Opposite Effect of Intracellular Ca2+ and Protein Kinase C on the Expression of Inwardly Rectifying K+Channel 1 in Mouse Skeletal Muscle*

The level of inwardly rectifying K+ channel 1 (IRK1) mRNA decreased upon denervation and increased during muscle differentiation in mouse skeletal muscle. To identify the mechanism(s) underlying the regulation of IRK1 mRNA expression, we examined its expression using the well differentiated C2C12 mouse skeletal muscle cell line as a model system. Since nerve-induced muscle activity results in contraction, it was questioned whether the changes in IRK1 expression might be relevant to the increased intracellular calcium that functions as a cytoplasmic messenger in excitation-contraction coupling. Indeed, activation of either l-type calcium channels or ryanodine receptors increased the level of IRK1 mRNA. More directly, ionomycin activated the IRK1 expression in time- and dose-dependent manners, which was abolished by treatment with EGTA. Genistein, a tyrosine kinase inhibitor, also abolished the stimulating effect of ionomycin. Meanwhile, activation of protein kinase C by 12-O-tetradecanoylphorbol acetate (TPA) markedly decreased the level of IRK1 mRNA, which required ongoing protein synthesis. Actinomycin D experiments revealed that ionomycin increased the half-life of IRK1 mRNA from 0.86 to 1.97 h, but TPA decreased it to 0.38 h. However, neither ionomycin nor TPA appreciably altered the rate of IRK1 gene transcription. Based on these observations, we conclude that intracellular calcium and protein kinase C are oppositely involved in the muscle activity-dependent regulation of IRK1 gene expression and that both act at the level of mRNA stability.

The level of inwardly rectifying K ؉ channel 1 (IRK1) mRNA decreased upon denervation and increased during muscle differentiation in mouse skeletal muscle. To identify the mechanism(s) underlying the regulation of IRK1 mRNA expression, we examined its expression using the well differentiated C2C12 mouse skeletal muscle cell line as a model system. Since nerve-induced muscle activity results in contraction, it was questioned whether the changes in IRK1 expression might be relevant to the increased intracellular calcium that functions as a cytoplasmic messenger in excitation-contraction coupling. Indeed, activation of either L-type calcium channels or ryanodine receptors increased the level of IRK1 mRNA. More directly, ionomycin activated the IRK1 expression in time-and dose-dependent manners, which was abolished by treatment with EGTA. Genistein, a tyrosine kinase inhibitor, also abolished the stimulating effect of ionomycin. Meanwhile, activation of protein kinase C by 12-O-tetradecanoylphorbol acetate (TPA) markedly decreased the level of IRK1 mRNA, which required ongoing protein synthesis. Actinomycin D experiments revealed that ionomycin increased the half-life of IRK1 mRNA from 0.86 to 1.97 h, but TPA decreased it to 0.38 h. However, neither ionomycin nor TPA appreciably altered the rate of IRK1 gene transcription. Based on these observations, we conclude that intracellular calcium and protein kinase C are oppositely involved in the muscle activity-dependent regulation of IRK1 gene expression and that both act at the level of mRNA stability.
It has been known that denervation influences many biophysical and biochemical properties of skeletal muscle fibers (1)(2)(3)(4)(5)(6)(7). The mechanisms by which denervation initiates these changes are still unclear. They may be caused by the loss of neurotrophic factors normally released from the nerve terminals (8,9). Alternatively, the electrical inactivity of the denervated muscle might be responsible, since direct electrical stimulation to the denervated muscle restores all passive electrical parameters of the membrane that were observed without denervation (10, 11). Considering the importance of calcium in the process of muscle contraction induced by neural activity, calcium may play a critical role in linking the biochemical and biophysical changes with muscle activity.
