the dendrites were labeled. The largeterminalsof themossyfibersof thedenta tegyrusgranuleneurons were heavily labeled, suggesting that the punc-tate labeling pattern represents calcium channel s innerve terminals. The pattern of immunostaining was cellspecific. The cell bodies of some pyramidal cells in layersII, III, and V of the dorsal cortex, Purkinje cells, andscattered cell bodies elsewhere in the brain were alsolabeled at a low level. The results define complementarydistributions of N- and l-type calcium channels in den-drites, nerve terminals, and cell bodies of most centralneurons and support disti nct functional roles in calcium-dependent electrical activity, intracellular calcium regu-lation, and neurotransmitter release for these two chan-nel types.IntroductionVoltage-gat ed calcium channels generate calcium-dependent action potentials in dendrites, allow cal-cium entry that initiates neurotransmit ter release andother intracellular regulatory processes, and play apivotal role in the control of neuronal firing patternsand excitability. Four physiologically and pharmaco-logically distinct classes of voltage-sensiti ve calciumchannels have been identified; these are designatedas T, L, N, and P (Bean, 1989; Lli nas et al., 1989; Hess,1990). L-type calcium channels mediate long-lastingcalcium currents and are specifically inhibited by di-hydropyridines and other organic calcium channelantagonists (Bean, 1989; Hess, 1990). In contrast,N-type calcium channel s are specifically and irrevers-

ibly inhibited by wconotoxin GVIA (w-CgTx) (Aosakiand Kasai, 1989; Plummer et al., 1989).

L-type voltagedependent calcium channels puri-fied from rabbit skeletal muscle transverse tubulesare a complex of five subunits: al, a2, B, y, and 8(Takahashi et al., 1987; Campbell et al., 1988; Catterallet al., 1988). cDNAs encoding each of these subuni tshave been cloned (Tanabe et al., 1987; Ellis et al., 1988;Ruth et al., 1989; Jay et al., 1990), and the a2 and 8subunits have been shown to be produced from asingle precursor by posttranslational processing (DeJongh et al., 1990; Jay et al., 1991). al subunits canfunction alone as voltage-gated calcium channel swhen expressed in Xenopus oocytes or mammaliancells (Perez-Reyes et al., 1989; Mikami et al., 1989), buttheir expression is increased and their functionalproperties are altered by coexpression of the othersubunits(Lacerdaetal.,1991;Singeretal.,1991;Varadiet al., 1991; Wei et al., 1991). Neuronal L-type calciumchannels have a similar subunit composition and arediscretely localized in the cell bodies and proximaldendrites of central neurons (Ahlijanian et al., 1990;Westenbroek et al., 1990). Neuronal N-type calciumchannels are not yet fully characterized biochemi-cally, but it is likely that they contain al-, a28, andB-like subunits (Ahlijanian et al., 1991; Sakamoto andCampbell, 1991; McEnery et al., 1991).

cDNAs encoding the al subunit of skeletal muscleL-type calcium channel have been used to identifyfour classes of cross-hybridizing cDNA clones encod-ing brain calcium channels, designated rbA, rbB, rbC,and rbD (Snutch et al., 1990). The rat brain class C andD gene products share approximately 75% amino acididentity with rabbit skeletal muscle calcium channelsand are closely related to L-type calcium channelsfrom other tissues (Tanabe et al., 1987; Ellis et al., 1988;Mikami et al., 1989; Perez-Reyes et al., 1989; Biel et al.,1990; Hui et al., 1991; Snutch et al., 1991; Seino et al.,1992; Williams et al., 1992). In contrast, the rbAand rbBisoforms are more distantly related to L-type calciumchannels (30%~44% amino acid identity) and are can-didates for neural-specific calcium channel isoforms(Starr et al., 1991; Dubel et al., 1992). A full-length rbBcalcium channel al subunit sequence that encodes aprotein of 2336 amino acid residues has been de-scribed. This protein was identified as the al subunitof an w-CgTx-sensiti ve N-type calcium channel by spe-cific immunoprecipitation of w-CgTx receptor sitesfrom rat brain with an anti-peptide antibody (CNB-1)recognizing a highly variable segment of its aminoacid sequence (Dubel et al., 1992). In the presentstudy, we have used the CNB-1 antibody to identifytwo different forms of the al subunit of the N-typechannel and to investigate its cellular and subcellulardistribution in rat brain.


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Resultslmmunoprecipitation of N-type Calcium Channelsby CNB-1 AntibodiesN-type calcium channels were labeled with [1251]o-CgTx under conditi ons in which only high affinity andessentially irreversible binding sites for o-CgTx areoccupied, solubilized with digitonin, and immuno-precipitated with increasing amounts of CNB-1 anti-bodies. As shown in Figure 1, the amount of immuno-precipitated N-type calcium channels increased to amaximum of 43% of the total o-CgTx receptor sitespresent. Dihydropyridine receptor sites on l-type cal-cium channels are not immunoprecipitated withCNB-1 antibodies under these conditions (Dubel etal., 1992). These results indicate that the CNB-1 anti-bodies recognize a specific subpopulati on of o-CgTxreceptor sites. Evidently, there are additional o-CgTxreceptor sites, possibly associated with other sub-types of N-type calcium channels, that are not recog-nized by the CNB-1 antibodies.Identification of Two Size Forms of the al Subunitof an N-type Cal cium Channellmmunoblotting of a partially purified glycoproteinfractionasdescribed in Experimental Procedureswit haffinity-purified CNB-1 antibodies revealed two im-munoreactive protein bands with apparent molecularmasses of 210 and 240 kd (Figure 2A). The specificit yof the interaction of CNB-1 antibodies with these bandswas tested by competition with the CNB-1 peptide (Fig-ure 2B). After preincubation with this peptide at 7 PM,no signal could be detected with the CNB-1 antibody,even if the blot was overexposed, as in Figure 28,to detect a low level of nonspecific interaction. Inaddition, neither band was recognized when probedwith nonspecific antibodies (data not shown). Takentogether, these results show that CNB-1 antibodies

interact specifically with two bands that likely corre-spond to two different size forms of rbB-I al subunits.

The specificity of the CNB-1 antibodies is furthersupported by comparisons of the amino acid se-quences of cloned calcium channels. The region ofthe al subunit chosen for production of anti-peptideantibodies is hypervariable among different calciumchannel subtypes, and the sequence of the CNB-1peptide is not conserved in any cloned calcium chan-nel whose sequence has been published or in a newcalcium channel al subunit related to rbB-I, whoseprimary structure has recently been determined(Soong and Snutch, 1992, Sot. Neurosci., abstract). Toprovide additional evidence on this point, we purifiedantibodies reactive against the carboxylll residues ofpeptide CNB-1 by epitope selection (see ExperimentalProcedures). These antibodies gave similar results inboth immunoblot and immunocytochemical experi-ments, supporting the specificity of the CNB-1 anti-bodies for calcium channel al subunits encoded bythe rbB-I gene. While we cannot exclude the possibil-ity that additional calcium channels whose primarystructures are not yet determined may contain anidentical or a cross-reacting antigenic epitope, it isunlikely that any new isoforms would have the highabundance and widespread distribution demon-strated below for rbB-I.

