Role for calcium from the sarcoplasmic reticulum in coupling muscle activity to nicotinic...

Role for Calcium from the Sarcoplasmic Reticulumin Coupling Muscle Activity to NicotinicAcetylcholine Receptor Gene Expression in Rat

Larry Adams, Daniel Goldman

Mental Health Research Institute and Department of Biological Chemistry, 205 Zina Pitcher Place,University of Michigan, Ann Arbor, Michigan 48109

Received 12 September 1997; accepted 29 December 1997

subunit RNA levels as a result of decreasing nAChRABSTRACT: Neurally evoked muscle electricalsubunit promoter activity. Finally, we show that thisactivity suppresses nicotinic acetylcholine receptordecreased promoter activity is mediated through the(nAChR) gene expression in extrajunctional domainssame DNA sequences that control activity-dependentof adult muscle fibers. It has been proposed that thisgene expression. Therefore, we propose that in ratregulation is mediated by calcium influx through volt-muscle, calcium release from the SR participates inage-dependent L-type calcium channels but bypassescoupling muscle depolarization to nAChR genethe sarcoplasmic reticulum in chick and mouse C2C12expression. q 1998 John Wiley & Sons, Inc. J Neurobiol 35: 245–cells. Here we report that in rat muscle calcium influx257, 1998through L-type calcium channels preferentially re-Keywords: muscle; nicotinic acetylcholine receptor;duced nAChR 1-subunit RNA via a post-transcrip-activity-dependent gene expression; calcium; sarco-tional mechanism. In contrast, calcium release fromplasmic reticulumthe sarcoplasmic reticulum (SR) suppressed nAChR

INTRODUCTION al., 1995; Altiok et al., 1995; Zhu et al., 1995),while the extrajunctional suppression of nAChRgene expression is mediated by nerve-induced mus-Synaptogenesis at the neuromuscular junction (NMJ)cle depolarization (Simon et al., 1992; Klarsfeldserves as an excellent system for studying how theand Changeux, 1985; Goldman et al., 1988).presynaptic neuron can control postsynaptic gene

expression. For example, muscle innervation results The molecular mechanism by which muscle ac-in the repression of nAChR genes (abgd) in extra- tivity suppresses nAChR gene expression is not welljunctional myonuclei and the induction of nAChR understood. However, it has been suspected thatgenes (ab1d) in endplate-associated myonuclei components of the signaling cascade that mediates(Klarsfeld et al., 1991; Sanes et al., 1991; Simon excitation–contraction coupling may also partici-et al., 1992; Gundersen et al., 1993; Hall and Sans, pate in coupling membrane depolarization to1993). This latter induction seems to be mediated nAChR gene expression. Although early reportsby nerve-derived ARIA (Sandrock et al., 1977), a suggested that calcium released from the sarcoplas-neuregulin (Falls et al., 1993), that binds epidermal mic reticulum (SR) can suppress nAChR proteingrowth factor (EGF) –like receptors (Moscoso et levels (Birnbaum et al., 1980; Pezzementi and

Schmidt, 1981), more recent experiments suggestthat calcium influx across the plasma membrane,

Correspondence to: D. Goldman through L-type channels, may be responsible forContract grant sponsor: NIHdecreasing nAChR gene expression in response toContract grant sponsor: Muscular Dystrophy Association

q 1998 John Wiley & Sons, Inc. CCC 0022-3034/98/030245-13 muscle activity (Klarsfeld et al., 1989; Huang and

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8P40 1953/ 8P40$$1953 04-22-98 15:21:14 nbioa W: Neurobio

246 Adams and Goldman

Schmidt, 1994; Huang et al., 1994). This result sug- Cell Culturegests that DNA sequences mediating activity-de-