Calcium is known to be involved in many cellular events as a second messenger. A regulatory role of calcium in the expression of sodium channels, acetylcholinesterase, and nicotinic acetylcholine receptor has been suggested (12)(13)(14)(15)(16). In addition, it has recently been shown that calcium influx blocks the expression of nicotinic acetylcholine receptor ␣-subunit gene in chick skeletal muscle (17). There are reports that coupling of the electrical activity with altered gene expression is mediated by protein kinase C (PKC) 1 pathway (18,19). Gonoi and Hasegawa (20) demonstrate by using a patch clamp method that innervation of skeletal muscle fibers plays a key role in the induction and maintenance of inwardly rectifying K ϩ currents in mouse flexor digitorum longus muscle. The resting potential of many excitable cells, including skeletal muscle, is determined by resting potassium conductance of IRK that shows inward rectification, allowing potassium ions to move more readily inward the cell membrane than outward. Katz (21) first describes inward rectification of the resting K ϩ conductance of frog skeletal muscle. Since then, electrophysiological properties of this conductance have been studied by a number of investigators (22,23), and subsequently the channel has been cloned from a mouse macrophage cell line (24).
The present work aims at elucidating the molecular mechanisms involved in the neural and developmental regulation of IRK1 expressions. Here we suggest that intracellular calcium and PKC oppositely regulate the expression of IRK1 mRNA in mouse skeletal muscle and both regulations are associated with mRNA stability.

EXPERIMENTAL PROCEDURES
Materials-[␣-32 P]dCTP and [␣-32 P]UTP and nylon membrane (Nytran) were purchased from NEN Life Science Products and Schleicher & Schuell, respectively. TPA, ryanodine, trifluoperazine, and Bay K 8644 were purchased from Research Biochemical Inc., and 8-bromo-cyclic GMP, dibutyryl cyclic AMP, genistein, and ionomycin were from Sigma. Culture dishes were purchased from Corning Glass, and other culture reagents were obtained from Life Technologies, Inc.
Cell Culture-C2C12 cells were plated at a density of 3 ϫ 10 4 cells/ml in growth medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution) and cultured at 37°C for 2 days. Differentiation from myoblasts to myotubes was induced by changing the growth medium with differentiation medium (Dulbecco's modified Eagle's medium with 2% horse serum and 1% antibiotic-antimycotic solution). Experiments were routinely done at 6 days after the medium change.
Surgery-For denervation studies, 8-week-old ICR mice weighing about 25 g were anesthetized with avertin (0.014 -0.018 ml of 2.5% avertin/g of body weight), and a 5-mm length of the right sciatic nerves at the upper thigh were cut out. The transection totally denervated the muscles of the lower leg. A sham operation was performed on the contralateral side of all denervated animals, and the contralateral innervated muscles were used as controls. At various times after the denervation, denervated and control muscles were isolated and prepared for RNA isolation.
Northern Blot Analysis-Total RNA was isolated from mouse skeletal muscles and from cultured C2C12 muscle cells using the guanidinium thiocyanate-acidic phenol method (25). RNA was dissolved in 0.5% SDS and denatured in the presence of 50% formamide, 2.2 M formaldehyde, 20 mM MOPS, 4 mM sodium acetate, 0.5 mM EDTA at 60°C for 5 min. Aliquots (15-30 g) of RNA were size-fractionated by electrophoresis on a 1% (w/v) formaldehyde-denaturing-agarose gel and were transferred to Nytran membranes by capillary blotting.
Nytran membranes were placed in a polyethylene heat-sealable bag and prehybridized with hybridization buffer (5 ϫ saline/sodium/phosphate/EDTA, pH 7.4, 5 ϫ Denhardt's solution, 0.5% SDS, 0.2 mg/ml fragmented, denatured salmon sperm DNA, and 50% formamide) at 42°C for 3 h. Hybridization was carried out at 42°C for 2 days with heat-denatured IRK1 cDNA probe (5 ϫ 10 6 cpm/ml). After hybridization, the membranes were washed twice in 2 ϫ SSC (1 ϫ SSC: 150 mM NaCl, 15 mM citrate, pH 7.0), 0.1% SDS at room temperature for a total of 20 min, then once in 0.1ϫ SSC, 0.1% SDS at 42°C for 20 min. Membranes were exposed to x-ray film at Ϫ70°C for 1-7 days. After autoradiography, the probe was stripped off the membrane by incubation in distilled water at 100°C for 10 min. Membranes were then rehybridized with other control probes under the same conditions. Northern blot experiments were repeated at least three times with reproducible results.