To confirm further the identity of the 240 kd and 210kd proteinsas N-type al subunits,their sedimentationbehavior during sucrose gradient centrifugation wascompared with that of the [1251]w-CgTx-labeled N-typeand the [3H]PN220-110-l abeled L-type calcium chan-nels (Figures 2C and 2D). The N-type calcium channelwas partially separated from the L-type calcium chan-nel by sedimentation (Figure 2D). Both bands de-tected by CNB-1 showed the same distributionthroughout the sucrose gradient, with the highestlevel of protein and in fraction 7 (Figure 2C). In two

CNB 1 - Antiserum [@I]

Figure 1. lmmunoprecipitati on of w-CgTx-Labeled N-type Calcium Channel s byCNB-1 AntibodiesMembranes from rat cerebral cortex werelabeled with 2 nM [2SI]~CgT~, sedi-mented, solubilized with digitonin, andcleared by centrifugation as described inExperimental Procedures. Protei n A-Seph-arose preincubated with antibodies fromthe indicated amount of antiserum wasadded to 100 PI aliquots, and the sampleswere treated as described in ExperimentalProcedures. Nonspecific immunoprecipi-tation was determined with a correspond-ing amount of nonimmune antiserum andsubtracted from each value obtained withCNB-1 antiserum. Nonspecific immuno-precipitation was less than 8% of maxi-mum. The total amount of labeled N-typechannelswasdetermined bythefilterassayand corresponds to 100%. A saturationcurve was fitted to the data according tothe equation y = 41.1 x [I - exp(-x/21.4)].


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A BI 5; - +

240,220,205- %- :205= i

0

C

fraction number

Figure 2. lmmunoblotti ng with CNB-1 De-tects Two Protein Bands That Corn&rate

2.0 z2E with N-type, but Not L-type, Calcium Chan-\ nels during Sucrose Gradient Sedimen-zE tation1.5 (A) Calcium channels were enriched fromb2 solubilized forebrain membranes by WGA: affinitychromatographyandsucrosegradi-cz ent centrifugation. A 1 ml sample was1.0 , treated with heparin agarose to concen-0 trate the calcium channels, extracted withI SDS sample buff er, separated by SDS-poly-

05 x acrylamide gel electrophoresis, blotted, in-:

cubated with CNB-1 antibodies that hadbeen affinity purified on CNB-1 peptide,I and detected with t he ECL method as de-J 0.0 A scribed in Experimental Procedures. TheI migration positions of CI- and B-spectrinand of myosin heavy chain are indicated atthe left side of the gel together with theirrespective molecular masses (in kilodal-tons).

(B) An immunoblot of a sucrose gradient fraction enriched with N-type channel (A) was probed wi th affinity-purified CNB-1 antibodiespreincubated in the absence (left lane, -) or presence fright lane, +) of 7 uM CNB-1 pepti de for 1 hr. Note that the blot was overexposedto demonstrate the complete absence of any antibody bound to the blot in the presence of the CNB-1 peptide. Molecular mass standardsare indicated beside the gel as in (A).(C)Calcium channels wereenriched from [3H]PN22Bl10-l abeled membranes, and different sucrosegradient fractions were immunoblot-ted with CNB-1 antibody as described in (A). Molecular mass standards are indicated at the left side of the gel.(D) Determination of the amount of PN22&110- and o-CgTx-labeled L-and N-type channels, respectively, in different sucrose gradientfractions. Bound [3H]PN22&110 was detected by scintillation counting. To determine the amount of N-type calcium channels, aliquotsof each fraction were incubated with 2 nM Pl]uKgTx, precipitated with polyethylene glycol, and filtered as described in ExperimentalProcedures. Note that both bands recognized by CNB-1 comigrate. They also cosediment with the uKgTx receptor. Two additionalexperiments yielded si milar results with an even more precise codistribution of the CNB-1 immunoreactivity and [lXl] oCgTx-bindingsites in the sucrose gradient.

experiments (data not shown), the immunoreactive240 kd and 210 kd bands comigrated precisely withthe o-CgTx binding activity throughout the peak. Ina third experiment, in which the 240 kd and 210 kdbands were particularly well resolved (Figures 2C and2D), these two bands also comigrated closely with thec&gTx binding activity, with a peak in fraction 7, butthere was more immunoreactivity in the leading frac-tions and less in the trailing fractions than expectedfrom the binding data. CNB-1 antibodiesdid not cross-react detectably with L-type al subunits, which hadtheir maximal concentrati on in fraction 9. Overall, theresults provide strong support for identification of

both the240 kd and the210 kd CNB-I-immunoreactiveproteins as o-CgTx-sensiti ve N-type calcium channelal subunits.localization of N-type Calcium Channels in theDorsal Cerebral CortexThroughout the rostral-caudal extent of the rat brain,al subunits recognized by the CNB-1 antibodies arelocalized mainly in dendrites, with less frequent stain-ing of cell bodies. In addition, punctate structures,which we interpret as presynaptic terminals (see be-low), are observed along the surface of dendrites andcell bodies. This pattern of immunoreactivity is illus-


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Figure 3. Localization of N-type Calci um Channels in the Dorsal Cerebral CortexSagittal sections of adult brain were stained wi th affinity-purified CNB-1 antibodies by the PAP method as described in ExperimentalProcedures. (A) Low magnification of the dorsal cortex stained with CNB-1 indicating the pattern of staining in various layers such asII, III, and V. (B) Control section stained with CNB-1 preadsorbed with CNB-1 peptide. (C) Higher magnification of cortical layers II andIII demonstrating dendritic staining pattern. (D and E) Localization of N-type calcium channels in dendrites located i n layers IV andV illustrating a sudden decrease in immunoreactivity observed as the dendrites reach tayers I and II. (F-H) Higher magnification of


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trated for the dorsal cerebral cortex in Figure 3. At lowmagnification, the pattern of CNB-1 immunostainingin the dorsal cortex is laminar (Figure 3A). This lamina-tion is the result of CNB-1 staini ng of the apical den-drites of pyramidal neurons residing in layer V (Figure3A, bottom band of stain) or in layers II and III (topband). The apical dendrites of these neurons ascendvertically through the cortex and terminate in the mo-lecular layer or layer I. A peptide block of the CNB-1antibody reveals that the staining is specific for therbB sequence (Figure 38). Localization of the CNB-1antibody in dendrites of pyramidal cells in layer Voften appears truncated, as the dendritic staining isvery intense in the proximal portions of the apicaldendrite and diminishes along the more distal por-tions (Figures 3D and 3E). This decrease of immunore-activity is often sudden and generally occurs wherethe dendrite traverses through the deeper half of layerIV, causing the laminated appearance observed atlower magnificati ons. By contrast, there is intense den-dritic staining throughout layers II and III. This stain-ing seems to represent both the dendrites of cellsresiding in layers II and III and the distal portions ofdendrites from deeper layers.