Rat primary muscle cells were isolated and cultured aspendent gene expression should also mediate thedescribed previously (Chahine et al., 1993). Cells wereeffects of calcium influx. Surprisingly, this wasplated on 60-mm collagen-coated culture dishes at a den-never tested.sity of 3 1 106 cells /mL. In general, these cells beganTherefore, we examined the effect various cal-to fuse by day 4 postplating and plates became confluent

cium-perturbing drugs had on nAChR RNA levels with contracting myotubes around day 6. At this time,and promoter activity in primary rat muscle cultures. cells were maintained in medium containing tetrodotoxinIn contrast to that reported for chick muscle (Huang (3 mg/mL) to prevent myotube contraction. Cells wereet al., 1994; Huang and Schmidt, 1994), calcium transfected on day 6 postplating and were treated withinflux through L-type channels preferentially re- various calcium-perturbing drugs about 48 h later. Cul-duced rat nAChR 1-RNA via a post-transcriptional tures were also treated with cytosine arabinoside (3 mg/

mL) for 24–48 h beginning immediately after transfec-mechanism. However, calcium release from the SRtion. Cells for RNase protection were treated with drugsdecreased all nAChR subunit RNAs by decreasingon day 8 postplating. Control cells were treated with vehi-transcription from their promoters. Most important,cle used to dissolve the drug.we show that these drugs mediate their effects by

acting through the same DNA elements previouslyshown to mediate activity-dependent control of geneexpression. Taken together, these results suggest Expression Vectors andthat in rat, muscle activity mediates its effects on Transfection AssaysnAChR gene expression, at least in part, by stimu-

The chick nAChR 850 bp a-subunit promoter was kindlylating calcium release from the SR. Although inprovided by J. P. Merlie (Merlie and Kornhauser, 1989).contradiction to some recent work (Huang andThis DNA was digested with BglI, blunted, and thenSchmidt, 1994; Huang et al., 1994), these results aredigested with HindIII to release a 116-bp a-promoterconsistent with older reports suggesting SR calciumfragment that was subcloned into SmaI /HindIII-digestedregulates nAChR protein levels (Birnbaum et al.,pXP1 (Nordeen, 1988). This generates an expression

1980; Pezzementi and Schmidt, 1981; Forrest et al., vector harboring an a-promoter fragment spanning nucle-1981). otides 0116 to /20 driving luciferase expression. The

a-subunit promoter mutants Mp and Mb have been pre-viously described (Piette et al., 1990). Mutations werecreated by site-directed mutagenesis using oligonucleo-MATERIALS AND METHODStide primers and confirmed by DNA sequencing. TheCMVCAT, mouse g-, and rat d- and 1-promoter/ lucifer-RNA Isolation and RNasease expression vectors have been previously describedProtection Assay(Gilmour et al., 1995; Walke et al., 1994, 1996). The ratd-promoter expression vector is a chimera containing theTotal RNA was isolated using the method of Chirgwin et

al. (1979). Antisense RNA probes used to detect muscle rat d-promoter’s 47-bp activity-dependent enhancer up-stream the minimal enkephalin (MEK) promoter (Walkecreatine kinase (MCK) and nAChR a, b, g, d, and 1-

RNAs were described previously (Chahine et al., 1993). et al., 1996). This construct is more active than thatlacking the MEK sequences. The MEK promoter was notRNase protection assays were carried out as previously

described (Walk et al., 1994; Saccomanno et al., 1992). regulated by calcium (Walke et al., 1994). CMVCATwas not influenced by ryanodine or (0)Bay K 8644 inThe MCK probe was included in every RNase protection

assay and served to normalize for different amounts of our muscle culture system (CAT activity ratio for ryano-dine/control was 0.99 { 0.12 averaged over 30 experi-RNA in the various samples. The MCK RNA also serves

to indicate if the cellular perturbations we imposed on ments; CAT activity ratio for Bay K 8644/control was1.1 { 0.12 averaged over seven experiments) and there-the cultured muscle cells had a general effect on myo-

genic factor activity, since the MCK RNA is induced by fore used for normalization purposes. Myotube cultureswere transfected using calcium phosphate DNA precipi-myogenic factors upon muscle differentiation in a similar

fashion as the nAChR genes. MCK RNA was not regu- tates as previously described by Chahine et al. (1992).CMVCAT was used at 3 mg/plate and the various nAChRlated by any of the conditions employed in this report.

RNase protection assays were repeated at least twice and wild-type and mutant expression vectors were used at10–20 mg/plate. Total DNA was adjusted to 30 mg/platequantitated by scanning densitometry. Specificity of pro-

tected bands was confirmed by hybridizing probes to using BSSK(/) (Stratagene) DNA. Luciferase and CATassays were performed as previously described (BrasiertRNA, which resulted in no protected fragments on the

gel. In addition, probe integrity was monitored by omit- et al., 1989; Neumann et al., 1987). Experiments wereperformed in triplicate and repeated at least twice.ting the RNase step during the RNase protection protocol.


