Journal of Neuroscience Research 32:593-404 (1992)

Isolation and Characterization of Periaxolemmal and Axolemmal Enriched Membrane Fractions From the Rat Central Nervous System V.S. Sapirstein, R. Durrie, B. Cherksey, M.E. Beard, C.J. Flynn, and I. Fischer Division of Neurobiology, The Nathan Kline Institute, Orangeburg (V.S.S., R.D., M.E.B., C.J.F.), Departments of Psychiatry (V.S.S.) and Physiology and Biophysics (B.C.), New York University Medical School, New York, New York; Department of Anatomy and Neurobiology, Medical College of Pennsylvania, Philadelphia (I.F.)

In this report, we describe the fractionation of crude axolemmal fractions from rat lower brainstem into subfractions enriched in markers for either periaxolemmal myelin or axolemma. These subfractions were isolated on density gradients as bands layering on 0.8M and 1.OM sucrose. Both subfractions consisted of unilamellar vesicles. Relative to myelin purified from the same starting material, the 0.8M subfraction was enriched in MAG, CNPase, carbonic anhydrase and Na+, K + ATPase but was extremely low in PLP and MBP. In addition, this fraction exhibited a protein profile distinct from myelin. The 1.OM fraction was also highly enriched in Na+, K + ATPase and had an overall composition similar to the 0.8M subfraction. However, it differed from the 0.8M subfraction by being low in MAG, CNPase, and carbonic anhydrase, but enriched in voltage-dependent Na+ channel, axon-specific fodrin, and MAP1B. Based on these characteristics we concluded that the 0.8M and 1.OM subfractions were highly enriched in periaxolemmal myelin and axolemmal membrane, respectively. Plasmolipinlo was unique with equally high levels in myelin and in the 0.8M and 1.OM subfractions. Both subfractions were enriched, relative to myelin, in the alpha subunit of the GTP binding protein, Go, and the alpha subunit common to all G proteins, GAll. Electrophysiology with membrane subfractions fused to lipid bilayers showed that both membranes contained sets of K + and CI- channels, which based on channel sizes and open times, are largely distinct from one another. 0 1992 Wiley-Liss, Inc.

Key words: myelinated axon, plasmolipin, periaxolemmal myelin, axolemma and ion channels INTRODUCTION In the past two decades there has been increasing appreciation that myelin is not an inert insulator of 0 1992 Wiley-Liss, Inc.

the axon, but based upon enzyme activities found in myelin, may in part be a participant in dynamic processes which control the function of myelinated fibers.7,9,12,19,24,28,32,42,45 Moreover, the identification of periaxolemrnal and paranodal myelin as the site of MAG and C N p a ~ e , ~clearly ' demonstrate that these domains represent a specialized region of the myelin complex. The concept of control of neuronal (axonal) excitability by adjacent myelin and oligodendroglial membrane is reinforced by recent observations of ion channels, in particular K + channels,3341in oligodendroglial plasma membrane, in uni- and bilamellar vesicles prepared from bulk m ~ e l i and n ~ ~in paranodal m ~ e l i n . ~ ~ Plasmolipin, a major oligodendrogliaYmyelin intrinsic membrane protein"734 has been shown to form K t channels in lipid bi1aye1-s~~ and also may be involved in myelin-axonal interactions. The presumed specialization of the periaxolemmal compartment suggests this region of the myelin complex possesses constituents specifically localized there for the purpose of influencing the function of the axon. In this study, we report a method for isolating enriched fractions of periaxolemmal myelin and axolemma, and describe initial studies on their biochemical and functional characterization. We demonstrate that periaxolemmal myelin

Received November 23, 1991; revised and accepted March 2, 1992. Address reprint requests to Dr. V.S. Sapirstein, Division of Neurobiology, Nathan Kline Institute, Orangeburg, NY 10962. Abbreviations: BTX, batrachotoxin; CNPase, 2'-3' cyclic nucleotide phosphohydrolase; EDTA, ethylenediamine tetra acetic acid; EGTA ethylene glycol bis(amino-ethyl-ether) N,N,N',N'-tetra acetic acid; G protein, GTP(guanosine triphosphate) binding protein; Hepes, 4-(2hydroxyethy1)-1-piperazinethanesulfonicacid; MAG, myelin associated glycoprotein; MAP, microtubule associated protein; MBP, myelin basic protein; Na+, K + ATPase, Na+ and K + stimulated adenosine triphosphatase; PLP, myelin proteolipid protein; SDS sodium dodecyl sulfate; TES, N-tris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid.