The staining of dendritic shafts has an uneven,patchy appearance (Figures 3F-3H). In addition, alongthe length of dendrites that are immunoreactive forN-type calcium channels, there is also staining ofsmall, punctate structures having the appearance ofsynapses on the dendrites (Figures 3D-3H). These syn-aptic structures are most easily observed along thelabeled dendrites in layers IV and V. They are oftennumerous and tend to lie within the boundaries ofthe immunoreactive dendritic shaft. These punctateaccumulations of immunoreactivity might representclusters of N-type calcium channels in the postsynap-tic membrane of the dendritic shaft and spines orclusters of channels in the presynaptic membrane ofthe nerve terminal. Examination at higher resolutionin the large terminals of the mossy fibers of the hippo-campus as described below suggests that they repre-sent presynaptic terminals.

Within the dorsal cort ex, there are also cell bodiesin layers V and VI that are immunoreactive for N-typecalcium channels. One example can be observed inFigure 3G (arrowheads). Staining in these cell bodiesis much less intense than that observed in the den-drites, as is illustrated by the intense staining of thedendrite passing by the cell body indicated in Figure3G. Staining of cell bodies by CNB-1 antibodies is lo-calized to mainly pyramidal -shaped neurons. Thisstaining is observed more clearly i n the confocal mi-croscope, as illustrated in Figure 7 below.

Localization of N-type Calcium Channe lsin the HippocampusIn the hippocampal formation, N-type calcium chan-nel immunoreactivity is also located predominantl yin dendrites. There is dense CNB-1 immunoreactivitythroughout the stratum radiatum in the apical den-drites of the CAI-CA3 pyramidal neurons (Figure 4A).In the CA3-CA2 region, there is a band of stainingin the stratum lucidum due to immunoreactive basaldendrites of the pyramidal neurons. This staining ofbasal dendrites is also present, but is not as evident,in the CA1 region. Dense staining of dentate granulecell dendrites is also observed in the molecular layerof the dentate gyrus (Figure 4A). A negative controldeveloped wi thout CNB-1 antibodies illustrating thespecificity of this staining is shown in Figure 4B.Higher magnifications reveal that there is relativelylittle staining of N-type calcium channels in the cellbodies of pyramidal neurons in CAI-CA3 (Figures 4cand 4d, arrows) as compared with the dense immuno-reactivity of the apical dendrit es (Figures 4C and 4D).Other cell types in the hippocampus and dentate gy-rus did not stain for N-type calcium channels at thelevel of development of the immunocytochemical re-action used in our experiments.

As in the dorsal cortex, dendrites stained by CNB-1antibodies are studded with punctate accumulationsof immunostain that appearto be localized at synapticsites (Figures 4C and 4D). Coronal sections throughthe hippocampus reveal much larger, more extendedstructures that are strongly immunoreactive forN-type calcium channels in the apical dendrites ofpyramidal cells in the CA3-CA2 region (Figure 4E). Thesize and distribution of these structures (Figure 4E,arrows) resemble those of the large nerve terminalsof the mossy fibers of the dentate granule cells thatform synapses on the dendrites of the CA3 and CA2pyramidal cells. Occasionall y, strongly stained nerveterminals can be visualized at the end of a long, lightlystained mossy fiber nerve ending (Figure 4E, arrow-heads). These results support the conclusion that thestrongly stained punctate structures revealed byCNB-1 antibodies are presynaptic terminals.localization of N-type Calcium Channel s in theForebrain and MidbrainN-type calcium channel staining is prominent through-outtheforebrainand midbrain.Thestainingobservedis associated mainlywith dendrites, and in some caseson the cell bodies, of many populations of neurons.Several examples are presented in Figure 5. We ob-served N-type calcium channel staining in dendritesof the glomeruli of the olfactory bulb and in the adja-

dendrites in layers IV and v showing staining of synaptic structures (arrows) along the shaft of immunoreactive dendrites and anoccasional selected cell body (arrowheads). Magnification: 62.5x (A); 62.5 x (B); 352~ (0; 320x 0; 452~ (0 320X (I% 800~ (cl;800x (H).


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Figure 4. Localization of N-type Cal cium Channels in the HippocampusSagittal (A-D) and coronal (E) sections stained wit h CNB-1 antibody by the PAP method as described in Experimental Procedures. (A)Low magnification of the hippocampus. (B) Control section i ncubated without CNB-1 antibodies. (C and D) Higher magnificati on ofdendritic staining in the CA1 and CA3 regions, respectively, using CNB-1 antibody. Small arrows point to the pyramidal cell bodies thatare lightly stai ned with CNB-1. (E) Higher magnification of CNB-I-immunoreactive terminals (large arrows) in the CA3 region. Not e thelightly immunoreacti ve nerve ending (arrowheads) and its associated terminal. Magnification: 22x (A); 22x (B); 320x (C); 320x (D);640x (E).


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Figure 5. Distribution of N-type Calcium Channels in Forebrain and Midbrain RegionsSagittal sections of adult rat brain were stained with CNB -1 antibody by the PAP method as described in Experimental Procedures. Thisfigure illustrates dendritic staining i n various regions, including olfactory bulb glomerulus and external plexif orm layer (A), pyriformcortex with little stai ning in layer I compared with layers II and III (B), caudate (C), and substania nigra at low (D) and high (E)magnifications. Note also the punctate staining of nerve terminals (arrows) in (A). Magnification: 200x (A); 160x (B); 220x (C); 64x (D);320x (EL

cent external plexiform layer, through which the den-drites of mitral cells, tufted cells, and granule cellsramify (Fi gure 5A). Large accumulations of stain atcomplex synaptic structures are also observed in thisregion (Figure 5A, arrows). In the pyriform cortex,CNB-1 antibody is localized to dendrites and synapticstructures in the deeper portion of layers II and III(Figure 5B). Interestingly, there was little staining inlayer I of the apical dendri tes of the pyramidal neu-rons located in layers II and III. Therewas also a mesh-work of dendrite staining observed in the caudate-putamen (Figure 5C). The immunostained dendriteshere are very fine in appearance and correspond insize to those described for cells in this region. Rela-tively light staining of cell bodies was observed inthe caudate-put amen. Extensive labeling of dendritesusing CNB-1 antibodies was also detected in the sub-

stantia nigra (Figures 5D and 5E). In this regi on therewas also occasional labeling of neuronal cell bodies.localization of N-type Calcium Channel s in theCerebellum and Brain StemIn the cerebellum, N-type calcium channels recog-nized by CNB-1 are found mainly on Purkinje cells(Figures 6A and 68). The staining was observed on thecell body of Purkinje cells and was relatively densealong the entire length of their dendrites (Figure 6A).The strongest immunoreactivityoccurred at the proxi-mal part of the dendrites, including the dendritic shaftand the first and second branch points; the branchpoints often exhibited the densest staining (Fi gures6A and 6B). At higher magnification, a punctate-likepattern of staining was apparent along the cell bodiesand dendrites of Purkinje cells. These punctate struc-


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Figure 6. Localization of N-type CalciumChannels in the Cerebellum and Hind-brainSagittal sections of adult rat brain werestained with CNB-1 antibody using the PAPmethod described in Experimental Proce-dures. (A) Low magnification of the cerebel-lar cortex demonstrating CNB-l-immuno-reactive Purkinjecell bodiesand dendrites.(B) Higher magnification of immunoreac-tive Purkinje cells demonstrating stainingin cell body and proximal dendrites anddendritic branch points. (C) Low magni-fication of granular layer demonstratingN-type calcium channel s in scattered Golgicells. (D) Higher magnification of the gran-ular layer illustrating relatively light CNB-1immunoreactivity in granule cells. (E) Lowmagnification of facial nucleus (lower por-tion) and adjacent reticular f ormation (up-per portion) demonstrating CNB-l-positivedendrites in these regions.(F) Higher mag-nification of the facial nucleus. Magnifica-tion: 250x (A); 400x (B); 160x (C); 220x(D); 80x (E-F).

tures may be either spines or terminals; nerve termi- 6C and 6D). The N-type calcium channels recognizednals seem most li kely in view of the staining pattern by CNB-1 antibodies were also observed, but in rela-in other brain regi ons. Golgi cells in the granular layer tively low density, on the cell bodies of granule cellsshowed immunoreactivitywith CNB-1 aswell (Figures in the granular layer (Figures 6C and 6D). The CNB-1


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antibodies did not appear to stain the cell bodies ofstellate and basket cells in the molecular layer.

