THE JOURNAL OF BIOLQGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 269, No. 44, Issue of November 4, PP. 2721627223, 1994 Printed in U.S.A.

Regulation of Acetylcholinesterase mRNA Stability by Calcium during Differentiation from Myoblasts to Myotubes*

(Received for publication, June 20, 1994, and in revised form, August 10, 1994)

Zhigang LuoS, Maria-Elena FuentesSQ, and Palmer Taylorn From the Department of Pharmacology-0636, University of California, Sun Diego, La Jolla, California 92093

The expression of acetylcholinesterase (AChE), nico- tinic acetylcholine receptors (nAChR), and their corre- sponding mRNAs increases dramatically during the con- version of myoblasts to myotubes in C2-Cl2 cells. The increase in expression of nAChR arises from transcrip- tional activation of the genes encoding the receptor sub- units, whereas stabilization of labile transcripts is pri- marily responsible for enhanced AChE expression. In a search for the signaling pathways responsible for stabi- lization of the AChE mRNA, we found that ryanodine, synthetic ryanodine receptor antagonists and L-type, but not N-type, Ca2' channel blockers inhibit the differ- entiation-induced expression of AChE mRNA, but not the nAChR mRNA. Selective inhibition of increased ex- pression of AChE is also evident. Inhibition by ryano- dine and nifedipine is additive suggesting different tar- get sites for the two Ca2+ channel ligands. Ryanodine binding sites can be detected in both myoblasts and myotubes, but they increase substantially during differ- entiation. Rates of AChE gene transcription are not al- tered by ryanodine and nifedipine, indicating that de- creased Ca2+ availability prevents stabilization of the mRNA normally seen with differentiation. Muscle cells still undergo elongation and fusion in the presence of ryanodine or L-type Ca2' channel antagonists. Ryano- dine block is fully reversible, indicating functional in- tegrity of the cellular expression system after the drug treatment. These findings indicate that intracellular ry- anodine-sensitive calcium channels and extracellular L- type Ca2+ channels link to play an important role in sta- bilizing AChE mRNA and suggest that transient increases in intracellular Ca2+ may be critical for the commitment of AChE expression during myogenesis.

Differentiation of stem cells into muscle can be analyzed as a series of discrete steps. The muscle determination genes of the MyoD family serve as primary determinants of the overall proc- ess (Weintraub et al., 1991). Upon expression of this family of genes, cells acquire the muscle lineage as myoblasts. At con- fluence and with the arrest of cell division, myoblasts fuse to become elongated multinucleated cells or myotubes.

Concomitant with the arrest of cell division and growth is the expression of a series of muscle specific genes important to cellular excitability and contraction (Hastings and Emerson, 1982). Certain of these gene products localize within the syn-

Grant GM 18360 (to P. T.). The costs of publication of this article were * This work was supported by United States Public Health Service

defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8 The first and second authors contributed equally to this study. 8 Present address: Dept. of Molecular Biology, Pharmaceutical Re-

7 To whom correspondence should be addressed. Tel.: 619-534-1366; search Group, Bristol-Myers Squibb, Princeton, NJ 08543-4000.

Fax: 619-534-8248.

apse, and these include proteins associated with transduction of signals for acetylcholine-mediated neurotransmission, the nicotinic acetylcholine receptor (nAChR)' and acetylcholines- terase (AChE). Both the nAChR and AChE show markedly increased expression of their mRNAs and gene products with muscle cell fusion. Clustering of the nAChR occurs, and these clusters become a potential focus for synapse formation (Car- taud and Changew, 1993; Hall and Sanes, 1993). Also associ- ated with synapse formation is the increased focal expression of AChE. In the neuromuscular junction, AChE is localized in the basal lamina of the synapse (cf. Massoulie et al., 1993).

Expression studies show that transcriptional enhancement is primarily responsible for the increased nAChR mRNA asso- ciated with muscle differentiation (Buonanno and Merlie, 1986; Evans et al., 1987; Baldwin and Burden, 1988; Wang et al., 1988; Frail et al., 1989). Parallel increases in AChE mRNA and protein also occur, but surprisingly, increases in AChE expres- sion do not arise from transcriptional activation, but rather stabilization of a rapidly turning over mRNA (Fuentes and Taylor, 1993). To examine further the mechanistic bases for the mRNA stabilization, we examined the role of release of intra- cellular Ca2+ and cellular excitability in the regulation of the AChE mRNA.

EXPERIMENTAL PROCEDURES Materials-'251-a-Bungarotoxin (lZ5I-a-BTX, specific activity: 14.5

pCUpg), L3H1ryanodine (specific activity: 63.5 Ci/mmol), and [a-3ZPlUTP (specific activity: -800 Ci/mmol) were purchased from DuPont NEN. FLA 365 was a gift from Dr. Isaac N. Pessah at University of Califor- nia, Davis. Ryanodine was purchased from Sigma and Calbiochem. Other chemicals were from Sigma. All cell culture supplies were from Fisher Scientific, and components of culture medium were from Life Technologies, Inc.

Calcium Antagonists and Other Drug Zkeatment-Stock solutions of nifedipine, verapamil and FLA 365 were made in dimethyl sulfoxide (Me,SO); stock solutions of ryanodine and diltiazem were made in phos- phate-buffered saline solution. These stock solutions were kept at 4 "C and diluted before each treatment. The final concentration of Me,SO was less than 0.01%, and the same amount of Me,SO was added to each control group. Me,SO at 0.01% did not influence the increase of AChE activity associated with fusion or the fusion process, per se. Tetrodotoxin (TTx) stock solutions (1 mg/ml) were made in acetate buffer, pH 4, and equal amounts of acetate buffer were added in parallel control culture dishes.