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step was included to preclude residual myelin contaminants, although usually little material was observed on this layer. The material on the 0.8M and 1.O M sucrose layers were defined as the 0.8M and 1.OM subfractions. These membranes were diluted in deionized water and centrifuged at 15,OOOg for 20 min to remove any small membrane contaminants. The extrinsic membrane proteins of these fractions were identified by treatment of the membranes with Na,CO,, as described p r e v i ~ u s l y , ~ ~ and the extracted proteins precipitated with 10% cold trichloroacetic acid. METHODS Protein determinations were performed on SDS Membrane Fractionation solubilized membrane preparations using the dye binding Myelin, periaxolemmal , and axolemmal enriched method of Bradford' with reagents from BioRad. All fractions were purified from rat lower brainstem dis- additional reagents were ultra-pure or molecular biologsected free of visible grey matter. These membrane frac- ical grade and were purchased from either BRL (Gaithtions were all isolated from the same preparation by a ersberg, MD) or Sigma Chemical Co. (St. Louis, MO). modification of the procedure of Detskey et al.13 originally formulated for the isolation of crude axolemmal Electron Microscopy Membrane fractions were collected by centrifugaenriched fractions. The modifications include the use of HEPES instead of TES buffer, the initial centrifugation tion and fixed for 60 min at 4°C in 2% glutaraldehyde step was in 0.9M sucrose (and was used as crude myelin) (Electron Microscopy Sciences, Ft . Washington, PA) rather than 1.OM and that after the first centrifugation and 2% p-formaldehyde (Polysciences, Wamngton, PA) step the floated membranes were rehomogenized in in 0.1M sodium cacodylate buffer, pH 7.4. Following 0.85M (BRL, Gaithersberg, MD) sucrose and recentri- fixation, the material was rinsed three times over 30 min fuged. The resultant floating membrane pads were in 0.1M sodium cacodylate, pH 7.4, containing 5.0% washed in 10 mM HEPES, pH 7.4, containing 5 mM sucrose, post-fixed in 2% osmium tetroxide (Electron EDTA. The membranes were then osmotically shocked Microscopy Sciences) in water, dehydrated through a in this buffer for 1.5 hr. The overnight discontinuous graded series of ethanols and propylene oxide, and emdensity gradient centrifugation step described in the orig- bedded in epoxy resin, LX-112 (Ladd, Burlington, VT). inal procedure was carried out on a gradient of 15, 24, Sections of embedded material (50-70 nm thick) were 28, 32, and 37% sucrose. The sucrose concentration is obtained using an MT-6 Ultramicrotome (RMC, Tucson, reported here as percent order to facilitate comparison AZ) equipped with a diamond knife and viewed after with the original procedure. The sucrose percent was staining with uranyl acetate, in a Philips CM-10 microdefined by density determined using a refractometer scope operating at 60-80 kV. (Fisher Scientific, Pittsburgh, PA). The myelin, sedimenting at the 15-24% sucrose Gel Electrophoresis and Western Blot Analysis All electrophoretic analyses were carried out acinterface, and crude axolemma, sedimenting at the 2832% sucrose interface, obtained by this procedure were cording to the procedure of Laemmli" using either a purified further. The myelin was osmotically shocked 4-18% acrylamide gradient or 14% isocratic gels. Acwith distilled water and sedimented twice at 12,OOOg in rylamide and bis-acrylamide were obtained from Naa Beckman Instruments (Wakefield, MA) Model 52-21 tional Diagnostics (Mannville, NJ) while all other eleccentrifuge to remove residual microsomal contaminants trophoretic reagents were purchased from BioRad and layered over 0.75M sucrose. The material was cen- (Riverside, CA). Molecular weight markers were prestrifuged in a SW-28 rotor at 75,008 for 1.5 hr in a Beck- tained standards from BRL: myosin heavy chain, 200K; man L7-55 ultracentrifuge. The band floating on 0.75M phosphorylase b, 97.4K; bovine serum albumin, 68K; ovalbumin, 43K; chymotrypsinogen, 25.7K; a-lactoglosucrose was taken as purified myelin. The axolemmal subfractions were purified from bulin, 18K; and lysozyme, 14.3K. crude axolemma after its lysis for 30 min in 5 mM TrisPreparation and scanning of Western blots were HC1, pH 8.3, containing 0.1 mM EDTA. The lysate was as previously described.33 Polyclonal antibodies to made isotonic by the addition of 1/4 volume of 40% Na+ ,K ATPase (used at 1:1,000) were a generous gift sucrose and was layered over 0.65M, 0.8M, and 1.OM of Dr. W. Stahl, University of Washington. Polyclonal sucrose and centrifuged at 75,OOOg for 1.5 hr. Since antibodies to MAG (used at 1:1,000) were provided by most myelin fractions have densities less than 0.65M this Dr. Colman, Columbia University; anti-CNPase mono-

is the myelin region to which proteins known to be important in dynamic membrane function, such as G proteins, Na + K ATPase, and carbonic anhydrase, are localized. Our method also allows for the isolation of an axolemmal enriched fraction which is distinct from the periaxolemmal myelin membrane. Lastly, we have adapted a modified patch clamp/lipid bilayer technique for the initial analysis of ion channel activity in the isolated membranes. +

+

Isolation of Axolemmal Fractions From Rat CNS

clonal antibodies (used at 1:lOO) were a gift of Dr. T. Sprinkle, VA Medical Center (Augusta, GA), and polyclonal antibodies to PLP (used at 1500) were provided by Dr. M. Lees of the Eunice Kennedy Shriver Center (Waltham, MA). Polyclonal antibodies to MAP- 1B were used at 1:1,000 and were prepared as previously described. l5 Antibodies to carbonic anhydrase (used at 1:500) were a gift of Dr. Wendy Cammer, Albert Einstein College of Medicine, Bronx, NY. Antibodies to axon-specific fodrin (used at 1500) were provided by Dr. Steven Goodman of the University of Southern Alabama (Mobile, AL). Polyclonal antibodies to the Na+ channel (used at 1:1,000) were a gift of Dr. K. Angelides, Baylor University School of Medicine (Houston, TX). Monoclonal antibodies to MBP (18 kDa) were purchased from Chemicon (Temecula, CA) and used 1:500. Plasmolipin polyclonal antibodies were prepared and used as described previously. 14333 Antibodies to the alpha subunit of the GTP binding protein, Go, and to the alpha subunit common to all GTP binding proteins, GA/1, were obtained from Dupont-New England Nuclear and were used at 1:1.000.