Calcium-Dependent nAChR Gene Expression 247

Drugs and Reagents

Ryanodine, thapsigargin, dantrolene, and the (/) and(0) enantiomers of Bay K 8644 were purchased fromRBI (Natick, MA). A23187 was obtained from Sigma(St. Louis, MO). Tetrodotoxin was purchased from Mo-lecular Probes (Eugene, OR). Ryanodine was preparedfresh as either a 1- or 10-mM stock as described pre-viously (Pezzementi and Schmidt, 1981). Dantrolene andthe (/) and (0) enantiamers of Bay K 8644 were pre-pared as 10-mM stocks in dimethyl sulfoxide (DMSO).Thapsigargin and A23187 were prepared as 1-mM stocksin DMSO.

RESULTS

Calcium-Dependent Regulationof nAChR RNA Levels

To determine if calcium influx across the plasmamembrane and/or efflux from the SR could regulatenAChR expression, we assayed nAChR RNA levelsin rat primary myotube cultures treated with variouscalcium-perturbing pharmacological reagents. Ry-anodine and thapsigargin increases calcium effluxfrom the SR by activating the ryanodine receptor(Meissner, 1968; Buck, 1992) and inhibiting SRcalcium transport ATPase activity (Sagara and In-esi, 1991), respectively. In contrast, Bay K 8644specifically acts on L-type calcium channels thatcontrol calcium influx across the plasma membrane

Figure 1 Calcium-dependent regulation of nAChR(Smilowitz, 1988; Reuter et al., 1985). We alsoRNA levels. Primary myotubes were treated with either

used the calcium ionophore A23187, which is ex-1.0 mM ryanodine, 150 nM thapsigargan, 20.0 mM Bay

pected to equilibrate calcium across all membranes. K 8644, or 1.0 mM A23187 for 48 h prior to harvestingSimilar to previously published results (Walke cells and isolating RNA. Muscle nAChR and creatine

et al., 1994), we found A23187 suppressed expres- kinase (MCK) RNA levels were revealed by RNase pro-sion of all five nAChR subunit RNAs (Fig. 1) . For tection assays. (Top left) Typical RNase protection assay.the 1-RNA, we had previously shown that A23187- The graphs are quantitation of multiple RNase protection

assays. Quantitation of RNase resistant hybrids was per-mediated suppression required calcium influx fromformed by scanning densitometry. nAChR RNA levelsextracellular stores (Walke et al., 1994). By usingwere normalized to MCK RNA levels to correct for dif-the L-type calcium channel agonist (0)Bay Kferent RNA yields between the different samples. RNase8644, we confirmed this result and showed that 1-protection assays were repeated at least twice with differ-RNA levels decreased approximately 75% by cal-ent samples of muscle RNA. Error bars are { standard

cium influx across the plasma membrane (Fig. 1) .deviation.

In contrast, (0)Bay K 8644 had no effect on b- org-RNA levels and reduced a- and d-RNA levelsonly by about 20% and 40%, respectively (Fig. 1) . (ryanodine and thapsigargin) and was reduced by

only 25–45%. We have assayed the response ofIn contrast to the relatively specific and dramaticaction of (0)Bay K 8644 on the 1-RNA, ryanodine the a-subunit RNA to 1 mM ryanodine in over 10

independent experiments. Although ryanodine gen-and thapsigargin caused a decrease in all the nAChRRNAs (Fig. 1) . The degree of repression varied erally causes a 75–85% decrease (Figs. 1 and 2; see

also Fig. 4) in a-RNA levels, we observed effects asfrom about 75–88% for the a- and g-RNAs to ap-proximately 50% for the b- and d-RNAs. Interest- small as 60% (see Fig. 7) . Although we do not

know the reason for this variability, it may reflectingly the 1-RNA was least responsive to these drugs