As previously described for forebrain and midbrainregions, complex patterns of dendritic and synapticstaining were observed throughout other regions ofthe hindbrain using CNB-1 antibodies. For example,fine dendrites and synaptic st ructures were immuno-stained by CNB-1 antibodies in the facial nucleus andthe adjacent reticular formation (Figures 6E and 6F).Similar immunoreactivity of some dendrites withCNB-1 was present throughout the hindbrain. In addi-tion, occasional staining using CNB-1 antiserum wasobserved in the cell bodies of scattered cell groups.Examination of Calcium Channel Distribution byConfocal MicroscopyThe punctate pattern of immunostaining by CNB-1antibodies was resolved more clearly with the avidin-biotin detection method and confocal laser micros-copy. In layer V of the cerebral cortex, punctate regionsof intenseCNB-1 immunoreacti vitywereobserved su-perimposed on general patchy staining of the den-dritic shaft (Figures 7A and 78). lmmunostaining ofneuronal cell bodies was less frequent and of gener-ally lower intensity, but when observed was also in apatchy pattern with superimposed punctate synapti cstructures, as illustrated for several neurons in layerV (Figures 76 and 7C). In the cerebellum, we observedpunctate staining on the Purkinje cell somata (Figure7D), along the proximal portion of the dendrites (Fig-ure 7D), and at branch points further out along thedendrites (Figure 7E). The optical sectioning capabilityof the confocal microscope allowed us to focus seri-ally through vertical sections of the cell and confirmthat the concentrati ons of calcium channels immuno-reactive with CNB-1 antibodies are located on the cellsurface of the soma and dendrites (compare Figures7E and 7F). In addition, there was general staining ofthe Purkinje cell dendrites (Figure 7F) similar to thatobserved with the peroxidase-ant iperoxidase (PAP)technique.Differential Subcellular Distribution of N-type andL-type Calcium ChannelsOur previous studies with a monoclonal antibodyagainst the a2 subunits of L-type calcium channelsshowed that they are primarily localized in neuronalcell bodies and proximal dendrites of central neurons(Ahlijanian et al., 1990; Westenbroeket al., 1990). Thus,their distribution is complementary to the distribu-tion of the N-type calcium channels studied here. Thispoint is illustrated for the cerebral cortex in Figure 8.L-type calcium channels are observed in cell bodiesand clustered at the base of major dendr ites (Figure8A). In contrast, N-type calcium channels are localizedprimarily in dendrites and associated synapses incompanion sections (Figure 8B). This differential dis-tributionofthesetwochanneltypessupportsthecon-elusion that they play distinct roles in signal pro-cessing and transduction in neurons.

DiscussionMultiple N-type Calcium Channelslmmunochemical experiments on N-type calciumchannels suggest that at least two different subtypesof this subunit are present in the rat brain. Immuno-precipitation of the solubilized high affinity w-CgTxreceptor with CNB-1 was incomplete; using a largeamount of CNB-1 antiserum, only 43% of the highaffini ty*CgTx receptors could be precipitated. Thus,it is likely that at least one more subtype of o-CgTx-sensitive N-type calcium channel is present and ac-counts for the remaining o-CgTx receptors. Theamino acid sequence corresponding to the CNB-1peptide or the accessibili ty of this site for antibodybinding may differ in another subtype(s), preventingeffective immunoprecipitati on by the CNB-1 anti-body. It also seems likelythatthe N-type channel com-plex can be formed by different a2 subunits becausesaturating concentrati ons of a monoclonal antibodygenerated against the a2 subunit of skeletal muscleL-type channel specifically immunoprecipitates only6%-8% of the w-CgTx recept ors after solubilizationfrom rabbit brain (Ahlij anian et al., 1991). These find-ings suggest that the remaining 92% of N-type chan-nels contain other a2 subunits not recognized by thisantibody. In agreement with these immunochemicalresults, a new subtype of brain calcium channel alsubunit related to rbB-I has been detected by cDNAcloning (Soong and.Snutch, 1992, Sot. Neurosci., ab-stract), and additional isoforms of the a2 subunitshave also been described (Williams et al., 1992; Kim etal., 1992). Thus, the biochemical properti es and sub-cellular distribution described in this report likely re-fer to a specific subset of o-CgTx-sensitive N-type cal-cium channels.Two Size Forms of al Subunits of N-typeCalcium ChannelsOur immunoblotting experiments strongly suggestthe existence of at least two dif ferent size forms of theal subunit(s) recognized by CNB-1 antibodies. CNB-1stains two protein bands with molecular weights ofapproximately 240 and 210 kd in immunoblots. Bothbands were specifically recognized, since the corre-sponding peptide completely blocks binding ofCNB-1 to both bands. During sucrosegradient centrif-ugation, which allows the separation of the PN220-110receptor from theo-CgTx receptor, both bands comi-grate exactly with each other and with the o-CgTxreceptor. It cannot be excluded that proteolysis of the240 kd form dur ing purification gives rise to the 210kd band, but this seems not to be the case. Extensiveprecautions to prevent proteolysis such as loweringthe temperature to nearly OC by precooling all glass-ware and rotors before use and inclusion of high con-centrations of a large variety of protease inhibitors,did not inhibit the appearance of the 210 kd band.Another approach to prevent postmortem proteolysiswas to perfuse the rats with calpain inhibitors 1 and 2


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Figure 7. Distribution of N-type Calcium Channels Viewed wi th the Confocal MicroscopeSagittal sections of adult rat brain were stained with CNB-1 antibody using the immunofluorescence technique as described in Experi-mental Procedures. (A) Section t aken from layers II and III of the dorsal cortex demonstrating dense, patchy staining of dendrites withpunctate staining of terminals along the length of the shafts. (B and C) Higher magnification of staining on the surface of pyramidalneuron somata a nd dendrites from cells in the dorsal cortex. Note as well the more dense staini ng of a dendrite adjacent to the punctatestaining along a cell body in (B). (D) Section from the cerebellum illustrating the dense CNB-1 immunoreactivit y along the cell bodyand proximal dendrite. (E and F) Sections from the molecular layer of the cerebellum demonstrating staining of terminals on the surfaceof the dendrites fE) as well as dense staining of Purkinje cell dendrites (F), as determined by stepping through the section at 2 Hrnincrements using the confocal microscope. Magnification: 820x (A-F); optical section thickness: 0.7 Wm.