595

ductances), and 200 mM NaCl (for Naf conductances). Total-event histograms of K conductances were prepared using pClamp software. Events were sampled at 10,00O/sec. and approximately 1 million events used to construct each histogram. Na+ channel analyses were facilitated by extending channel open times by the addition of BTX (at a final concentration of 120 nM), along with the membrane ves’ was a generous icles, to the cis side of the b i l a ~ e r . ~BTX gift of Dr. John Daly of the Laboratory of Bioorganic Chemistry, NIDDK (Bethesda, MD). Channel conductances are reported in pico-siemans (pS) calculated as p s = pA/v. +

RESULTS Electron Microscopic Analysis We have isolated membrane subfractions of crude axolemmal enriched fractions and assessed them by electron microscopy. The subfractions are defined by the sucrose concentration on which they were reisolated, i.e., 0.8M and 1.OM (see Methods). The fractions were routinely examined by electron microscopy and both Electrophysiology were found to be comprised of unilamellar membranes In experiments involving electrophysiologic meaand vesicles (Fig. 1). Although a rare myelin profile was surements the initial homogenate contained 5 mM pobserved, the micrographs indicated a very low level of mercaptoethanol. Preliminary experiments indicated this contamination by compact myelin and no observable miwas necessary to retain active ion channels. Electrophystochondria. Approximately 8 g wet weight of starting iologic analysis of ion channel activity was carried out by material yielded between 0.4 and 0.8 mg of protein of the fusion of membrane subfractions, resuspended in each subfraction. The yield of myelin from this amount 0.4M sucrose, with lipid bilayers coating the surface of of tissue was about 30 mg. glass pipettes.” Membrane suspensions were added to the cis side of the bilayer formed on the surface of the patch clamp pipette. The final concentration of the mem- Electrophoretic Analysis brane subfractions on the cis side of the bilayer was The electrophoretic profiles of the subfractions approximately 13 pg/ml. Micropipettes with a tip diam- were obtained and compared with that of isolated myeeter of approximately 1 p m (Corning 7052 glass, Corn- lin, (Fig. 2A,B). The 0.8M and 1.OM subfractions coning NY) were pulled using a Kopf Model 720 Electrode tained numerous bands in the 30-100 kD range that were Puller (Tujunga CA). Lipid bilayers were formed using a low or absent from myelin, while the major myelin bands 1: 1 mixture of phosphatidyl ethanolamine and phosphati- in the 14-25 kD region of the gel corresponding to PLP dyl serine (Sigma Chem. Co., St. Louis, MO.) using the and the MBPs were greatly reduced. Although the pro“tip-dip” technique of Coronado and Latorre. l 1 Volt- tein profiles of the 1.OM and 0.8M subfractions were ages were applied via an Axon Instruments (Foster City remarkably similar, several high molecular weight bands CA) A12120 patch-clamp amplifier with a lOOGOhm were only present in the 1.OM fraction. To determine to head stage. The bathing solution was held at ground. The what extent these high molecular weight bands repredata obtained in the channel studies were amplified to sented neuronal cytoskeletal proteins of similar molecu100 mV/pA and the membrane current was recorded on lar weights, we first assessed whether these were part of a Neurocorder DR-384 (NeuroData, New York) for sub- the extrinsic protein network (Fig. 2B, lane 3) The resequent analysis. The data were filtered at 1 kHz and sults indicated that proteins whose molecular size were digitized at a rate of 25,000 samples/sec. The digitized similar to those of fodrin and MAP-1B are quantitatively records were then analyzed by computer using the removed by Na,CO, and could be recovered from this pClamp program (Axon Instruments). Experiments were extract, lane 4. Their identification as fodrin and MAPperformed using symmetrical bathing solutions of 150 1B was confirmed by Western blot analysis (see Fig. 6). mM of KCl (for K f conductances), CsCl (for C1- con- The identification of N a f , K + ATPase (Fig. 4A) and

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and MBP were very low in the two subfractions and were often barely detectable at the membrane levels tested making relative quantitation of these proteins in these fractions difficult. However, based upon data using higher membrane concentrations (data not shown) we estimate that the levels of these proteins in the 0.8M subfraction would indicate compact myelin contamination to be less than 10%.

Fig. 1, Electron microscopic analysis of membrane subfractions. Electron microscopy was carried out as described in Methods. A: The 0.8M subfraction. B: The 1.OM subfraction. X 8.000.

actin and tubulin were also determined by Western blot analysis, data not shown. The distribution of specific proteins in the 0.8 and 1.OM subfractions and the myelin from which these membranes were isolated were determined by Western blot. The low level of many marker proteins in individual fractions made it difficult to obtain relative quantitative assessments of all the proteins studied. However, estimates for selected markers are given as relative area units from scans of Western blots. A complete summary of the Western blot data presented in this section is in Table I.