Figure 2 Concentration-dependent effect of ryanodine on nAChR RNA levels. Primary myo-tubes were treated with various concentrations of ryanodine for 48 h prior to harvesting cellsand isolating RNA. Muscle nAChR a-, 1-, and g-subunits and creatine kinase (MCK) RNAlevels were revealed by RNase protection assays. (Top) Representative RNase protectionassays. (Bottom) Quantitation of RNase protection assays. Relative RNA levels were deter-mined by scanning densitometry and dividing nAChR RNA values by MCK RNA values (seeMaterials and Methods) . RNase protection assays were repeated at least twice with differentsamples of muscle RNA. Error bars are { standard deviation.

slight variations in our ryanodine stock solution Forty-eight-hour exposure to ryanodine resulted ina concentration-dependent decrease in nAChR pro-and/or the developmental stage of our primary mus-

cle cultures. These changes in nAChR RNA levels moter activity (data shown for the 116-bp a-pro-moter) (Fig. 3) , similar to the response of the en-in response to ryanodine and thapsigargin are simi-

lar to those previously documented for active and dogenous RNA. Maximal inhibition was observedinactive myotubes (Chahine et al., 1993). at 1 mM ryanodine, which decreased a-promoter

Ryanodine is unusual in that it stimulates calcium activity by approximately 66%, while 100 mM ryan-release from the SR at concentrations around 0.1– odine had little effect (Fig. 3) . Also, 1 mM ryano-10 mM and begins to block the release of SR cal- dine suppressed expression from the 388-bp g-pro-cium at concentrations above 10 mM (Meissner, moter (63% reduction), 102-bp d-promoter (72%1968; Buck et al., 1992; Delbono and Chu, 1995). reduction), and 5-kb 1-promoter (69% reduction).This concentration-dependent effect of ryanodine Within 6 h following exposure to 1 mM ryano-on calcium release was also reflected in a- and g- dine, the a-promoter was already suppressed ap-RNA levels (Fig. 2) . In contrast, the 1-RNA re- proximately 50% (Fig. 4, bottom). However, wesponded similarly to low (1-mM) and high (100- did not detect a significant decrease in a-RNA untilmM) concentrations of ryanodine (Fig. 2) , which 12 h after drug treatment (Fig. 4, top). These resultsmay indicate a nonspecific effect on this RNA. are not surprising since transcriptional changes usu-Nonetheless, these experiments and others (Fig. 1) ally precede changes in RNA levels which are gov-consistently revealed that ryanodine was less effec- erned by both transcription and stability.tive at reducing 1-RNA levels compared to the other In contrast to ryanodine, (0)Bay K 8644 hadnAChR RNAs. only a small effect on nAChR a-promoter activity,

resulting in approximately a 20–25% decrease afterCalcium Release from the SR 48 h (Fig. 5, top). In addition, none of the otherSuppresses nAChR Gene Expression nAChR promoters were regulated by (0)Bay K

8644 at concentrations ranging from 1 to 30 mMTransient transfection assays were used to examinethe effect of ryanodine on nAChR promoter activity. (data only shown for the 1-promoter) (Fig. 5, bot-



Figure 3 Concentration-dependent effect of ryanodineon nAChR a-subunit promoter activity. Primary musclecultures were cotransfected with a 116-bp chick a-pro-moter/ luciferase expression vector and CMVCAT.Transfected cells were treated with varying concentra-tions of ryanodine for 48 h prior to harvesting andassaying reporter gene expression. The CMVCAT vectoris not regulated by ryanodine and was used for normaliza-tion purposes. Bar graphs are the average of triplicatetransfections normalized to CAT activity. Error bars are{ standard deviation.

tom). This result suggests that the specific effect of(0)Bay K 8644 on nAChR 1-subunit RNA (Fig. 1)

Figure 4 Time course of nAChR RNA and promoterlikely results from a post-transcriptional regulation.activity changes in response to ryanodine. (Top) Pri-mary myotubes were treated with 1.0 mM ryanodinefor 0, 6, 12, 24, and 48 h. Muscle creatine kinaseDNA Sequences Mediating the Effects(MCK) and the nAChR a-subunit RNA (a ) levelsof Ryanodine Map to Those That Arewere determined by RNase protection assays (repre-Necessary for Activity-Dependentsentative autoradiogram shown above) and quantitatedRegulationby scanning densitometry. Relative RNA values are