Hybridization Probes-Preparation of hybridization probes was performed as described previously (26). The total RNA isolated from mouse skeletal muscle was reverse-transcribed in the presence of random hexamer (Boehringer Mannheim). For a polymerase chain reaction cloning of the IRK1 cDNA from mouse skeletal muscle, reverse-transcribed products were used as templates. The 5Ј and 3Ј primers were 5Ј-CGAGACCCAGACAACCAT-3Ј and 5Ј-TCCCCCATCACTATCGTT-3Ј, corresponding to the 411-428 and 793-810 nucleotide sequences of IRK1 cDNA as described previously (24). The fragments (400 base pairs) obtained were ligated into the EcoRV site of pBluescript KS(ϩ), and sequences were analyzed. The sequences of the cDNA fragments were identical to those of IRK1 in J774 mouse macrophage cell line (24). 32 P-labeled antisense DNA probes were synthesized from linearized plasmids containing IRK1 fragments using Taq polymerase (Promega) and 3Ј primer.
Nuclear Run-on Analysis-Nuclei isolation and nuclear run-on transcription assays were performed as described by Greenberg and Bender (27). After cells were harvested by centrifugation, the pellets were resuspended in lysis buffer (10 mM Tris, pH 7.4, 3 mM CaCl 2 , and 2 mM MgCl 2 ), centrifuged for 5 min at 500 ϫ g and resuspended in the same lysis buffer containing 0.5% Nonidet P-40. The cells were then broken in a Dounce homogenizer, and nuclei were sedimented at 500 ϫ g for 5 min. The nuclei were resuspended in 50 mM Tris-HCl, pH 8.3, containing 40% glycerol, 5 mM MgCl 2 , and 0.1 mM EDTA and mixed with equal volume of 2 ϫ reaction buffer containing 10 mM Tris-HCl, pH 8.0, 5 mM MgCl 2 , 0.3 M KCl, 5 mM dithiothreitol, 1 mM each ATP, GTP, and CTP and 100 Ci of [␣-32 P]UTP and incubated at 37°C for 30 min for in vitro transcription. Radiolabeled mRNA was isolated and hybridized for at least 36 h at 65°C to slot-blotted membranes containing 5 g of linearized plasmid containing IRK1 or GAPDH insert. After extensive washing with 2 ϫ SSC, membranes were exposed to x-ray film at Ϫ70°C.

Neural and Developmental Regulation of IRK1 mRNA Ex-
pression-It has been demonstrated using a patch clamp method that innervation of skeletal muscle fibers plays a key role in the induction and maintenance of inwardly rectifying currents in mouse flexor digitorum longus muscle (20). To further clarify the role of innervation on IRK1 expression, the effect of chronic denervation on the alteration in the IRK1 mRNA level was examined by Northern blot analysis. Within 1 day after the denervation, the mRNA level in slow twitch soleus muscle (soleus) was dramatically reduced, whereas that in the contralateral muscle remained unchanged (Fig. 1, upper  panel). Similar data were obtained for denervated fast twitch extensor digitorum longus muscle (EDL), although the reduction in the mRNA level was not as obvious as that in denervated soleus. Interestingly, the IRK1 mRNA level in the contralateral EDL was significantly higher than that seen in the same muscle without denervation (Fig. 1, lower panel). Although the reason for this change is not known at present, it is possible that the increase in mRNA level in contralateral EDL may be attributed to the increased use of the contralateral leg. Analogous observations have been made in cat and frog skeletal muscles. Steinbach (28) demonstrates the increase in neuromuscular junctional size in fast twitch muscles contralateral FIG. 1. Changes in the level of IRK1 mRNA in soleus and EDL muscles after denervation. The muscles of 8-week-old mice were denervated by transecting the sciatic nerve as described under "Experimental Procedures." After the denervation, total RNAs were isolated from the denervated and contralateral innervated muscles at the indicated days. They were then hybridized with IRK1, GAPDH, and 18 S rRNA probes. Each sample represents RNA from the muscles of 5 animals.