Distribution of Compact Myelin Proteins The distribution of compact myelin-associated proteins in purified myelin and the 0.8M and 1.OM subfractions was assayed by determining PLP and MBP levels by Western blot analysis. The 0.8M and 1.OM subfractions appeared to contain little of these proteins (Fig. 3A,E) compared to the myelin fraction. The level of PLP

Distribution of Periaxolemmal Myelin Proteins In contrast to the compact myelin markers, analysis of MAG, a periaxolemmal myelin protein41 (Fig. 3C), showed a very different pattern with the 0.8M subfraction being the site of enrichment in this protein. Unlike many other markers, ie. PLP and MBP, which made relative quantitation difficult, MAG levels were more easily assessed (Fig. 4A,B). Lanes 1 and 2 in Figure 4B represent the MAG level in 20 pg of two preparations of the 0.8M subfractions prepared in parallel along with the scans of these Western blots (Fig. 4A). The integrated area of the scans were 966-975 relative area units. Twenty micrograms of myelin, lanes 3 and 4, gave a much weaker (and variable) signal with an integrated area of the corresponding scans in 4A giving values of 133-180. Analysis of lesser amounts (10 pg) of the 0.8M subfraction, lanes 5 and 6, and analysis of the corresponding scans gave an integrated area of 520-544 units. Comparison of the MAG in 20 pg of the myelin fraction with 20 pg of the 0.8M subfraction gave an enrichment of this protein in the subfraction of 6.25-fold while comparison of the myelin with 10 pg of the 0.8M subfraction gave an enrichment of 6.86-fold. The lower enrichment when compared with 20 p g of the 0.8M sample probably suggests we are close to saturation of the Western blot signal in this sample. The nearly 7-fold enrichment of MAG in the 0.8M subfraction relative to myelin represents a twofold increase in the degree of enrichment compared to crude myelin (Fig. 5), which was only 3-3.5-fold. Comparison of 10 and 20 pg of the 0.8M subfraction, lanes 1 and 2 (relative area units of 210 and 405, respectively) with 25 and 50 pg of crude myelin, lanes 3 and 4 (relative area units of 173 and 316, respectively) indicate a 33.5-fold enrichment of MAG in the 0.8M subfraction compared to crude myelin. The one-half lower enrichment in MAG relative to crude myelin suggests that onehalf of the MAG signal has been removed in purifying myelin. Based on the relative signal of MAG in the 0.8M fraction and crude myelin and the level of total protein recovered in these fractions (35 mg and 0.6 mg in the crude myelin and 0.8M fraction, respectively), it appears that about 20% of MAG has been recovered in this unilamellar fraction.

Isolation of Axolemmal Fractions From Rat CNS

B.

A.

1

2

Mr X

1 2

3

597

lo''

4

Fig. 2. Electrophoretic profile of myelin and unilamellar subfractions. Electrophoresis was carried out on a 4-18% acrylamide gradient gel using 50 p g protein per sample. A: Two examples of myelin isolated as described in Methods. B: lane 1, the 0.8M subfraction; lane 2, the 1 .OM subfraction; lane 3,

intrinsic membrane proteins after extraction of the 1 .O subfraction with Na,CO,; lane 4, TCA precipitate of Na,CO, extracted proteins. Lines to the left of lane 3 and to the right of lane 4 correspond to proteins apparently enriched in the intrinsic and extrinsic protein pools, respectively.

The oligodendroglial plasma membrane and putative periaxolemmal-paranodal my elin marker , CNPase4' (Fig. 3D) gave a distribution similar to that found for MAG with an enrichment in the 0.8M subfraction of close to sixfold (relative area units in myelin and the 0.8M subfraction were 173 and 1,021 , respectively). We also determined the distribution of the oligodendroglial and myelin protein, plasmolipin. We found that, unlike the marker proteins examined above, plasmolipin did not preferentially segregate into any of the membrane fractions (Fig. 3F, Table I). To determine the levels of plasmolipin more quantitatively, we probed Western blots of plasmolipin in the axolemmal fraction and compared it to a plasmolipin standard curve as previously described.33 Based on this analysis (data not shown) plasmolipin was found to represent close to 5% of the total membrane protein in this fraction, which compares favorably to that previously reported for plasmolipin in myelin" and confirms the data in Figure 3F, indicating equivalent amounts of this protein in axolemma and in myelin.

Distribution of Plasma Membrane Proteins To further analyze the composition of the 0.8M and 1.OM subfractions, we also examined the general plasma membrane marker Nai , Kt ATPase, and the oligodendroglial specific, membrane-bound carbonic anhydrase. The Na+ , K + ATPase level was high in both subfractions but low in purified myelin (Fig. 3A). In contrast, the oligodendroglial specific protein, carbonic anhydrase, was about three fold higher in the 0.8M subfraction (Fig. 6A) compared to myelin and 5.5-fold higher than the 1.OM subfraction with respective area units of the scans being 724, 218, and 131. Distribution of Neuronally Enriched Proteins To assess the contribution of neuronal membrane to the 1.OM and 0.8M subfractions, we determined the distribution of neuronally enriched (presumably axolemmal) proteins. We examined the sodium (Na+) channel and two neuronally enriched cytoskeletal proteins, axonspecific fodrin and MAP-1B. The data clearly show that

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TABLE I. Distribution of Protein Markers in Subfractions of the Myelinated Axon Membrane fraction"

Myelin PLP MBP MAG CNPase Carbonic Anhydrase N a +K + ATPase Na+ Channel MAP- 1B Fodrin Plasmolipin Go GA/1

++++ ++++ + + ++ +

* * -t

++++ + +

0.8M subfraction

1 .OM subfraction

+

+ t +

?