and leupeptin before decapitation. All three reagents Based on these results, we conclude that two sizeare thought to cross cell membranes and should block forms of the al subunit are probably present in situ.proteolysis before homogenizati on of the tissue. This Two different size forms of the al subunit with ap-treatment did not reduce the amount of al subunit in parent molecular masses of 190 kd and 210 kd werethe 210 kd band in comparison with the 240 kd band, also found for the skeletal muscle L-t ype channel inas would have been expected if degradation of the al purified preparati ons (De Jongh et al., 1989,199l) andsubunit by proteases occurred during purification. in intact skeletal muscle cells (Lai et al., 1990). Map-


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Figure 8. Comparison of N and L-type Calcium Channel Localization in the Dorsal CortexSagittal sections of adult rat brain were stained with M ANC-1 or CNB-1 antibody using the PAP method as described in ExperimentalProcedures. This figure illustrates the differences in localization in the dorsal cortex of L-type calcium channels (A), which are mainlyin the cell soma and proximal dendrites using MANC-1, compared with N-type calcium channels (B), which are mainly in the dendriteswith less staining in the cell body. Magnification: 410x.

ping with anti-peptide antibodies showed that theC-terminal end of the smaller form i s truncated, whilethe larger form contains the complete sequence en-coded by the cloned cDNA. Consensus sequences forphosphorylati on by cAMPdependent kinase in theC-terminal region of the full-length form are rapidlyphosphorylated, suggesting the possibility of differ-ential regulation of the two size forms by proteinphosphorylati on (Lai et al., 1990; Rotman et al., 1992).cDNAs encoding the major truncated form have notbeen detected, suggesting that the two forms of theskeletal muscle L-type al subunit are produced byproteolytic processing in vivo. The 210 kd form of theneuronal w-CgTx-sensit ive N-type al subunit ob-served here might also differ from the 240 kd form attheC-terminal. Infact,cDNAsthatdefinerbBproteinswith three different C-termini have now been identi-fied. These include t he original full-length cDNA of262 kd and two truncated versions of approximately190 kd and approximately 204 kd (Dubel and Snutch,unpubli shed data). The 210 kd protein that we ob-served may correspond to the cDNA of approximately204 kd; the 240 kd band that we observed may repre-sent the predicted full-length 262 kd protein. The 5%-10% underestimate of true molecular masses by SDS-polyacrylamide gel electrophoresi s is similar to thatobserved for al subunits of skeletal muscle L- typecalcium channels (De Jongh et al., 1991).

cDNAs encoding several forms of the P-type chan-nel as well as one of the neuronal L-type al subunitshave been identified by molecular cloning and se-quencing. In all cases, the isoforms are identical ex-cept for C-terminal ends of different length, whi chare probably created by differential splicing of mRNA(Hui et al., 1991; Mori et al., 1991; Starr et al., 1991;Williams et al., 1992). Therefore, the 240 kd and 210 kdforms of the neuronal N-type al subunit may also becreated bydifferential splicing, butwecannotexcludethe possibili ty that a specific proteoly-tic processingstep occurs in vivo to create the 210 kd form from the240 kd form. Since the al subunit of an o-CgTx-sensitive calcium channel is phosphorylat ed by bothCAMP-dependent protein kinase and protein kinaseC in vitro (Ahlijanian et al., 1991) and there are severalconsensus sequences for phosphorylati on in the C-ter-minal region, the two size forms of this subunit maybe differentially regulated by protein phosphoryla-tion, as has been postulated for skeletal muscle L-typecalcium channels.Broad Regional Expression of an N-type CalciumChannel in the BrainNorthern blot analysis showed that the rbB-I tran-script is expressed only in neuronal cells and is widelydistributed in the CNS (Dubel et al., 1992). Consistentwith these results, we find that the CNB-1 antibodies


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label calcium channel s throughout the brain, includ-ing the frontal and dorsal cerebral cortex, hippocam-pus, cerebellum, midbrain, hindbrain, and medulla.Whilethere is regional variation in thedensityof label-ing and in the fraction of neurons that are immunore-active for the CNB-1 antibodies, most gray matter ar -eas are stained above background, suggesting a broad,if not ubiquitous, expression of the rbB gene in differ-ent brain regions.

Our results localizing the protein products of a sin-gle N-type al subunit can be compared with previousstudies localizing binding sites for w-CgTx. In autora-diographic localization of N-type calcium channelsusing pz51 ]w-CgTx, regions of high binding site densityincluded the striatum, hippocampus, molecular l ayerof the dentate gyrus, frontal cortex, and cerebellarmolecular and granular layers (Kerr et al., 1988; Take-mura et al., 1988,1989). These are all regions where weobserved dense staining of N-type calcium channelswith the CNB-1 antibodies. Compared with our find-ings, a recent study using a monoclonal antibody too-CgTx itself suggests a wider di stribution of N-typecalcium channels in the cerebellar cortex, especiallyin the molecular layer (Fortier et al., 1991). It is proba-ble that this monoclonal antibody recognizes w-CgTxbound to all forms of the N-type calcium channel, incontrast with the CNB-1 antibody, which immunopre-cipitates less than half of brain o-CgTx receptor sites,representing a specific subtype(s) of N-type channels.Thus, it is likely t hat other subtypes of o-CgTx-sen-sitive N-type calcium channels are differentially ex-pressed and possibly differentially localized in centralneurons.N-type Calcium Channels in Dendrites and Cell BodiesElectrophysiological studies show that the ion cur-rents measured in different regions of single neuronsare distinct and highly specialized. Information aboutthe spatial distribution of ion channel subtypes in dif-ferent neuronal compartments is required for a clearunderstanding of electrical signaling in neurons. Thedistribution of voltage-dependent ion channels overthe somatic and dendritic membranes of neurons hasprovided evidence for multiple classes of calciumchannels in these neuronal compartments (Crill andSchwindt, 1983; Llinas, 1984). In dendrites, both lowvoltage-acti vated T-type calcium channels and highvoltage-acti vated calcium channels are thought toparticipate in the generation of calcium-dependentaction potenti als that are conducted along the den-drite and play an important role in processing infor-mation from the multiple synaptic inputs to the den-dritic arbor (Lli nas and Sugimori, 1979). Repetitiveactivation of synaptic input to the distal dendrites ofhippocampal pyramidal cells causes dramatic i n-creases in cytosolic calcium levels throughout thedendritic arbor through activation of voltage-gatedcalcium channels (Regehr et al., 1989). Previous stud-ies using cerebellar slices have suggested that the den-dritic arbor is an important site of calcium-dependent

electrogenesis and calcium ent ry into Purkinje cells(Llinas and Sugimori, 1980; Tank et al., 1988) and thatdendritic calcium el ectrogenesis is localized at hotspots near or at dendritic bifurcations (Llinas and Sugi-mori, 1979). The dendrites are also prominent sites ofcalcium entry, causing increases in cytosolic calciumin Purkinjecells (Tank et al., 1988). What calcium chan-nel subtypes are responsible for the calcium-depen-dent action potentials and cytosolic calcium tran-sients in the dendrites of central neurons?