FIG. 2. Developmental changes of IRK1 mRNA expression both in vivo and in vitro. A, total RNAs from the lower hind limb muscles of mice of different postnatal ages were sequentially probed with indicated probes. Each sample represents RNA from the muscles of 3-10 animals at the same stage of development. MHC, myosin heavy chain. B, total RNAs were isolated from differentiating C2C12 muscle cells at 1, 3, 5, and 7 days. Change in IRK1 expression during in vitro myogenesis was analyzed by Northern blot. The days indicate the time after the medium change from growth medium to differentiation medium for myogenic differentiation. to denervated muscles of cats. In addition, denervation results in an increase in multiple innervation of muscle fibers in contralateral muscles of frog (29). Other works have also shown that muscle twitch time was altered following contralateral denervation (30,31).
We also examined whether IRK1 expression is regulated during muscle development. When Northern blot analysis was carried out using total RNAs in lower leg muscles obtained from mice at different ages, the amounts of IRK1 mRNA increased with muscle development (Fig. 2A). Furthermore, the level of IRK1 mRNA from C2C12 mouse muscle cell line also increased as the myogenic development proceeded (Fig. 2B). These results clearly demonstrate that the IRK1 expression is developmentally regulated.
Calcium Is Responsible for Links between Muscle Activity and Increase of IRK1 mRNA Level-To characterize the factors involved in the neural regulation of IRK1 expression, a series of experiments were carried out in vitro using cultured C2C12 myotubes. Because nerve-induced muscle activity results in muscle contraction, elements of ECC may be shared by the mechanism that links the muscle activity with IRK1 gene regulation. The major components involved in ECC are voltagedependent calcium channels (L-type calcium channels) in transverse tubule membrane and ryanodine receptors in sarcoplasmic reticulum membrane (32). As shown in Fig. 3, chronic depolarization of C2C12 myotubes upon treatment with 40 mM extracellular potassium stimulated the expression of IRK1. Activation of L-type calcium channels by treatment with Bay K 8644 together with 40 mM potassium further increased the amount of IRK1 mRNA. However, the increased expression of IRK1 by depolarization could be reversed upon treatment with L-type calcium channel blocker D600, although the mRNA level is still higher than that seen without any treatment. Because both ryanodine and caffeine have been well known to affect sarcoplasmic reticulum calcium release, they have been used in the assessment of sarcoplasmic reticulum function in controlling cytoplasmic calcium concentrations. Ryanodine in nanomolar concentrations keeps the ryanodine receptor channels to an open state (33), and caffeine activates the ryanodine receptor channels by increasing channel opening probability (34). As shown in Fig. 3, caffeine and ryanodine increased the IRK1 mRNA level. These results suggest that elevation of intracellular calcium level that is associated with ECC may mediate depolarization-IRK1 gene activation coupling.
Consistent with this suggestion, calcium ionophore ionomycin was also found to increase the expression of IRK1 mRNA in a dose-dependent manner (Fig. 4A). Upon treatment of C2C12 myotubes with 0.5 M ionomycin, the expression was increased in a time-dependent manner up to a maximum level at about 2 h after the treatment (Fig. 4B). Moreover, the ionophore elevated the level of IRK1 mRNA only when free calcium was present in the extracellular environment (Fig. 4C).