++++ ++++ ++++ ++++

++ + + ++++ ++++ ++++

A.

+

+ ++++ ++++ ++++ ++++ ++++ ++++ ++++

"Highest concentration is defined as + + + + ; moderate level is defined as + + , low level is defined as + , barely detectable levels are defined as +.

the two cytoskeletal proteins (Fig. 6B,C) were very enriched in the 1 .OM subfraction compared to either myelin or the 0.8M subfraction. The Na+ channel showed a distribution similar to fodrin and MAPlB with the highest concentration found in the 1.OM subfraction; the 0.8M subfraction was markedly reduced in this protein and myelin appeared to have very low levels of this protein (Fig. 6D). We used MAPlB to assess the recovery of the 1.OM subfraction from crude myelin (Fig. 7A,B), as we had done for MAG. The data indicate that MAPlB is enriched approximately 12-fold in the 1.OM fraction compared to crude myelin (42 relative area units compared to 5 13 area units). If we assume the recovery of the axolemmal fraction is as high as 0.8 mg protein and the crude myelin is about 30 mg then this 12-fold enrichment represents about a 30% recovery of this MAPlB containing fraction from the crude myelin.

Additional Characteristics of Membrane Subfractions The myelin complex has been shown to contain several GTP binding proteins (G proteins).6 To obtain a more refined view of the distribution of these proteins in the myelin complex, we have analyzed two members from this class of proteins. The G protein complex consists of several classes of subunits including that class which is defined as the alpha subunits which itself is heterogeneous. Two prominent members of this class is the alpha subunit specific for Go and the alpha subunit common to all G protein complexes, G N l . We found significant reactivity towards antibodies to both of these proteins in the periaxolemmal myelin (0.8M) and axo-

C. D.

E.

F. 2

3

Fig. 3. Composite Western blot analysis of membrane marker proteins. Electrophoresis was carried out on a 4-18% gradient acrylamide gel. The separated proteins were transferred to nitrocellulose and the levels of individual proteins analyzed as described in Methods using specific antibodies as defined in A-F. Lane 1, myelin; lane 2,0.8M subfraction; lane 3, 1.OM subfraction. A: PLP; lane 1 (5 pg protein), lane 2 (20 pg protein), and lane 3 (20 pg protein). B: NafKfATPase, 25 pg protein per sample; C, MAG, 25 pg protein per sample; D, CNPase, 25 pg protein per sample; E, MBP, 3 pg of myelin protein, and 10 pg of protein from the individual subfractions; F, plasmolipin, 2 pg of protein per sample.

lemma1 (1 .OM) enriched fractions (Fig. 8A,B). However, the levels of Go and GA/l were extremely low in the myelin from which these membranes were isolated (Fig. 8A,B).

Isolation of Axolemmal Fractions From Rat CNS

599

A.

A. B.

A

C.

MAG

B.

1

2

3

4

5

6

D.

Fig. 4. Western blot analysis and quantitation of MAG in myelin and periaxolemmal myelin. MAG was analyzed by Western blot as described in Methods and the blots scanned as described previou~ly.~~ A: The three scans correspond to the data illustrated in lanes 1, 3, and 5 in B. B: Lanes 1 and 2 are Fig. 6. Composite Western blot analysis of carbonic 2o pg Of protein Of periaxolemmal lanes and are 2o drase and selected neuronal proteins. Electrophoresis was carand lanes and are I*g Of protein ried Out O n a 4-18% acrylamide gradient gel using 25 pg % of protein of periaxolemmal myelin. protein in A and 50 pg of protein per sample in B-D. Lane 1, myelin; lane 2, 0.8M subfraction; lane 3, 1 .O subfraction. A: Carbonic anhydrase. B: MAP-1B. C: Fodrin. D: Na+ channel.

1

2

3

''

MAG

1 2

3 4

Fig. 5 . Comparison of MAG in periaxolemmal myelin and crude myelin. Lanes 1 and 2 represent 10 and 20 pg protein of periaxolemmal myelin; lanes 3 and 4 represent 25 and 50 pg of protein of crude myelin prepared as described in Methods.