Our previous immunocytochemical studies showthat L-type calcium channels recogni zed bythe mono-clonal antibody MANC-1 are predominantl y localizedin cell bodies and proximal dendrites of hippocampalpyramidal cells and most other major classes of cen-tral neurons (Ahlijanian et al., 1990; Westenbroek etal., 1990, Figure 8A). Thus, it is unlikely that they arethe major calcium channels involved in generation ofcalcium-dependent action potentials in dendrites. Incontrast, our present results with an antibody thatrecognizes a specific subtype(s) of the w-CgTx-sensi-tive N-type calcium channel s demonstrat e that thischannel subtype is localized along the entire lengthof the dendrites of a wide range of neurons. The local-ization of these N-type calcium channels throughoutthe dendrite is complementary to the restricted local-ization of L-type calcium channels in cell bodies andproximal dendrites. A majority of dendrites in thebrain are immunoreactivefor CNB-1 antibodies. Fromthis subcellular distribution, we conclude that theo-CgTx-sensiti ve N-type calcium channels encodedby the rbB gene are important contributors to den-dritic action potential s and to voltage-gated calciuminflux into dendrites, whereas L-type calcium chan-nels primarily mediate calcium influx into proximaldendrites and cell bodies of most neurons.

In cerebellar Purkinje cells, we found an increaseddensity of CNB-I-immunoreacti ve calcium channelsat branch points in dendrites. A similar pattern of den-dritic staining was observed by Fortier et al. (1991)through detection of specifically bound w-CgTx withan anti-o-CgTx antibody. Thus, a locally high densityof o-CgTx-sensiti ve calcium channels may contributeto the generation and conduction of calcium-depen-dent action potentials at these branch points. Theimportance of dendritic branch points in calcium sig-naling in the Purkinje cell is also suggested by theobservation that the ryanodine-sensiti ve and inositoltrisphosphate-act ivated intracellular calcium releasechannels are localized in dendritic shafts and appearto be more abundant at main branch points of thedendritic arbor (Ellisman et al., 1990; Walton et al,,1991). The common sites of localization of cell surfaceand intracellular calcium channel s suggest that thedendritic branch points play critical roles in the calci-um-dependen t integration of dendritic signals.Although CNB-1 antibodies recognize primarilycal-cium channels in dendrites, a limited number of dis-crete subpopulati ons of neuronal cell bodies are alsoimmunoreactive. For example, the cell bodies of a


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Characterization of N-type Ca* Channel al Subunit1111

few pyramidal neurons in the dorsal cortex and mostPurkinje cells in the cerebellum are immunoreactivefor CNB-1 antibodies, whereas hippocampal pyrami-dal neurons possess relatively little CNB-1 immunore-activity. Physiological studies of calcium currents indissociated neurons detect o-CgTx-sensiti ve calciumchannels in PC12 cells, sympathetic ganglion neu-rons, dorsal root ganglion neurons, and many centralneurons (Reynolds et al., 1986; Hirning et al., 1988;Plummer et al., 1989; Usowicz et al., 1990; Regan et al.,1991). However, dissociation of these neurons causesbreakage and resorption of the proximal region ofthe dendrites into the cell body. Therefore, studies ofdissociated cells may overestimate the level of N-typecalcium channels present in the cell body, dependingon how much of the dendrites is resorbed. Alterna-tively, other N-type calcium channels not recognizedby CNB-1 may be localized more predominantly tocell bodies.

The distribution of calcium channels recogni zed byCNB-1 on dendrites and cell bodies of most centralneurons is uneven. This patchy distribution is similarto that observed by Jones et al. (1989) for fl uorescentlylabeled o-CgTx on hippocampal pyramidal cells indissociated cell culture. In that case, immobilizationof the o-CgTx-labeled calcium channels in the planeof the membrane was demonstrated by photobleach-ing experiments. The patchy distribution observed onneurons in vivo in our experiments may also be main-tained by immobilization of the channels t hrough in-teraction with cytoskeleton or other cellular compo-nents.N-type Calcium Channels at Synaptic Si tesIn addition to the uneven distribution of N-type cal-cium channel s along the surface of dendrites and cellbodies observed with CNB-1 antibodies, sharply de-fined punctate localizations were also detected at syn-aptic sites. A wide body of literature has providedevidence that N-type calcium channels are localizedin nerve terminals. o-CgTx was shown to inhibit neu-rotransmitter release at the neuromuscular junctionin frogs (Kerr and Yoshikami, 1984; Koyano et al., 1987;Sano et al., 1987; Crinnell and Pawson, 1989), ando-CgTx-sensiti ve N-type calcium channels have beenlocalized to the presynapti c active zones by fluores-cent labeling with rhodamine-label ed o-CgTx (Robi-taille et al., 1990; Cohen et al., 1991). o-CgTx alsoblocks the major presynaptic calcium channel in chickciliary gangl ion (Stanley and Coping, 1991). In con-trast, w-CgTx is much less effective at the neuromus-cular j unction of mammals (Sano et al., 1987; Wessleret al., 1990; Protti et al., 1991). o-CgTx inhibits voltage-dependent calcium flux and transmitter release in ratbrain synaptosomes (Reynolds et al., 1986; Woodwardet al., 1988; Xiang et al., 1990) and blocks release ofacetylcholine, substance P and calcitonin gene-related peptide from peripheral nerve terminals(Lundy and Frew, 1988; Seabrook and Adams, 1989;Maggi et al., 1990) as well as norepinephrine, vaso-

pressin, and acetylcholine from central nerve termi-nals (Miller, 1987; Dayanithi et al., 1988; Dutar et al.,1989; Lipscombe et al., 1989; von Spreckelsen et al.,1990). Our immunocytochemical results provide di-rect morphological evidence for the presence ofN-type channels in the nerve terminals forming syn-apses on many central neurons. We observed punc-tate staining that resembled nerve terminals along cellbodies and dendrites of cortical and hippocampal py-ramidal neurons, on cerebellar Purkinje cells, and onthe dendrites of many other classes of neurons. It islikely that these areas of punctate staining are locatedat synapses, and their appearance suggests a localiza-tion in the presynaptic terminal rather than the post-synaptic membrane of dendritic spines or shafts. Thisconclusion is clearest at the synapse bet ween themossy fibers of the dentate granule neurons and thehippocampal CA3 pyramidal cells. The punctate local-izations of CNB-1 immunoreactivity in the mossy fiberterminal field are unusually large and have the ex-tended morphology of the mossy fiber terminals.Moreover, the immunochemical reaction product fillsthe presynaptic terminals and reveals their orienta-tion and shape, allowing a clear identification. Elec-tron microscopic studies are currently in progress inorder to define the distribution of calcium channelswithin these nerve terminals at higher resolution, butthe conditions of fixation and processing of tissue forelectron microscopic examination have so far pre-vented specific immunocytochemical labeling by theCNB-1 antibodies.