The above data suggest that calcium release from nAChR a-subunit RNA levels normalized to MCKRNA levels. (Bottom) Primary muscle cultures werethe SR can suppress nAChR gene expression. Be-cotransfected with the 116-bp chick a-promoter / lucif-cause calcium release from the SR is an activity-erase construct and a CMVCAT vector ( for normaliza-dependent process, and because the nAChR promot-tion purposes ) and treated with 1.0 mM ryanodine forers are also regulated by muscle activity, it seemedfor 2, 6, 12, 24, and 48 h prior to harvesting andreasonable to hypothesize that the same DNA ele-assaying reporter gene expression. The normalized lu-ments mediating activity-dependent expression alsociferase value for control (0 h) is 13,041 { 2201 light

mediated the response of these genes to ryanodine. units. Bar graphs are the average of triplicate transfec-Three nAChR subunit gene promoters (a, g, and tions normalized to CAT activity. Asterisks above his-

d) have been characterized in sufficient detail to tograms indicate statistical significance at p õ 0.05identify DNA residues necessary for activity-depen- using Student t test. Error bars are { standard devia-dent regulation [Fig. 6(A)] . The 116-bp a-subunit tion.



promoter contains two E boxes necessary for activ-ity-dependent regulation in primary chick myotubesand the mouse C2C12 cell line (Bessereau et al.,1994; Su et al., 1995). Mutation of either E-box[Mp or Mb in Fig. 6(A)] partially influenced activ-ity-dependent gene expression, while mutation of bothsequences completely abrogated this regulation (Bes-sereau et al., 1994; Su et al., 1995). Here, we showthat both E-boxes must also be mutated to block thesuppressive effects of ryanodine [Fig. 6(B)].

We had previously identified a 46-bp region ofthe g-subunit gene promoter (nucleotides 0114 to0159) that is necessary for its suppression by mus-cle activity (Gilmour et al., 1995). A minimal g-promoter construct, pX2BG76 [Fig. 6(A)] , lackingresidues 0114 to 0159 is not regulated by muscleactivity. However, when nucleotides 0114 to 0159are placed upstream of this minimal promoter(pX2BGE76) [Fig. 6(A)] , activity-dependent ex-pression is restored (Gilmour et al., 1995). Like-wise, pX2BGE76 promoter activity is suppressedby ryanodine, while pX2BG76 is not [Fig. 6(B)] .

Finally, we had also previously reported the iden-tification of a 47-bp enhancer (d 047/01) that isnecessary and sufficient to mediate the effects ofmuscle activity on d-subunit gene expression(Walke et al., 1996). This enhancer appears to becomposed of three elements (SV, Sp, and E), eachof which when mutated completely abrogates regu-lated expression in response to muscle activity(Walke et al., 1996). Expression vectors were cre-ated in which the wild-type or mutant 47-bp en-hancer were placed upstream of the minimal en-kephalin (MEK) promoter (Walke et al., 1996)[Fig. 6(A)] . The MEK promoter is not regulatedby calcium (Walke et al., 1994) or muscle activity(Walke et al., 1996). The wild-type construct (d047/01) showed approximately a 75% reductionin activity in response to ryanodine, while the vari-ous mutants (d 047/01 SV mut, Sp mut, and Emut) were insensitive to ryanodine [Fig. 6(B)] .

We also tested these wild-type and mutated pro-moter constructs for responsiveness to thapsigargin.As reported for ryanodine, wild-type promoterswere repressed by thapsigargin, while mutant pro-moters were not (L. Adams and D. Goldman, un-published observation). Therefore, elements medi-

Figure 5 Effect of (0)Bay K 8644 on nAChR a- and1-subunit promoter activity. The luciferase gene, placedunder control of the 116-bp chick a-promoter or the 5-kb 1-promoter, was transiently transfected into myotubesalong with the CMVCAT vector (for normalization pur- and assaying reporter gene expression. Bar graphs areposes) . Cells were then treated with 30.0 mM (0)BayK the average of triplicate transfections normalized to CAT8644 or buffer for 2, 6, 12, 24, and 48 h prior to harvesting activity. Error bars are { standard deviation.



ating nAChR promoter repression by calcium re- mediate activity-dependent expression of thesegenes. Therefore, we propose that in rat, SR calciumlease from the SR map to those that mediate

activity-dependent expression. is a source of cytoplasmic calcium that contributesto reduced nAChR gene expression in active com-pared to inactive muscle.The Effects of Ryanodine on nAChR