Electrophysiology We have begun to define the biological activity of the subfractions by characterizing the ion channel activity present in the membranes by a modified patch clamp/ lipid bilayer technique as described in Methods. Totalevent histograms prepared from both the periaxolemmalenriched and axolemmal-enriched fractions indicated that the K conductance population differences exist in these two membrane fractions (data not shown). Specifically, fusion of the periaxolemmal myelin fraction with the lipid bilayer was accompanied by ion channel-like conductance events (Fig. 9A). In the presence of symmetrical KCl solutions two easily distinguishable channel-like K + conductances with amplitudes of 30 pS (i) and 100 pS (ii) were observed and a third, less prevalent +

one, 50 pS (iii). Most single channel events were rapid with mean open times of 1-2 msec. However, the 100 pS conductance events exhibited longer open times of up to 15 msec (iv). The axolemmal subfraction gave a decidedly different pattern (Fig. 9B) with most of the K + channel activity predominated by 100 pS (i), 150 pS (ii), and 180 pS (iii) channel openings with occasional 30 pS (iv) openings. Both membranes appeared to express a K + channel of about 100 pS; however, unlike the periaxolemmal membrane none of these axolemmal events exhibited the 15 msec extended open times. Even more striking were the differences found for the C1- channels in the two subfractions (Fig. 10A,B), measured with symmetrical CsCl solutions. The axolemma1 subfraction (Fig. 10B) contained a C1- conductance of 30 pS (i) and exhibited a mean open times of 10 msec (i) and 30 msec (ii). The C1- channels in the periaxolemma1 fraction (Fig. IOA) were larger and appeared to consist of four types having amplitudes of 100 pS (i) and 180 pS (ii) as well as the 30 pS (iii) channel seen in the axolemmal subfraction. An occasional opening of 220 pS (iv) was also observed. On the basis of Western blot analysis, the axolem-

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4

Mr x 10”

!I

A.

8

+43 Go

+30

1

2

3

4

B. +43 !L /

GAII

J B.

+30 1

MAP?b

1

2

Fig. 7. Western blot analysis and quantitation of MAPlB in axolemma and crude myelin. MAPlB analysis was as described in Methods. A: Scans of Western blots illustrated in B . B: Lane 1 is 50 pg of protein of the axolemmal subfraction and lane 2 is 50 pg of crude myelin prepared as described in Methods.

ma1 subfraction was enriched in the Na+ channel. We have confirmed the existence of Na+ channel activity in the axolemmal enriched fraction by electrophysiologic measurement. Addition of axolemmal enriched membrane vesicles to the lipid bilayer system in the presence of 200 mM NaCl, but the absence of BTX, failed to induce measurable channel activity. However, addition of BTX to the bath (120 nM, final concentration) extended the channel opening times to 1-2 msec, allowing detection of Na+ conductance changes (Fig. 11). At holding potentials of 40 mV or higher, voltage-dependent Na+ channels were predominated by single channel conductances in the 25 pS range as has been reported previously from membrane fusion studies for the BTX sensitive Na+ channels.6 Analysis of periaxolemmal myelin under the same conditions showed this Na+ channel activity is also present, but although these data are not quantitative, the frequency of activity appeared to be much reduced (data not shown).

2

3

4

Fig. 8. Western blot analysis of GTP binding proteins. Membrane proteins were separated by electrophoresis on 4-18% polyacrylamide gels and transferred to nitrocellulose as described in Methods and analyzed using antibodies to the alpha subunit of Go and to the common alpha subunit, GAI1. A: Analysis of Go alpha. B: Lane 1 is myelin; lane 2 is the 0.8M subfraction; lane 3 is the 1.OM subfraction, and lane 4 is the crude axolemma prior to subfractionation. All fractions were analyzed using 25 pg of protein. B: Analysis of GA/l. Lane 1 is myelin; lane 2 is the 0.8M subfraction; lane 3 is the 1.OM subfraction; and lane 4 is the crude axolemma prior to subfractionation. All fractions were analyzed using 25 pg of protein.

DISCUSSION The purpose of this study was to obtain, from crude myelin, relatively pure fractions of periaxolemmal myelin and axolemma so as to identify the loci of biological activities in the myelinated axon of the CNS. Subfractionation studies such as these never yield truly purified fractions, however, the data presented here indicate that membrane fractions have been obtained which are highly enriched in marker proteins representative of these membrane domains. When one examines the distribution of classic compact myelin proteins such as PLP and MBPs, it is clear that they are essentially present only in our “pure” myelin fraction and are extremely low in the periaxolemmal and axolemmal subfractions . In contrast, periaxolemmal and paranodal markers are effectively removed from myelin and segregate into the 0.8M subfraction which we have designated periaxolemmal-enriched.

Isolation of Axolemmal Fractions From Rat CNS

601

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I S""..,

Fig. 9. Potassium channels in periaxolemmal and axolemmal enriched membrane fractions measured in vitro. Changes in K conductance were monitored across lipid bilayers bathed by symmetrical KCI (150 mM) as described in Methods. Currents in pic0 amps were recorded at -50 mV holding potential with the bath (cis) held at ground. The unit conductances were calculated as reciprocal ohms (pS) and were defined as pS = QAIV. A: Conductance changes induced by the fusion of the 0.8M subfraction with the lipid bilayer. B: Conductance changes induced by the fusion of the 1.O subfraction with the lipid bilayer. The designation of specific events (i-iv) are as defined in the text.

A.