The CNB-1 antibodies label only a small fraction ofnerve terminals in most brain regions. Since CNB-1antibodies immunoprecipitate less than half of solubi-lized o-CgTx receptors, other forms of N-type chan-nels that we do not detect may be located in the unla-beled nerve terminals. In addition, calcium channelsother than theo-CgTx-sensiti ve N-type forms are pres-ent in axon terminals and participate in neurotrans-mitter release that is blocked by spider venoms. TheseP-type calcium channels may also serve an importantrole in calcium influx and transmitter release at manycentral synapses (Hillman et al., 1991; Mintz et al.,1992; Uchitel etal.,1992;Venemaetal. , 1992). Determi-nation of the relative roles of calcium channel sub-types in release of different classes of neurotransmit -ters in distinct neuronal groups will be an importantstep toward understanding the specificity and regula-tion of transmitter release.Experimental ProceduresMaterialsTwo-month-old Sprague-Dawley ratswereobtained from Bantinand Kingman (Bellevue, WA). [3H]PN220-110 (3200 GBq/mmol)and [ZSl]o-CgTx (81.4 TBq/mmol) were purchased from New En-gland Nuclear-DuPont, digitonin from Gallard-Schlesinger, theECL detection kit for immunoblotti ng from Amersham, and pro-tein A-Sepharose, heparin agarose, wheat germ agglutinin&VGA), CNBr-activated Sepharose 4B, and 3,3diaminobenzi-dine tetrahydrochloride from Sigma. WGA was coupled to theCNBr-activated Sepharose according to the manufacturefs in-


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structions. Goat anti-rabbit IgC and rabbit PAP were obtainedfrom Zymed; the biotinylated anti-rabbit IgG and avidin D fluo-rescein were obtained from Vector. All other reagents were ob-tained from commercial sources.Partial Purification and lmmunoprecipitati on of NeuronalCalcium ChannelTo prevent proteolysis during the procedure, all steps were car-ried out as close to 0C as possible. Ultracentrifuge rotors wereprecooled on ice, and all procedures were carried out at 4OC oron ice. The following protease inhibitors were included in allbuffers: pepstatin A, leupeptin, and aprotinin (1 pglml); phenyl-methanesulfonyl fluoride (0.2 mM); benzamidine (0.1 mglml);and calpain inhibitor I and II (8 pg/ml each). For one preparation,typically 10 rat brain cortices and hippocampi were homoge-nized in 110 ml of 320 mM sucrose with a glass-Teflon homoge-nizer (12 strokes at - 1000 rpm). After a short centrifugati on (5000rpm, 2 min, SS 34 rotor) the supernatant (Sl) was incubated with370 kBq [3H]PN220-110 for 30 min in the dark. The l abeling al-lowed the convenient detection of L-type calcium channelsthroughout the purification. The membranes were pelleted(50,000 rpm, 35 min, 70. 1 Ti rotor) and solubilized with 170 mlof 1.2% digitonin, 300 mM KCI, 150 mM NaCI, 10 mM sodiumphosphate buffer (pH 7.4) for 20 min. The inclusion of 300 mMKCI increased thesolubilization efficiency of both PN220-IlOando-CgTx receptors from about 25% to about 50%-60%. Unsolubi-lized material was sedimented by centrifugation as before, andthe supernatant (S3) was slowly poured over a40 ml WGA-Sepha-rosecolumn (50 ml/hr). Littleor no bound [3H]PN220-110could bedetected in the flowthrough, indicating that the L-type calciumchannels are completely adsorbed by the WGA. I n contrast, 50%of thew-CgTx receptors were found in the flowthrough. This wasalso the case when the ionic strength of S3 was reduced by a2-fold dilution with 10 mM sodium phosphate buffer, when theresin was incubated with S3 for 1 hr, and when only one-thi rdof 53 was applied to the WCA column. These findings indicatethat only about 50% of the solubilized N-type calci um channelshave sufficient affinity for WCA under these experimental condi-tions to be effectively bound by the WGA-Sepharose column.The column was washed with 300 ml of 0.1% digitonin, 75 mMNaCI, 50 mM sodi um phosphate, 10 mM Tris-HCI (pH 7.4) at aflow rate of 150 mllhr, and bound calcium channels were elutedwith 100 mM N-acetyl-D-glucosamine in the same buffer at a flowrateof50mI/hr.Twomilliliterfractionswerecollected. PN220-110receptors were detected by scintillation counting of 30 ~1 sam-ples, and w-CgTx receptors by postlabeling and filtration assayswith [251]o-CgTx. The maximaof theelution peaks for both recep-tors overlapped exactly, but the w-CgTx receptor showed a broadtail, which could not be narrowed by increasing the N-acetyl-o-glucosamine concentration to 250 mM or by eluting at a lowerflow rate. These findings are indicative of a high affinity of theadsorbed fraction of N-type calcium channels to WGA. Up to 4ml from several calcium channel peak fractions was loaded ont o5%-20% (w/w)sucrosegradients(36mlcontainingO.l% digitoninand buffered with 10 mM Tris-HCI [pH 7.41) and centrifuged(45,000 rpm, 2 hr 15 min, 50V Ti rotor). Two milliliter fractionswere collected, frozen in liquid N?, and stored at 80C.Determination of [3H]PNZ20-1 10 and [251]wCgTx ReceptorsReceptor-bound [3H]PN220-110 was determined by direct scintil-lation counting of samples that were essentially free of unboundligand, such as the sucrose gradient fractions and WGA fractionseluted with N-acetyl-D-glucosamine. Receptor-bound [H]PN220-110 in samples containing free ligand was determined by thesame filter bindi ng assay as described for [*51]o-CgTx receptorsbelow.

To measure w-CgTx binding by the filtration assay, [251]w-CgTxwas added to 30-50 pl aliquots of sucrose gradient or columnfractions to give a final concentration of 1 nM for the 1251-labeledligand and incubated for 15 min at room temperature. Sampleswere put on ice for at least 5 min, 2 mg of bovine serum albuminwas added, and the samples were precipitat ed with 4 ml of ice-

cold 10% polyethylene glycol (average molecular weight, 8000)in 10 mM MgCI, , 10 mM Tris-HCI (pH 7.4) for 5 min. T he sampleswere filtered through Whatman CF/C filters, and the filters werewashed three times with 4 ml of the polyethylene glycol solutionand counted in a gamma counter.

To determine what percentage of the labeled channels wouldbe retained on the filters, S3 was incubated with [251]w-CgTx. Theunbound ligand was removed by WGA-Sepharosechromatogra-phy. Calcium channels were eluted with N-acetyl-o-glucosamineas described above, and aliquots were gamma count ed eitherdirectly or after polyethylene glycol precipitation. About 72% ofthe total amount of [ZSI]W-CgTx receptor could be detected onthe filters. All values obtained by filtration assays were correctedfor 72% efficiency of precipitation accordingly.lmmunoprecipitationFor immunoprecipitation, Sl was incubated with 3.7 kBq[*5l]CgTx for 30 min on ice, diluted IO-fold with phosphate-buffered saline (PBS; 0.05 M sodium phosphate, 150 mM NaCI,2.7 mM KCI [pH 7.4]), solubilized, and centrifuged as describedabove. Protein A-Sepharose was swollen and washed wit h PBS,incubated with either CNB-1 antiserum or a nonimmune serumfor 4 hr at room t emperature, and washed three ti mes with PBScontaining 0.1% digitonin. The protein A-Sepharose antibodycomplexwas added to an aliquot of the [1251]w-CgTx-labeled, solu-bilized calcium channel and incubated for 4 hr at 4OC. The com-plex was collected by centrifugation, washed three times with0.1% digitonin in PBS, and counted.lmmunoblotti ng of Calcium ChannelsTo concentrate calcium channels, 1 ml aliquots of different su-crose gradient fractions were incubated with 40 pl of heparinagarose, which bi nds neuronal L-type and N-type calcium chan-nels (Sakamoto and Campbell, 1991) for 2-3 hr at 4OC. The resinwas washed three t imes with 0.1% digitonin in PBS and extractedwith 40 ~1 of 3% SDS, 20 mM dithiothreitol, 10% sucrose, 125 mMTris-HCI (pH 6.8) containing protease inhibitors as describedabove, for 30 min at 50C~60X