The observation that activation of ryanodine re-Gene Expression Are Blockedceptor calcium channels in the sarcoplasmic reticu-by Dantrolenelum caused decreased nAChR gene transcription,while activation of sarcolemmal L-type calciumDantrolene inhibits calcium release from the SR

(Ellis and Carpenter, 1972; Nelson et al., 1996). channels specifically affected 1-RNA expressionpost-transcriptionally, suggests that specific signal-We used this drug to determine whether we could

suppress the effects of ryanodine on nAChR gene ing cascades are linked to specific channel types.However, we have not ruled out the possibility thatexpression. If ryanodine mediates its effects on

nAChR expression by stimulating calcium release these two different calcium channels mediate theireffects on nAChR gene expression by raising intra-from the SR, then dantrolene should reduce this

effect. However, if ryanodine mediated its effects cellular calcium to different levels. The idea of dis-tinct signaling cascades being coupled to particularindependently of calcium release from the SR, dan-

trolene might be observed to have little effect. calcium channels is not new. For example, calciuminflux through NMDA receptors and L-type calciumIndeed, in the presence of dantrolene, myotubes

exhibited a dramatically reduced ryanodine re- channels mediate their effects on c- fos promoteractivity by activating distinct signaling pathways insponse compared to myotubes that were not treated

with dantrolene (Fig. 7) . Increasing amounts of neurons (Ghosh et al., 1994).Therefore, we recognize the possibility that adantrolene blocked the ryanodine-dependent sup-

pression of nAChR RNA and promoter activity in specific source of intracellular calcium might playan important role in mediating activity-dependenta dose-dependent manner up to approximately 75%

of control (Fig. 7) . In addition, dantrolene alone gene expression. Our results are surprising in lightof recent studies indicating that calcium fluxwas able to modestly increase nAChR RNA and

promoter activity by approximately 20% and 40%, through L-type channels, but not from SR stores,suppresses nAChR gene expression in chick and therespectively. In contrast, use of the L-type calcium

channel antagonist (/)Bay K 8644 in place of dan- mouse muscle cell line C2C12 (Huang and Schmidt,1994; Huang et al., 1994). The reasons for thistrolene in the above experiments had no effect on

nAChR gene expression (L. Adams and D. Gold- apparent discrepancy are not known. However, it ispossible that the rat and chick nAChR genes re-man, unpublished observation). These experiments,

along with the use of other SR-acting drugs such spond to calcium-dependent signaling emanatingfrom different calcium sources. In addition, itas thapsigargin, support the idea that suppression of

nAChR RNA and promoter activity by ryanodine should be kept in mind that the C2C12 cell line isa transformed muscle cell that may have acquiredis largely a result of calcium release from the SR.

However, we cannot rule out the possibility, albeit many changes leading to its transformed phenotype.Differences between this cell line and the primaryremote, that dantrolene could increase nAChR RNA

and promoter activity by another, unknown mecha- rat muscle cultures used in this study may includesignaling cascades and SR development (Grassi etnism.al., 1994). In fact, the observation that ryanodinewas unable to block depolarization-triggered cal-cium release from the SR and the partial effective-DISCUSSIONness of dantrolene in C2C12 cells (Huang et al.,1994) are consistent with this latter possibility.The major findings reported here are: (a) L-type

channel agonist Bay K 8644 had no effect on RNase protection and transient transfectionassays revealed a relatively robust effect of ryano-nAChR promoter activity, yet specifically sup-

pressed the nAChR 1-subunit RNA levels; (b) dine and thapsigargin on nAChR gene expression(Figs. 1, 4, and 6). Our observation that in additionnAChR RNA levels and promoter activity are sup-

pressed by treatment with drugs that have been dem- to decreasing a-, g-, and d-RNA and promoter ac-tivity ryanodine also suppressed 1-promoter activityonstrated to release calcium from the SR; and, fi-

nally, (c) nAChR promoter sequences mediating (69% reduction) is consistent with transgenic stud-ies showing the 1-promoter is regulated by musclethis suppression map to those previously shown to