Tissue Cultures"C2-Cl2 cells (from American Type Culture Collec- tion) were stored at -70 "C and cultured at 37 "C, with 5% CO, in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum, 0.5% chick embryo extract, and 1% penicillin, streptomycin, and am- photericin B stock solution (Antibiotic-Antimycotic; Life Technologies, Inc.). Cells were passed either two or three times before plating. Dif- ferentiation from myoblasts to myotubes was induced at about 70% of confluence by replacing the high serum medium with Dulbecco's modi- fied Eagle's medium containing 2% horse serum and 1% of the above antimicrobial stock solution.

The abbreviations used are: nAChR, nicotinic acetylcholine recep- tor; AChE, acetylcholinesterase; BTx, bungarotoxin; ?Tx, tetrodotoxin; bp, base paids); kb, kilobase pair(s1; snRNA, small nuclear RNA.

27216

Calcium and Acetylcholinesterase mRNA Stabilization 27217

Determination of AChE Activity-AChE was extracted from C2-Cl2 cells in 0.01 M sodium phosphate buffer containing 1 N NaC1, 1% Triton X-100, 0.01 M EGTA, and a spectrum of protease inhibitors (Silman et al., 1978). Enzyme activity was determined at room temperature as described by Ellman et al. (1961) in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.3 m~ 5,5'-dithiobis-(2-nitrobenzoic) acid, 0.5 m~ acetylthiocholine iodide, and 0.05-0.1 ml of cell extract.

Determination of nAChR-The densities of cell surface nAChR were monitored by binding of '251-a-BTx to intact cells cultured in six-well plates at room temperature. Cells were washed with differentiation medium and incubated for 10 min in the presence or absence of 10 m~ carbamylcholine chloride. lZ5I-a-BTx at 10 n~ was added directly to each well and incubated for 2 h. Cells were washed three times gently with K+-Ringer's buffer (140 mM KC1, 5.4 m~ NaCl, 1.8 mM CaCl,, 1.7 mM MgCl,, 25 mM HEPES, 0.03 mg/ml bovine serum albumin, pH 7.41, and 1 ml of 1 M NaOH was added to each well to lyse the cells. Cell lysates were counted in a y counter, and protein was assayed in the cell lysates using BCA reagents (Pierce).

RNA Extraction and Protection Assay-Total RNA was extracted from C2-Cl2 cells as described by Chomczynski and Sacchi (1987) and stored at -20 "C. mRNAs encoding AChE and the y-subunit of nAChR were quantitated by RNA protection as described by Gilman (1989). For a probe ofAChE mRNA, a 456-bp mouse cDNA fragment encoding exons 4 and 6 of AChE gene was subcloned in Bluescript SK I1 plasmids and linearized with XhoI. This cDNA served as template for in vitro syn- thesis of the radioactive antisense cRNA probe. For a probe hybridizing with mRNA of the nAChR y-subunit, a 1.7-kb mouse cDNA (kindly provided by Drs. Jim Boulter and Stephen Heinemann, Salk Institute, San Diego, CA), cloned in pSP-65 plasmid and linearized withXho1, was used. To normalize sample loading, the entire human U1 snRNA gene, cloned in pSP-65 and linearized with HindIII (Howe and Steitz, 1986), was used. Molecular sizes of the protected mRNA were estimated by electrophoresis on polyacrylamide gels, and protected mRNA was quan- titated by densitometry (UltroScan XL, Pharmacia Biotech Inc.) or on a radioanalytical system (AmbisTM, San Diego, CA).

Nuclear Run-on nanscription Assay-Nuclei isolation and nuclear run-on transcription assays were performed as described by Greenberg and Bender (1989). Cells were washed two times and harvested in ice-cold phosphate-buffered saline. After centrifugation for 5 min at 500 x g, the pellets were resuspended in lysis buffer (10 m~ Tris, pH 7.4, 3 m~ CaCl,, and 2 mM MgCl,), centrifuged at 500 x g for 5 min and resuspended again in the same buffer containing 0.5% Nonidet P-40. Cells were then broken in a Dounce homogenizer, and nuclei were sedimented for 5 min at 500 x g. Approximately 5 x lo7 nuclei were resuspended in 200 pl of 50 mM Tris-HC1, pH 8.3, (containing 40% glycerol, 5 m~ MgCl,, and 0.1 mM EDTA, frozen in liquid nitrogen, and stored at -70 "C.

Nuclei in a volume of 200 p1 were thawed and mixed with 200 p1 of 2 x reaction buffer containing 10 mM Tris-HC1, pH 8.0,5 mM MgCl,, 0.3 M KCl, 5 mM DTT, 1 mM each of ATP, GTP, and CTP, and 10 pl of [U-~~PIUTP. Radiolabeled mRNA was transcribed, isolated, and hybrid- ized for at least 36 h at 65 "C to slot blots containing 5 pg of plasmid DNA. The plasmid (Bluescript SK 11) containing a 2.1-kb AChE insert was linearized with EcoRI, and the control plasmid was linearized with EcoRI or KpnI. The pSP-65 plasmid vectors containing a 1.7-kb frag- ment of the coding region of mouse nAChR y-subunit were linearized with BamHI or KpnI. Plasmids containing a 2.3-kb cDNA sequences encoding a HSP family protein (O'Malley et al., 1985) and pEMSVmyo8 containing a 1.5-kb myogenin cDNA(kind1y provided by Dr. Eric Olson, M. D. Anderson Hospital) were linearized with HindIII. Bluescript vec- tors containing a 1.4-kb DNA fragment of mouse a-tubulin were linear- ized with KpnI. For detecting the antisense and sense AChE mRNA, control M13 phage DNA and that containing a 2.3-kb single strand sense or antisense AChE cDNA insert, respectively, were used. After extensive washing with 2 x SSC, radioactivity was determined by au- toradiography and densitometry.