I 2Snn.c

Fig. 10. Chloride channels in periaxolemmal and axolemmal enriched membrane fractions. Changes in C1- conductances were monitored as described in Figure 9 except the lipid bilayer was bathed in symmetrical solutions of CsCl (150 mM). Currents in pA were recorded at -50 mV holding potentials. A: The conductance changes induced by the fusion of the 0.8M subfraction with lipid bilayer. B: The conductance changes induced by the fusion of the 1.OM subfraction with the lipid bilayer. The designation of specific events (i-iv) are as defined in Results. Similarly, neuronal proteins, which from white matter should represent axonal proteins, are relatively unique to our 1.OM fraction, defined as axolemmal enriched.

Fig. 11. Identification of BTX sensitive Na channels in the axolemmal enriched membrane fraction. Conductance changes in pA were obtained as described in the legends to Figures 9 and 10 except that the symmetrical bathing solutions was 200 mM NaCl. BTX sensitive sodium channels were obtained by addition of the toxin simultaneously with the membrane vesicles to the cis side of the bilayer at a final concentration of 120 nM .

These data are summarized in Table I. The designation of relative levels in this table are not meant to be quantitative but are presented to summarize the distribution of proteins and their relative enrichment in myelin and the two unilammelar subfractions . Based on these findings we propose that the method provides for highly enriched fractions of compact myelin, periaxolemmal myelin, and axolemma. By analyzing the distribution of various proteins among these fractions one may be able to assign a localization to less well defined species. Two applications of this method are our findings on the distribution of carbonic anhydrase and GTP binding proteins. GTP binding proteins transduce receptor mediated events, including the generation of second messengers and the direct control of ion channel a ~ t i v i t ywhile , ~ carbonic anhydrase is considered important in controlling ionic equilibria (see below). The localization of these proteins to the periaxolemma1 domain are consistent with the view that this membrane region is the site of dynamic activity within the myelin complex. It should be noted, however, that although carbonic anhydrase is much higher in the periaxolemmal fraction than myelin the degree of enrichment is only about one-half that seen for the MAG and CNPase. This suggests that it is not restricted to the periaxolemmal domain but is present throughout the myelin complex. Plasmolipin is present in both the periaxolemmal and axolemmal fractions and has been shown to form voltage dependent K + channels in ~ i t r with o ~ ~single channel conductances in the 80-100 pS range. Although we can not as yet ascribe any of the K + conductances reported in the present study to plasmolipin, both periaxolemmal and axolemmal subfractions possess K channels of this size. Plasmolipin was unique among the proteins analyzed in that it showed no preferred distri+

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bution within the membranes from the myelin complex. It should be noted that only oligodendrocytes express this protein34 and the presence of plasmolipin in axolemmal fractions may reflect the movement of this protein from the oligodendroglial/myelin to the neuronal compartment. The high level of plasmolipin in white matter coated vesicles,35 in which most other myelin proteins are absent may be evidence that an endocytic event incorporates a select periaxolemmal domain into the axon as has been observed in electron microscopic studies.26337 The 1.OM subfraction, which we are designating axolemmal enriched, appears to be low in myelin and periaxolemmal myelin markers. On the other hand, there appears to be an enrichment in this fraction of the predominantly neuronal proteins, Na channels, axonspecific f ~ d r i n and , ~ ~the neuron-enriched cytoskeletal protein MAP-1B .36 This fraction, like the periaxolemma1 myelin, appears to be enriched, relative to myelin, in G proteins and also possesses K + and C1- channel activity. An important feature of the axolemmal fraction was the apparent enrichment of MAP- 1B. Comparison of the relative amounts of MAP-1B and tubulin in the axolemma1 preparation and in the soluble fraction from white matter, indicates the level of MAP-1B far exceeds that which would result from microtubule association with the membrane and that MAP-1B distributes more like fodrin and ankyrin (data not shown). This raises the possibility that MAP-1B may act as a membrane cytoskeletal protein in the axon and may be a site of interaction between the membrane and the cytoplasmic cytoskeleton. Recent molecular cloning experiments in fact demonstrate that MAP-1B may share structural homology2' with membrane cytoskeletal proteins such as spectrin (fodrin) and not with the other microtubule associated proteins such as MAP-2 and TAU.25 Recent data on ion channel activity in cultured oligodendrocytes3 and patch clamp studies on paranodal m ~ e l i nsuggest ~ ~ that the ionic milieu of the axon may in part be regulated by ion channel activity in the myelin complex. The presence of ion channel activity in periaxolemmal myelin may represent a specialized example of glial buffering of the neuronal ionic milieu.I7 The initial in vitro demonstration of Kf and C1- channels in our periaxolemmal enriched fraction also supports this view. The enrichment of carbonic anhydrase in the periaxolemmal enriched fraction indicates that this membrane domain may utilize pH regulation and Hf transport to regulate extracellular ion concentrations as we had previously p o ~ t u l a t e d ' ~and ~ ~which ~ * ~ ~has been shown to be an important factor in control of neuronal firing. l6 We can not directly compare the channel activity +