Figure 6 nAChR promoter elements mediating the effects of ryanodine map to sequencesthat are necessary for activity-dependent gene expression. (A) Schematic representation ofa-, g-, and d-promoter expression vectors used in this study. a116 contains the 116-bp chicka-promoter driving luciferase expression. DNA sequence changes in mutants Mp and Mb areindicated with arrows (Piette et al., 1990). gpX2BG169 contains a 150-bp g-promoter drivingluciferase expression. gpX2BG76 is a minimal 57-bp g-promoter driving luciferase expression.gpP2BGE76 is a fusion construct in which g-promoter sequences0159 to0114 were ligated togpX2BG76 (Gilmour et al., 1995). d-47/01 contains the d-promoter muscle-specific, activity-dependent enhancer upstream of the minimal enkephalin (MEK) promoter. Nucleotide changesin mutants SV, Sp, and E are indicated by arrows (see Walke et al., 1996, for details) . (B)The luciferase gene, placed under control of wild-type or mutated fragments of the nAChRa-, g-, and d-subunit gene promoters were transiently transfected into myotubes along withthe CMVCAT vector (for normalization purposes) . Cells were then treated with ryanodine (1mM) or buffer for 48 h prior to harvesting for luciferase and CAT activity. For each construct,luciferase values represent averages from three different dishes of myotubes and are reportedas a percentage of control cells treated with buffer (100%). The normalized luciferase valuesfor controls, expressed as light units are: a-116 Å 150,477 { 9008; a-116 Mb Å 15,270 { 137;a-116 Mp Å 33,020 { 4445; a-116 Mbp Å 6816 { 2165; g-pXP2BG169 Å 8984 { 3164;g-pXP2BGE76 Å 119 { 20; g-pXP2BGE76 Å 20 { 4; d-47/01 Å 15,636 { 2236; d-47/01SV mut Å 282 { 27; d-47/01 Sp mut Å 249 { 18; d-47/01 E mut Å 328 { 24. Error barsare { standard deviation.



Figure 6 (Continued)

activity in vivo (Gundersen, 1993). However, be- Finally, the modest effect of calcium influx onnAChR a- and d-RNAs may reflect a change incause this promoter directs expression to the syn-

apse and is less responsive to muscle activity than promoter activity that is compensated by an oppos-ing change in RNA stability. This does not appearthe other nAChR promoters, we suggest that other

regulatory cascades may also contribute to 1-pro- to be likely, since the a-promoter was repressed byonly 20–25% following 24–48 h of drug treatment,moter activity and RNA expression profiles. Indeed,

it has recently been shown that the 1-gene is regu- while the 550-bp d-promoter, like the 1-promoter,was not regulated by (0)Bay K 8644 (data notlated by a Ras-dependent signaling cascade (Tansey

et al., 1996; Si et al., 1996). shown). These data suggest a post-transcriptionalmechanism of regulation of these RNAs by calciumIn contrast to the relatively robust effect of ryano-

dine on nAChR RNA levels, (0)Bay K 8644 was influx. However, we cannot rule out the possibilitythat elements mediating a response to (0)Bay Kmore restricted in its effects, causing a large de-

crease in 1-RNA with somewhat lesser of an effect 8644 are not included in our 550-bp d- and 5 kb 1-promoter constructs.on the a- and d-RNAs (Fig. 1) . This may indicate

that these latter RNAs are also regulated by a cal- Might treating muscle cells with calcium-per-turbing agents for periods extending beyond a fewcium influx-dependent signaling pathway. How-

ever, their responsiveness to this pathway may be hours cause such a large or long-lasting change inintracellular calcium that it results in nonspecificless than the 1-RNA owing to differences in their

DNA/RNA calcium-responsive elements. Alterna- effects due to perturbation of multiple regulatoryevents? We do not think so, because our RNasetively, if calcium influx is large enough, it might

begin to impinge upon signaling molecules that nor- protection and transient transfection assays argue infavor of specificity. First, RNase protection assaysmally respond to calcium changes occurring in close

proximity to the SR, modestly activating its signal- showed only drugs (ryanodine and thapsigargin) ,stimulating release of calcium from the SR suppressing cascade and resulting in a relatively modest

RNA change. Similar arguments could also be ap- nAChR gene expression, while the L-type calciumchannel activator specifically affected the adult-typeplied to the relatively modest effect of ryanodine

on 1-RNA expression. specific 1-RNA (Fig. 1) . Second, we showed that



there is a characteristic ryanodine dose response thatfollows the effect of ryanodine on the ryanodinereceptor (Meissner, 1968; Buck et al., 1992) (Figs.2 and 3). Third, we were able to attenuate the effectsof ryanodine on nAChR RNA and promoter activityby adding dantrolene (Fig. 7) , which inhibits cal-cium release from the SR. Fourth, time-courseassays suggest the effect of ryanodine on nAChRpromoter activity is already observable by 6 h afterdrug treatment (Fig. 4, bottom). All these resultsargue for a specific action of ryanodine on nAChRgene expression via its effect on calcium releasefrom the SR.