After separation by SDS-polyacrylamide gel electrophoresis,the proteins were transferred onto nitrocellulose, which wasblocked by incubation for 2 hr with 1 0% skim milk powder inPBS (SM-PBS). Blots were incubated with affinity-purified CNB-1antibodies in 10% milk powder in 20 mM Tris, 0.15 M NaCl (pH7.4) (TBS) for 1-2 hr. The blots were washed with SM-PBS (fivechanges), incubated with horseradish peroxidase-protein A di-luted I:1000 in SM-PBS containing 0.05% Tween-20, washed for5-6 hr with 0.05% Tween-20 in PBS (eight to ten changes) anddeveloped with the ECL reagent.Synthetic PeptidesPeptide CNB-1 (KYRHHRHR DRDKTSASTP A) and CNB-2 (NNDR-DKTSASTPA), corresponding to residues 851-867 and 857-867,respectively, of the al subunit of rat brain N-type calcium chan-nel, plus N-terminal lysineand tyrosineor asparagineextensionswere synthesized by the solid phase method (Merrifield, 1963).The peptides were then purified by reverse-phase high pressureliquid chromatographyonaVydac218TPlOcolumn.Theidentityof the purified peptide was verified by amino acid analysis.Preparation of AntibodiesThe purified peptide CNB-1 was coupled through amino groupsto bovine serum albumin with glutaraldehyde (Orth, 1979), dia-lyzed against PBS, emulsified in an equal volume of Freundscomplete (initial injection) or incomplete adjuvant, and injectedin multiple subcutaneous sites on New Zealand Whi te rabbit sat 3 week i ntervals. Antisera were collected after the secondinjection and tested by enzyme-linked immunosorbent assay us-ing microtiter plates with wells coated with 0.5 pg of peptide(Posnett et al., 1988). Antibody CNB-1 was affinity-purified using2 pmol of the CNB-1 or CNB2 pepti de that was first coupled toCNBr-activated Sepharose. The antiserum (2.0 ml) was bound tothe column at room temperature for 5 hr and washed with TBS.


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Bound IgG was eluted with 5.0 M M&I, and dialyzed against TBSusing a Centriprep-30 (Amicon). Antibodies purified by affinitychromatography on CNB-1 or CNB2 give comparable results.lmmunocytochemistryTwelve or more adult Sprague-Dawley rats were used; all wereanesthetized with sodium pentabarbital and intracardially per-fused wit h a solution of 4% paraformaldehyde in PB (0.1 M so-dium phosphate [pH 7.41) containing 0.34% L-lysine and 0.05%sodium m-periodate (McLean and Nakane, 1974). The brainswere removed from the cranium and postfixed for 2 hr. Thebrains were then sunk i n successive solutions of lo%, 20%, and30% (w/v) sucrose in PB at 4OC over 72 hr. Sagittal and coronalsections (25-35 pm) were cut on a sliding microtome.

Free-floating sections were processed for immunoctyo-chemistry with the indirect PAP technique (Sternberger, 1979) orby immunofluorescence. Tissue samples processed usi ng thePAP techniquewere rinsed in PB for 16-24 hrand then pretreatedwith the following alcohol series: 70% ethanol for 5 min, 100%methanol with 0.3% HzOz for 10 min, and PB S for 1 hr. Sectionswere then incubated in affinity-purified CNB-1 (diluted 1:15) for1 hr at room temperature followed by 36 hr at 4OC. All antiserawere dil uted in PBS containing 0.1% Triton X-100, 1% normalgoat serum. The sections were processed as follows at roomtemperature, except when otherwise indicated: rinsed for 1 hrin PBS containing 0.1% Triton X-100, incubated in goat anti-rab-bit I gG diluted I:30 f or 1 hr at 37C, rinsed for 1 hr in PBS con-taining 0.1% Triton X-180, incubated in rabbit PAP diluted I:100for 1 hr at 37OC, rinsed in PBS for 15 min, rinsed for 5 min inPB, rinsed in TB (0.1 M Tris-HCI [pH 7.4]), treated with 0.04%3,3-diaminobenzidi ne and 0.003% HZ02 in TB for IO min, rinsedin TB for 10 min, and ri nsed in PB for 10 min. The sections weremounted on subbed glass slides, dehydrated, cleared in xylene,and coverslipped. Some sections were stained with MANC-1 us-ing the PAP method as described previously (Ahlijanian et al.,1990; Westenbroek et al., 1990).Free-floating sections t hat were processed for immunofluores-cence were first rinsed in PB for 10 min, then in PBS for 1 hr.Sections were incubated in affinity-purified CNB-1 antibody (di-luted 1:15) for 1 hr at room temperature followed by 36 hr at4OC. All antisera were dil uted in the same diluent as above. Thesections wer e processed as follows at room temperature, exceptwhen otherwi se indicated: rinsed for 1 hr in PBS containing 0.1%Triton X-100, incubated in biotinylated anti-rabbit IgG dilutedI:150 for 2 hr at 37OC, rinsed for 1 hr in PBS containing 0.1%Triton X-100, incubated in avidin D fluorescein diluted I:150 for2 hr at 37C, rinsed in PBS for 15 min, and finally rinsed in PBfor I5 min before being mounted and coverslipped using Bio-media gel mount.AcknowledgmentsThis work was supported by grant PO1 NS20484 (PhilipSchwartzkroin, P. I.) from the National Institutes of Health, by agrant f rom the Office of Naval Research (Peter Schwindt, P. I.) toW. A. C., by the W. M. Keck Center for Advanced Studies ofNeural Signaling at the University of Washington, and by grantsfrom the Medical Research Council of Canada and the HowardHughes Medical institute I nternational Research Scholar Pro-gram to T. P. S. T. P. S. is a recipient of a fellowship from theAlfred P. Sloan Research Foundation, and J. W. H. is a recipientof a postdoctoral fellowship from the Deutsche Forschungsgem-einschaft. We thank Ms. Renee Costello for excellent technicalassistance. Peptides were synthesized in the Molecular Pharma-cology Facility, University of Washington. We thank Ms. Anita A.Colvin and Dr. Karen S. De Jongh for assistanceand expertise inpeptide synthesis. Confocal microscopy was carried out in theImaging Facility of the W. M. Keck Center for Advanced Studiesof Neural Signaling. We thank Ms. Paulette Brunnerfor excellenttechnical assistance.

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