Measurement of Ryanodine Binding Sites in C2-Cl2 Cells- Ryanodine binding sites in C2-Cl2 cells were assessed from the binding of L3H1ryanodine to crude sarcoplasmic reticulum fractions (Mitchell et al., 1983; Pessah et al., 1985). C2-Cl2 cells were washed, harvested, homogenized, and centrifuged at 2,000 x g for 10 min at 4 "C in pyro- phosphate buffer, pH 7.1 (20 m~ sodium pyrophosphate, 20 mM sodium phosphate, 1 m~ MgCl,, 0.5 mM EDTA, and 10% sucrose), to remove nuclei and unbroken cells. Membrane fractions were obtained from consecutive centrifugations of the above supernatants at 10,000 x g for 15 min and 27,000 x g for 45 min at 4 "C. The pellets were resuspended in assay buffer (40 mM Tridmaleate buffer, pH 7.1, containing 10% sucrose and 60 p~ CaCl,). Approximately 200 pg of protein were added

0 ' I I I I I

-3 -2 -1 0 1 Log [Drug1 ( I 4

ine inhibition of differentiation-induced AChJ3 expression in FIG. 1. Concentration dependences of ryanodine and nifedip-

C2-Cl2 cells. C2-Cl2 myoblasts at approximately 70% of confluence were differentiated into myotubes for 3 days in low serum medium in the presence of ryanodine (0) and nifedipine (M) as described under "Experimental Procedures." AChE was extracted and activity was de- termined by the method of Ellman et al. (1961). Data are presented as percentage of control within each experiment, and the values reported are means 2 S.E. averaged from 6-22 independent determinations.

to 0.25 ml of assay buffer containing increasing concentrations of L3H1ryanodine up to 250 n ~ . The highest concentration of L3Hlryanodine in the binding mixtures was 24 MI, and higher concentrations were achieved with unlabeled ryanodine. After incubation for 90 min at 37 "C with shaking, the suspensions were diluted with 5 ml of ice-cold assay buffer, rapidly filtered through GFlF glass fiber filters under vacuum, and counted. Specific binding was the difference between the total bind- ing and the nonspecific binding determined in the presence of 1 p~ un- labeled ryanodine. B,, and Kd were determined by Scatchard analysis.

RESULTS

Znfluence of Ryanodine and L-type Calcium Channel Antago- nists on Differentiation-induced Expression of AChE and nAChR-To examine the role of Ca2+ channels in differentia- tion-induced expression of AChE and nAChR, AChE activity and cu-BTx binding sites of nAChR were monitored during mus- cle differentiation in the presence or absence of ryanodine, ry- anodine receptor antagonists, and Ca2+ channel antagonists. As shown in Fig. 1, treatment with both ryanodine and nifedipine for 3 days caused similar concentration-dependent inhibitions of differentiation-induced expression of AChE. Ryanodine was more potent, with a half-maximal inhibition at approximately 20 nM. Nifedipine's half-maximal inhibition occurred at ap- proximately 3 1.1~.

During the course of muscle differentiation, AChE increased substantially in C2-Cl2 cells over a 3-day period (Inestrosa et al., 1983; Fuentes and Taylor, 1993) (Fig. 2). Treatment with 100 n~ ryanodine inhibited the differentiation-induced in- crease ofAChE (Fig. 2 A ) . Inhibition was evident as early as the first day of differentiation and continued over the 3-day course of muscle differentiation. Removal of ryanodine from the dif- ferentiation medium after day 2 showed not only that the block was reversible but also that a more rapid onset of expression could be induced than was evident without the initial block. Treatment for 3 days with the ryanodine receptor antagonist, FLA 365, at 10 p~ caused similar blockade (70%) in the differ- entiation-induced AChE expression (Fig. 2 4 ) .

Treatment with 5 p~ nifedipine, an L-type Ca2+ channel an- tagonist, during muscle differentiation resulted in inhibition of expression of AChE similar to that seen with ryanodine (Fig. 2B). Verapamil or diltiazem, two different types of L-type Ca2+ channel antagonists, at 5 VM caused similar inhibitions of

27218 Calcium and Acetylcholinesterase mRNA Stabilization

E 3 E X m II

.c

Y- O

a, m + a C a, 2 a, a

I I

0 1 2 3 0 1 2 3

Days in D i f f e r e n t i a t i o n Medium FIG. 2. Influence of calcium channel ligands on AChE and

nAChR expression during myoblast to myotube differentiation in C2-Cl2 cells. Cells were differentiated for 3 days in the presence or absence of different Ca2+ channel ligands. Cells from 3 plates/ experimental point were harvested at the designated time, and either AChE activities or nAChR binding sites were determined as described under "Experimental Procedures." A, AChE activity in C2-Cl2 cells treated with ryanodine or FLA 365.0, control; 0, 100 n~ ryanodine; ., FLA 365. B, AChE activity in C2-Cl2 cells treated with nifedipine. 0, 100 nM ryanodine present initially and washed out at day 2; A, 10 PM

control; 0, 5 p nifedipine. C , AChE activity in C2-Cl2 cells treated with diltiazem, verapamil, and o-conotoxin. 0, control; 0, 5 1.1~ dilti- azem; a, 5 p~ verapamil; A, 1 p w-conotoxin. D, nAChR binding sites in C2-Cl2 cells treated with ryanodine. 0, control; 0,100 n~ ryanodine. AChE activities or a-BTx binding sites on the nicotinic receptors at day 3 of differentiation were chosen as maximum (100%) values. Values reported are means * S.E. averaged for at least three independent determinations.