Based on the results described above, and be-cause calcium is released from the SR during mus-cle contraction, we propose that calcium releasefrom the SR participates in coupling muscle depo-larization to nAChR gene expression in rat. To sup-port this contention, we determined if the sameDNA regulatory elements mediating nAChR pro-moter activity in response to muscle depolarizationalso mediate the effects of ryanodine and thapsigar-gin. Indeed, for the a-, g-, and d-subunit genepromoters, where elements mediating activity-de-pendent expression were mapped, these same activ-ity-dependent sequences were also necessary forregulation by ryanodine and thapsigargin (Fig. 6;data shown only for ryanodine) .

However, it should be noted that the 116-bp a-promoter construct that was used in the above pro-moter study has been shown to be regulated bymuscle activity in a muscle-specific fashion (Merlieet al., 1994). In this case, transgenic animals harbor-ing a 111-bp a-promoter driving CAT expressionshowed activity-dependent expression in tibialis andextensor digitorum longus muscle but no expression

Figure 7 The effects of ryanodine on nAChR RNA in the soleus muscle. It was concluded that the lackand promoter activity are inhibited by dantrolene.

of activity-dependent expression in soleus muscle(Top) Primary myotubes were treated with 1.0 mMwas secondary to the more severe loss of expressionryanodine and /or 10 and 30 mM dantrolene for 48 h.in this muscle (Merlie et al., 1994). In addition,Muscle creatine kinase (MCK) and the nAChR a-sub-this same promoter, when placed in an adenovirusunit RNA (a ) levels were determined by RNase protec-vector, was suppressed by muscle activity in celltion assays ( representative autoradiogram shown at

top) and quantitated by scanning densitometry ( top) . culture but was unresponsive to muscle activity inRelative RNA values are nAChR a-subunit RNA levels vivo (Bessereau et al., 1994). Because the musclesnormalized to MCK RNA levels. (Bottom) Primary assayed in this latter study were not reported, it ismuscle cultures were cotransfected with the chick 116- difficult to determine if this lack of an activity-bp a-promoter / luciferase construct and CMVCAT (for dependent response in vivo was a result of the typenormalization purposes) . Following transfection, cells of muscle assayed. Nonetheless the a- and d-pro-were treated with 1.0 mM ryanodine and /or 10 and 30

moters used in this study clearly contain elementsmM dantrolene for 48 h prior to harvesting and assaying

mediating activity-dependent gene expression inreporter gene expression. Bar graphs are the averagevivo (Merlie et al., 1994; Tang et al., 1994; Walkeof triplicate transfections. Percent luciferase activityet al., 1996).represents normalized luciferase values reported as a

Might calcium influx also contribute to activity-percentage of the control cell value. Error bars are{ standard deviation. dependent expression of nAChR genes in rat skele-



This work was supported by grants from the Nationaltal muscle? This is a difficult question to answer atInstitutes of Health and Muscular Dystrophy Associationthis point. We clearly see some relatively modest(awarded to DG). The authors thank Dr. P. Gardner forregulation of the nAChR RNAs by calcium influx,providing the mouse g-promoter clones, Dr. J. P. Merlieand this may contribute to activity-dependent sup-for providing the chick a-promoter, W. Walke and M.

pression of these genes in vivo. However, we haveSapru for technical assistance, K. Williams and N. Go-

not been able to map this response to the nAChR burdhun for their help in DNA preparations and cell cul-promoter elements already identified as participat- ture, and Dr. R. Hume for critical reading of the manu-ing in activity-dependent control of gene expres- script.sion. One possibility is that promoter elements re-sponding to calcium influx are not contained withinthe promoter fragments used in this study. REFERENCES

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