that we observe in this study with those reported from patch clamp studies on cultured oligodendrocytes.2,3,38,22These studies were performed on cultured cells, using whole cell or excised patch techniques and were concentrated by necessity on the oligodendrocyte cell soma. The present study used membranes that had gone through an extensive purification procedure and much of the channel activity has probably been dissociated from cellular control mechanisms. The existence of such control processes has been shown in oligodendrocytes; for example Barres2 points out that C1- channel events can be measured in excised patches free of cellular factors but not in whole cell patch analysis. Our technique will hopefully be made comparable to these in situ studies through subsequent analysis of differential sensitivity to specific channel blockers. Uniand bilamellar vesicles have been previously23 prepared from multilammellar myelin and have been shown to possess K + channels. However, due to the nature of that preparation the authors felt it was unclear if these vesicles were derived from compact myelin or from other membrane domains. We feel our method is useful for comparing potential channel activity in well defined membrane fractions and in the present instance clearly helps distinguish the periaxolemmal and axolemmal enriched fractions. Total event histograms, although not ideal for the statistical analysis of this kind of single channel data, indicate that the population of K f channels in the two membranes are distinct. The presence of Naf channel activity measured in the presence of BTX confirms the identification of this channel by Western blot analysis. The Na+ channels measured here are similar, in both amplitude and open times, to previous reports using BTX induced channel openings in membrane/lipid bilayer fusion experim e n t ~Western . ~ ~ blot analysis indicates the Na channel protein is present, although at lower levels, in the periaxolemmal membrane. This is probably due to a combination of some cross contamination, as seen with other axolemmal markers such as MAPlB and axonspecific fodrin (Table I) and a low level expression of this channel in glia.*' In summary, we have presented a method for the isolation of fractions enriched in periaxolemmal myelin and axolemma. These fractions are distinct from myelin and with respect to important marker proteins, represent different membrane domains. Based on the methods employed here, these membranes appear to be the site of G proteins within the myelin complex and a site of transport enzyme and ion channel activity. Further characterization of these ion channels and the factors which may regulate them will hopefully allow for a greater appreciation of myelin-axonal interactions. +

Isolation of Axolemmal Fractions From Rat CNS

ACKNOWLEDGMENTS The authors wish to acknowledge the technical assistance of Charles Nolan, Gladys Gray-Board, and Roz Cohen. The authors would also like to thank Sidney Bernstein for his assistance in preparing illustrations. This work was supported by grants NS2.5950, AG09480, and BNS9011859. REFERENCES 1. Angelides KJ, Elmer LW, Loftus D, Elson E (1988): Distribution and lateral mobility of voltage-dependent sodium channels in neurons. J Cell Biol 106:1911-1925. 2. Barres BA, Chun LLY, Corey DP (1988): Ion expression in white matter glia: I. Type 1 astrocytes and oligodendrocytes. Glia 1: 10-30. 3. Barres BA, Chun LLY, Corey DP (1990): Ion channels in vertebrate glia. In Cowan WM, Shooter EM, Stevens CF and Thompson RF (eds): “Annual Review of Neuroscience,” Palo Alto, CA: Annual Reviews, Inc., pp 441-474. 4. Birnbaumer L, Abramowitz J, Yatani A, Okabe K, Mattera R, Graf R, Sanford J, Codina J, Brown AM (1990): Critical Rev Biochem Mof Biol 25:225-243. 5. Bradford MM (1976): Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254. 6. Braun PE, Horvath E, Bernier L (1990): Identification of GTPbinding proteins in myelin and oligodendrocyte membranes. J Neurosci Res 26:16-23. 7. Cammer W, Fredman T, Rose AK, Norton WT (1976): Brain Carbonic Anhydrase: Activity of isolated myelin and the effect of hexachlorophene. J Neurochem 27:165-171. 8. Casey PJ, Gilman AG (1988): G protein involvement in receptoreffector coupling. J Biol Chem 263:2577-2580. 9. Chakrabati AK, Yoshida Y, Powers JM, Singh I, Hogan EI, Banik NI (1988): Calcium activated neutral proteinase in rat brain myelin and subcellular fractions. J Neurosci 20:351-358. 10. Cochary EF, Bizzozero OA, Sapirstein VS, Nolan CE, Fischer I (1990): Presence of the plasma membrane proteolipid (Plasmolipin) in myelin. J Neurochem 55:602-610. 11. Coronado R, Latorre R (1983): Phospholipid bilayers made from monolayers on patch clamp pipettes. Biophys J 43:231-236. 12. Deshmukh DS, Kuizon S, Bear WD, Brockerhoff H (1982): Polyphosphoinositide mon-and diphosphoesterase of three subfractions of rat brain myelin. Neurochem Res 7:617-626. 13. Detskey PZ, Bigbee JW, DeVries GH (1988): Isolation and characterization of axolemma-enriched fractions from discrete areas of bovine CNS. Neurochem Res 13:449-454. 14. Fischer I, Sapirstein VS (1986): Characterization and biosynthesis of the plasma membrane proteolipid protein in neural tissue. J Neurochem 47:232-238. 15. Fischer I, Romano-Clark G (1990): Changes in microtubule-associated protein MAPlB phosphorylation during rat brain development. J Neurochem 55:328-333. 16. Jendelova P, Sykova E (1991): Role of glia in K+ and pH homeostasis in the rat neonatal spinal cord. Glia 4:56-63. 17. Kuffler SW, Potter DD (1964): Glia in the leech central nervous system: physiological properties and neuron-glia relationship. J Neurophysiol 27:290-320. 18. Laemmli UK (1970): Cleavage of structural proteins of the head of bacteriophage T4. Nature 227:680-685. 19. Lees MB, Sapirstein VS (1983): Myelin-associated enzymes. In

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