AChE expression. Furthermore, the potency of inhibition of AChE expression by nifedipine, verapamil, and diltiazem cor- related well with the rank order of binding affinities at their respective sites (Janis et al., 1987). However, treatment for 3 days with 1 p~ o-conotoxin, an N-type Ca2+ channel antagonist with the capacity to block Ca2+ uptake in neuronal cells com- pletely at this concentration (Rivier et al., 19871, did not inhibit AChE expression (Fig. 2C). Hence, inhibition appears selective for L-type CaZ+ channels.

During differentiation from myoblasts to myotubes, the in- crease in nicotinic receptors on the cell surface roughly paral- lels the expression of AChE (Inestrosa et al., 1983) (Fig. 2, C and D ) . The maximum density of cell surface nAChR during the 3-day differentiation was 280 f 14 fmollmg protein, which agreed closely with the maximum binding sites of the receptors reported for C2-Cl2 cells (Inestrosa et al., 1983). Treatment with 100 nM ryanodine during muscle differentiation had little or no effect on the differentiation-induced expression of nAChR (Fig. 2 0 ) .

If the sites of action for ryanodine and L-type Ca2+ channel blockers are distinct sites on a sequential pathway, one would expect at least additive effects on the expression ofAChE when both drugs are applied. On the other hand, if both drugs act on common target sites, one would expect less than additive an- tagonism. To examine the relationship between the ryanodine and Ca2+ channel blockers on the expression of AChE during muscle differentiation, C2-Cl2 cells were treated with 20 nM ryanodine, 3 p~ nifedipine, or a combination of both ligands. At

A B C FIG. 3. Additive effects of ryanodine and nifedipine on differ-

entiation-induced AChE expression in C2-Cl2 cells. Cells were differentiated for 3 days in the presence or absence of 20 IIM ryanodine or 3 PM nifedipine or the combination of the two agents. AChE was extracted and activity was determined as described. Data are presented as percentages of control activity in each experiment and values re- ported are means 2 S.E. averaged over 15-21 independent determina- tions. A, 20 nM ryanodine; B, 3 p~ nifedipine; C, 20 nM ryanodine and 3 p~ nifedipine.

these concentrations, each ligand alone blocks the expression of AChE about 40-50%. When both drugs were applied, there was an additional 50% inhibition of the remaining differentiation- induced AChE expression, indicating that the effects of ryano- dine and nifedipine were roughly additive (Fig. 3).

Effects of Ryanodine and L-type Calcium Channel Antago- nists on mRNA of AChE and nAChR-To examine whether inhibition of AChE expression by blocking the Ca2+ channels results from reduction in the levels of transcripts, mRNAs of AChE and the y-subunit of nAChR were monitored after treat- ment with different agents. As shown in Fig. 4 and summarized in Table I, low levels of AChE and y-subunit of nAChR mRNAs were detected before muscle differentiation. The mRNAs en- coding both AChE and the y-subunit of nAChR increased sub- stantially after 3 days of differentiation. Treatment with 100 nM ryanodine for 3 days inhibited the differentiation-induced increase of AChE mRNA. The AChE transcripts returned to normal levels within 24 h when ryanodine was removed at day 2 by replacement with fresh medium. Similar reductions in AChE mRNA, but of smaller magnitude, were observed after treatment with 10 p~ FLA 365 (the ryanodine receptor antago- nist) for 3 days. However, identical exposures of the cells to ryanodine and FLA 365 did not cause significant alterations of mRNA encoding the y-subunit of nAChR.

Treatment with 5 p~ nifedipine during muscle differentia- tion also caused substantial inhibition of differentiation-in- duced expression of the AChE mRNA (Fig. 5 A ) , but not in the mRNA encoding the y-subunit of nAChR (Fig. 5B). Similar in- hibitions of expression of AChE mRNA were also evident after treatment with 10 p diltiazem and verapamil, L-type Ca" channel antagonists with different sites of actions (Table I).

Ryanodine and L-type Calcium Channel Antagonists Do Not Influence Danscription of AChE and nAChR Genes-To deter- mine whether inhibition of the mRNA increase normally asso- ciated with muscle cell differentiation was due to the inhibition of transcription or interference with mRNA stabilization, we examined the rates of mRNA production by run-on transcrip- tion. Treatment with 100 I" ryanodine (Fig. 6) or 5 p~ nifedip- ine (Fig. 7) for 3 days did not appreciably influence rates of transcription of the AChE or the y-subunit of nAChR genes. A small increase in myogenin transcription after treatment with nifedipine was evident.

Calcium and Acetylcholinesterase mRNA Stabilization 27219

516 -

394 - 344 -

298 -

220 - 200 -

154 -

nAChR

AChE

u1

FIG. 4. Representative autoradiogram showing ryanodine and FJA 365 inhibition of the differentiation-induced increase of AChE transcripts in C2-Cl2 cells. C2-Cl2 cells were differentiated in the presence or absence of 100 nhl ryanodine or 10 PM FLA 365 for 3 days, after which RNA was extracted and analyzed by RNA protection as described. The AChE probe extended between bp 1651 and 2107. Protection by a mRNA species splicing between exons 4 and 6 should yield a 456-bp band. The receptor probe extended between bp 1168 and 1716 in the cDNA and the nAChR mRNA should protect a 548-bp species. To standardize for loading differences, an U1 snRNA probe was included, which gave a doublet of protected bands near 160 bp. To ensure complete RNA digestion, protection was examined in the pres- ence of transfer RNA.

Expression of Ryanodine Receptors during Differentiation of C2-C12 Cells-To examine whether Ca2+ release sites are lim- iting for mRNA expression and increase during differentiation, ryanodine binding sites in crude fractions of sarcoplasmic re- ticulum in C2-Cl2 cells were measured at the start of differ- entiation and 3 days thereafter. We found a small, but detect- able, number of ryanodine binding sites in the myoblasts, which increased about 4-fold upon differentiation (Fig. 8). How- ever, the maximum number of binding sites in the differenti- ated C2-Cl2 cells was substantially lower than that found for a similarly purified sarcoplasmic reticulum fraction from rat leg muscle. The latter was found to be about 750 fmoVmg protein (data not shown).

Influence of Cellular Excitability on Expression of AChE and Its mRNA Levels in C2-Cl2 Cells-To examine whether cellular excitability through action potentials plays a role in the expres- sion of AChE by contributing to the stabilization of its mRNA, AChE activity and mRNA levels were examined after treat- ment with 10 p~ TTx during muscle differentiation. Although the influence of TTx on AChE expression may be dependent on the levels of contractile activity of mature myotubes,2 block of

* Z. Luo, unpublished observations.

excitability by TTx caused only a small (20-30%) inhibition in the differentiation-induced increase of AChE mRNA and activ- ity at day 3, and was without significant influence a t days 1 and 2 in the differentiation medium (Fig. 9, A and B) . The same treatment did not inhibit the expression of mRNA of nAChR y-subunit over the entire time course (Fig. 9 0 .

To examine whether the effects of TTx were independent of those of ryanodine, mRNA levels of AChE and nAChR in C2- C12 cells were monitored after treatment for 3 days with 10 p~ TTx, 20 nM ryanodine, or a combination. As shown in Table I, 20 nM ryanodine caused approximately 50% inhibition of the dif- ferentiation-induced expression of AChE mRNA. Blocking the cellular excitability by TTx did not cause additional inhibition of the expression of AChE mRNA, nor was the expression of AChE changed (data not shown).

Nifedipine-induced Inhibition of Expression of AChE mRNA Can Be Reversed by Cycloheximide-Since differentiation-in- duced expression of AChE is due to the stabilization of the transcripts and short term cycloheximide treatment enhances the AChE mRNA stabilization through superinduction after initiation of differentiation (Fuentes and Taylor, 19931, we ex- amined whether inhibition of protein synthesis by cyclohexi- mide can diminish the effects of nifedipine. As indicated in Fig. 10, treatment with 5 p~ nifedipine for 3 days caused about 70% inhibition of the differentiation-induced expression of AChE mRNA. Treatment of C2-Cl2 cells with 10 PM cycloheximide for 6 h prior to extraction of total RNA increased the AChE mRNA 2-3 fold when compared with cells treated with only nifedipine for 3 days. Thus, superinduction of AChE mRNA by an inhib- itor of protein synthesis is still evident in the presence of L-type Ca2+ channel inhibitors.

DISCUSSION

Despite parallel increases on nAChR and AChE mRNAs dur- ing differentiation of C2-Cl2 cells from myoblasts to myotubes, the former mRNA levels increase from activation of transcrip- tion (Buonanno and Merlie, 1986; Baldwin and Burden, 1988; Evans et al., 1987; Wang et al., 1988), while the latter arises from mRNA stabilization (Fuentes and Taylor, 1993). Distinct regulatory events were shown by comparative rates of run-on transcription in differentiating C2-Cl2 cells and by expression of transfected reporter genes linked to the respective promoter regions (Li et al., 1993). Moreover, only AChE shows superin- duction of mRNA expression upon treatment with cyclohexi- mide after initiation of differentiation (Fuentes and Taylor, 1993). The linkages between the differentiation pathway to transcriptional activation in the case of the nicotinic receptor subunits and to stabilization of the AChE mRNA are poorly understood. Previous studies indicated that alteration of intra- cellular Ca2+ and block of Ca2+ channels and cellular excitability influence the expression of AChE in rat and chick primary skeletal muscle cultures (Rubin, 1985; Decker and Berman, 1990), suggesting possible roles of intracellular Ca2+ on AChE expression.

T-tubules in skeletal muscle contain the highest densities of L-type Ca2+ channels (Janis et al., 1987). However, in contrast to cardiac or smooth muscle, less than 5% of these channels appear to be involved in the mobilization of Ca2+ (Schwartz et al., 1985). These Ca2+ channels serve primarily as voltage-sen- sors coupling membrane depolarization to the release of Ca2+ from intracellular stores through ryanodine-sensitive calcium channels in the sarcoplasmic reticulum (Schneider and Chan- dler, 1973; Adams et al., 1990; Lu et al., 1994). Dihydropyri- dines, L-type Ca2+ channel antagonists, block intramembrane charge movement and Ca2+ release from sarcoplasmic reticu- lum with similar voltage and dose dependences (Rios et al., 1992). Furthermore, microinjection of a cDNA encoding dihy-

27220 Calcium and Acetylcholinesterase mRNA Stabilization TABLE I

mRNA levels of AChE and AChR in C2-Cl2 cells after treatment with Ca2+ channel ligands

U1-protected bands. Data are presented as percentage of maximum (100%) mRNAlevels taken from control cells differentiated for 3 days. Unless The labeled antisense mRNA probes protected by the mRNA were analyzed by densitometric analysis normalized with respect to the density of

otherwise specified, values reported are the means 2 S.E. averaged for a t least four independent determinations.

mRNA levels from protected mRNA probes (B of control)

Control 3-Day Ryanodine FLA Nifedipine

5 pM 10 pM undifferentiated untreated 2o nbl Diltiazem (10 Verapamil (IO

p w pal) 100 nal (10 pal)

AChE 6 . - 2 100 53 t 4 24 .- 5 39 .- 5 4 2 2 14 1 3 2 6 2 8 2 9 31 2 12 nAChR 21 t 5 100 107 2 12 101 2 13 128 2 15 97" ND * ND ND

a Data from Fig. 5B. * ND, not determined.

01 I I I I I

0- 0 1 2 3

Days o f D i f f e ren t i a t i on

FIG. 5. Inhibition of the differentiation-induced increase of AChE mRNA in C2-Cl2 cells by nifedipine. Cells were differenti- ated for 3 days in the presence or absence of 5 VM nifedipine, total RNA was extracted, and AChE mRNA was determined as described. Repre- sentative autoradiograms analogous to Fig. 4 were quantitated using U1 snRNA to normalize for loading differences. A, effects of nifedipine on levels ofAChE transcripts during differentiation. 0, control; 0, nife- dipine. B, effects of nifedipine on levels of nAChR y-subunit transcripts during differentiation. 0, control; 0, nifedipine.

dropyridine receptors into muscle cultures from muscular dys- genic mice restores intramembrane charge movement, slow Ca2' current, and excitation-contraction coupling (Numa et al., 1990; Tanabe et al., 1988; Adams et al., 1990).

Electron microscopy indicates that L-type Ca2' channel re- ceptors on the membranes of T-tubules and ryanodine receptors on the membranes of sarcoplasmic reticulum lie in close appo- sition (Block et al., 1980; Franzini-Armstrong, 1970). Recent studies demonstrate that the purified cytoplasmic loop region of the dihydropyridine receptor a, subunit specifically activates skeletal muscle ryanodine-sensitive Ca2+ release by increasing the channel opening probability and the affinity of ryanodine binding (Lu et al., 1994). Immunofluorescence labeling indi- cates that the temporal appearance and subcellular distribu- tion of a,-subunit of dihydropyridine receptors and ryanodine receptors are very similar, if not identical, throughout biogen- esis of skeletal T-tubules and triads (Yuan et al., 1991). Taken together, these findings strongly suggest that ryanodine recep- tors and L-type Ca2+ channels regulate Ca2+ movement and

A

1111 JI Sense ] AChE Antisense

M13

ynAChR

a-Tubulin

SK

0.oL J

Sense Antisense AChE AChE y-nAChR

FIG. 6. Effects of ryanodine on rates of transcription of AChE and nAChR genes measured by run-on transcription analysis. [a-"PIUTP-labeled transcripts were synthesized in nuclei isolated from C2-Cl2 cells maintained for 3 days in differentiation medium in the presence or absence of 100 nM ryanodine and hybridized to slots con- taining 5 pg of the appropriate cDNAas described. Control Bluescript I1 SK (SK) plasmids or M13 phages ( M 1 3 ) or those containing cDNA sequences for AChE, nAChR y-subunit, and a-tubulin were used. A, representative autoradiogram. E , quantitation of the signals by densi- tometric analysis. Open bars, control cells; hatched bars, treated cells. Densities were normalized with respect to a-tubulin mRNA density. Values reported are means .- S.E. averaged from at least four independ- ent determinations.

excitation-contraction coupling in skeletal muscle through an intimately coupled mechanism.

Ryanodine- and Dihydropyridine-sensitive Calcium Chan- nels Play a Role in Regulation of AChE Expression during Myogenesis-As indicated in Fig. 1, treatment both with ryan- odine, at concentrations that hold the ryanodine-sensitive channels in an open state (Lattanzio et al., 1987; Meissner, 1986; Smith et al., 1988) and eventually deplete intracellular Ca2+ stores as suggested by Hwang et al. (1987), and with nifedipine resulted in a dose-dependent inhibition of differen- tiation-induced AChE expression. Similar blockades were

Calcium and Acetylcholinesterase mRNA Stabilization 27221

AChE AChR

FIG. 7. Effects of nifedipine on rates of transcription of AChE and nAChR genes measured by run-on transcription analysis. [a-3'P]UTP-labeled transcripts were synthesized in nuclei isolated from C2-Cl2 cells maintained for 3 days in differentiation medium in the presence or absence of 5 phi nifedipine and hybridized to slots contain- ing 5 pg of the appropriate cDNA as described. Control Bluescript I1 SK (SKI plasmids or those containing cDNA sequences for AChE, HSP-70, nAChR y-subunit, and myogenin (Myo) were used. A, representative autoradiogram. B, quantitation of the signals by densitometric analysis. Open burs, control cells; hatched burs, treated cells. Densities were normalized with respect to HSP-70 mRNA density. Values reported are means f S.E. averaged from at least three independent determinations.

n 7 "0 100 200 300

[ 3H-Ryanodine] (nM) FIG. 8. Representative specific binding of [%]ryanodine to

crude sarcoplasmic reticulum fractions of C2-Cl2 cells. Sarco- plasmic reticulum fractions were isolated from C2-Cl2 cells before and after differentiation, and binding assays were performed as described. W, undifferentiated myoblasts a t day 0; 0, differentiated myotubes a t day 3. The B,, and Kd values estimated were 70 fmoVmg protein and 110 nM for undifferentiated myoblasts and 272 fmoVmg protein and 66 nM for differentiated myotubes, respectively. Data are representative of three determinations from separate sets of plates for the myotubes. Owing to the necessity of pooling over 60 15-cm plates for sufficient detection, only a single determination was made for the undifferenti- ated cells a t day 0.

achieved by the ryanodine antagonist FLA 365 and other L- type Ca2+ channel antagonists, which recognize distinct sites on these channels (Fig. 2). Thus, both ryanodine- and L-type Ca2+

100 -

50 -

0- 0 1 2 3

Days of Differentiation FIG. 9. Influence of "x treatment on AChE expression and

mRNA levels of AChE and y-subunit of nAChR during myogen- esis in C2-Cl2 cells. C2-Cl2 cells were differentiated for 3 days in the presence or absence of 10 p~ "x. Cells were harvested at designated time, and either AChE activity or mRNA levels of AChE and y-nAChR were determined as described. A, AChE activity. 0, control; (01, TTx- treated. B, AChE mRNA levels before (0) and after (0) TTx treatment. C, mRNA levels of the y-subunit of nAChR before (0) and after (0) TTx treatment. Data are presented as percentages of control values from cells differentiated for 3 days and values reported are means S.E. averaged from three to six independent determinations.

channels functionally couple to regulate AChE expression dur- ing myogenesis.

The onsets of ryanodine and dihydropyridine antagonism preceded slightly the onset of muscle fusion, which appeared around day 2 of differentiation (Fig. 2). Thus, Ca2+ channel regulation of AChE expression appears not only in differenti- ated myotubes, but also in differentiating myoblasts. This ob- servation is in accord with several findings. 1) Slow Ca2+ cur- rents (CaffYey et al., 1989) and dihydropyridine receptor mRNA (Marks et al., 1991) appear in early differentiating, unfused C2-Cl2 cells and BC3H1 muscle cells; 2) ryanodine binding sites (Fig. 8) and skeletal muscle ryanodine receptor mRNA (Arai et al., 1992) are detected in undifferentiated or early differentiating C2-Cl2 cells and fetal rabbit skeletal muscle; 3) the temporal appearance of ryanodine and dihydropyridine re- ceptors and their mRNAs occurs in parallel during differentia- tion and also precedes T-tubule formation (Marks et al., 1991; Yuan et al., 1991). Additive inhibition of AChE expression by ryanodine and nifedipine (Fig. 3) suggests distinct target sites for the two Ca2+ channel ligands.

27222 Calcium and Acetylcholinesterase mRNA Stabilization

1 5 0 k

" A B C D

duced expression of AChE mRNA in the presence of nifedipine. FIG. 10. Cycloheximide superinduction of differentiation-in-

C2-Cl2 cells were differentiated for 3 days in the absence or presence of 5 PM nifedipine. Ten pg/ml cycloheximide was added 6 h prior to RNA extraction. mRIVA levels were determined by RNA protection assays as

ized to U1 snRNA-protected bands. Data are presented as percentages described. Signals were quantitated using densitometry and normal-

of control values (100%) taken from cells differentiated for 3 days and values reported are means of two independent determinations. A, con- trol cells; B, cells treated with cycloheximide 6 h before harvesting at day 3; C, cells treated with nifedipine at day 0; D , cells treated with nifedipine at day 0 followed by cycloheximide 6 h before harvesting at day 3.

Ryanodine is sufficiently hydrophilic to allow its removal during the course of treatment. Ryanodine-treated C2-Cl2 cells retained their functional integrity in terms ofAChE expression as demonstrated by virtually full recovery of AChE activity (Fig. 2 A ) and mRNA levels (Fig. 4) upon removal of ryanodine. This clearly indicated that ryanodine action was reversible and not cytotoxic. C2-Cl2 cells still underwent elongation and fu- sion in the presence of ryanodine or L-type Ca2+ channel an- tagonists, and, except for the absence of spontaneous contrac- tion that frequently occurs in differentiated myotubes, there were no obvious phenotypic changes. In addition, ryanodine treatment did not cause parallel changes in nAChR (Fig. 2 0 ) ; its expression was shown to be associated with muscle fusion in C2-Cl2 cells (Inestrosa et al., 1983; Evans et al., 1987). Fur- thermore, other studies in our laboratory indicated that ryan- odine did not block the increase in AChE mRNA in differenti- ating neuronal cells derived from P19 pluripotent stem c e k 3 Therefore, ryanodine's influence on gene expression appears specific to AChE in skeletal muscle, and is not secondary to nonspecific inhibition of muscle fusion and cellular excitability.

Ryanodine- and Dihydropyridine-sensitive Calcium Chan- nels Play a Role in Stabilization ofAChE mRNA-Since differ- entiation-induced expression of AChE is mainly due to stabili- zation of the transcripts during myogenesis (Fuentes and Taylor, 19931, the inhibition of AChE expression by these Ca2+ channel ligands may result from the alteration in the mRNA stability, thereby diminishing transcript levels. Indeed, inhibi- tion of the differentiation-induced increase in AChE expression by ryanodine and L-type Ca2+ channel antagonists paralleled reductions in mRNAlevels. That message levels of nAChR were not affected by ryanodine and nifedipine treatment (Figs. 4 and 5B and Table I) revealed selectivity for the AChE mRNA.

Inhibition of differentiation-induced increase in AChE mRNA levels by ryanodine and L-type Ca2+ channel antago- nists may result from two possible mechanisms: (a) blocking the increase in rate of transcription of AChE gene or (b) pre- venting the stabilization of AChE transcripts. Since treatment

B. Coleman, unpublished observation.

with 100 nM ryanodine or 5 p~ nifedipine for 3 days during myogenesis did not cause any significant changes in transcrip- tional rates of AChE and nAChR genes (Figs. 6 and 7), the inhibition of AChE expression by ryanodine and L-type Ca2' channel antagonists is due to inhibition of stabilization of the AChE mRNA that normally occurs during differentiation.

Mechanisms of the Calcium Channel Regulation of AChE Expression"C2-Cl2 cells appear to contain far smaller quan- tities of ryanodine receptors than do mature skeletal muscles. Moreover, a substantial increase in the levels of ryanodine re- ceptor mRNA (Arai et al., 1992) and the number of ryanodine receptors (Fig. 8) occurs upon growth arrest and fusion of myo- tubes. Assuming that a functional L-type ryanodine-sensitive Ca2+ channel complex parallels the presence of ryanodine bind- ing sites, in the early portion of this time period AChE mRNA stabilization may be limited by the number of functional Ca2+ channels and, hence, Ca2+ availability from this subcellular organelle. Acomparison of the rate ofAChE accumulation after initiation of differentiation (day 0 to day 1, Fig. 2 A ) and after ryanodine wash out following ryanodine-arrested mRNA stabi- lization (Fig. 4) shows the latter to be more rapid. This could be a consequence of more functional channels at the latter stage of development.

Ryanodine and L-type Ca2+ channel blockers are inhibitory to AChE expression at an early stage of differentiation (days 1 and 21, whereas these ligands only block the spontaneous con- tractions that become evident after day 2 in fully differentiated C2-Cl2 cells (Fig. 2). However, even though Na' currents are found in an early stage of differentiation in (32412 cells (Caf- frey et al., 1989), TTx has little influence on AChE mRNAlevels and AChE activity until day 3 in differentiation medium (Fig. 9, A and B). The inhibition of AChE mRNA and activity induced by TTx in differentiated myotubes is far less than that induced by ryanodine and nifedipine treatment. Therefore, mechanisms underlying the antagonism of the differentiation-induced in- crease in the expression of AChE by ryanodine appear to be distinct from a general block of cellular excitability by TTx. This is further supported by the finding that TTx treatment does not alter the ryanodine inhibition of AChE expression when both agents are added together (data not shown). The small TTx inhibition of AChE expression in differentiated C2- C12 cells may be due to the interference with intracellular Ca2+ mobilization induced by membrane depolarization and Na+/ Ca2+ exchange. However, if the primary source of intracellular Ca2+ control in the differentiating myoblast comes from newly synthesized ryanodine channels, the effects of "x would be expected to be minimal.

Dissociation of cellular excitability and the availability of cellular Ca" was also seen in primary muscle cells. Rubin (1985) reported that blocking the spontaneous contraction by veratridine, a drug that activates instead of blocks Na' chan- nels as TTx does, resulted in increased expression of AChE. Membrane depolarization by high extracellular K+ did not an- tagonize the influence of TTx on AChE expression. Taken to- gether, these findings suggest that action potential and me- chanical components of muscle contraction are not essential to differentiation-induced expression of AChE. Instead, changes in the intracellular signals are critical for AChE expression. These could include changes in Na' influx andor secondary alterations in intracellular Ca2+ through Na+/Ca2+ exchange in mature muscle fibers as in Rubin's study or primary changes of intracellular Ca2+ in differentiating muscle cells as seen here.

Alternatively, other intracellular components, as yet unde- fined and secondary to muscle activity or intracellular Ca2+ availability, may be important in the regulation ofAChE during myogenesis. Since inhibition of protein synthesis by cyclohex-

Calcium and Acetylcholinesterase mRNA Stabilization 27223

imide is able to stabilize AChE mRNAin differentiating C2-Cl2 cells, it is likely that labile destabilizing factors are involved in regulating AChE mRNA (Fuentes and Taylor, 1993). Superin- duction of AChE mRNA by cycloheximide in the presence of nifedipine (Fig. 10) may arise from two possible mechanisms. If Ca2+ transients and cycloheximide are acting on a common pathway to stabilize AChE mRNA, then Ca2+ may play a role in regulating expression of destabilizing factors important in AChE mRNA turnover. On the other hand, if cycloheximide and Ca2+ act through independent pathways, the inhibitory effects of nifedipine on AChE mRNA accumulation may result from either blocking a stabilizing factor or activating a destabilizing factor critical for AChE mRNA turnover. In either case, our findings point to unidentified trans-acting factors, which can be regulated by Ca2+, playing important roles in AChE mRNA stabilization.

Although the precise mechanism of AChE mRNA stabiliza- tion and the involved cis elements are not understood, our study demonstrates that intracellular Ca2+ plays a role in AChE mRNA stabilization during myogenesis. This finding is consistent with reports that intracellular Ca2+ is involved in mRNA stabilization in other cell systems (Bartlett et al., 1992; Hahn et al., 1991; Wodnar-Filipowicz and Moroni, 1990; Ohmura and Onoue, 1990). In addition, our findings support a linkage between ryanodine-sensitive Ca2+ channels and L-type Ca2+ channels in mobilizing intracellular Ca2+ that affects AChE mRNA stabilization.

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