Genistein Abolishes the Ionomycin-induced Increase of IRK1 mRNA Level-In the nervous system, activity-induced increase in cytoplasmic calcium activates various tyrosine kinase pathways (35). To test whether a certain tyrosine kinase pathway is involved in the increased IRK1 mRNA level by ionomycin, C2C12 cells were incubated with various concentrations of genistein, a tyrosine kinase inhibitor, in the presence of 0.5 M ionomycin for 2 h. As demonstrated in Fig. 5A, genistein inhibited the increase in IRK1 mRNA by ionomycin in a dose-dependent manner, whereas basal IRK1 mRNA level still remained unchanged after the genistein treatment. Therefore, it is likely that tyrosine kinase is somehow involved in calciumdependent IRK1 expression.
Calcium activates a variety of cellular processes. For example, the activation of calcium/calmodulin-dependent protein kinase is a common mechanism mediating the effects of increase in intracellular calcium concentration (36). The calmodulin antagonists, trifluoperazine, however, had no effect on the expression of IRK1 in both control and ionomycin-treated myotubes (Fig. 5B). Several second messenger pathways associated with muscle electrical activity have been demonstrated. Nestler et al. (37) demonstrate that the cyclic GMP level increases upon electrical stimulation. In addition, the suppression of expression of embryonic-type nicotinic acetylcholine receptor genes by muscle activity can be reversed by increasing intracellular cAMP (38). However, neither dibutyryl cAMP nor 8-bromo-cGMP up to 0.5 mM showed any effect on the level of IRK1 mRNA in both control and ionomycin-treated cells (Fig.  5B). These findings exclude the possibility of involvement in the signaling pathway of cyclic nucleotides or calmodulin for the regulation of IRK1 expression.
Activation of PKC Decreases the Level of IRK1 mRNA-PKC activity has been reported to increase in active muscle fibers (39). Huang et al. (18,19) show that a calcium-requiring nuclear PKC mediates depolarization-acetylcholine receptor gene inactivation coupling. To test if the IRK1 expression is also regulated by PKC, C2C12 cells were treated with a phorbol ester, TPA, that is an activator of PKC. Against our expectation, TPA decreased the IRK1 expression in time-and dose-dependent manners (Fig. 6, A and B), and this effect could be prevented upon co-treatment with staurosporine or calphostin C, PKC inhibitors (Fig. 6C). Furthermore, TPA reduced the stimulating effect of ionomycin on the IRK1 expression (Fig.  6C). These results clearly demonstrate that PKC is involved in the decrease in IRK1 mRNA level.
Ionomycin and TPA, both, Act at the Level of IRK1 mRNA Stability-Studies were performed to assess whether ionomycin and TPA alter transcription rate or stability of IRK1 mRNA. First, transcriptional regulation was tested by nuclear run-on analysis. As shown in Fig. 7, treatment of 0.5 M ionomycin or 0.5 M TPA did not appreciably influence the rate of transcription of IRK1 gene. Then, to determine whether ionomycin or TPA alters the stability of IRK1 mRNA, we examined the decay of mRNA levels when the cells were incubated with actinomycin D. As shown in Fig. 8, the level of IRK1 mRNA decayed rapidly in control cells, the half-life being 0.86 Ϯ 0.12 h. Treatment of ionomycin increased the half-life of IRK1 mRNA to 1.97 Ϯ 0.21 h, whereas TPA decreased it to 0.38 Ϯ 0.18 h. The results imply that IRK1 mRNA level is regulated at posttranscriptional level.
Down-regulation by TPA of IRK1 mRNA Level Requires Ongoing Protein Synthesis-To determine whether the regulation of IRK1 mRNA expression requires protein synthesis, C2C12 cells were incubated with 250 M cycloheximide followed by treatment with TPA or ionomycin. As shown in Fig. 9, treatment with cycloheximide abolished the inhibitory effect of TPA on the IRK1 expression. Moreover, the block of protein synthesis further stimulated the increase of ionomycin-mediated IRK1 mRNA, although basal IRK1 mRNA level remained unchanged. Thus, only the PKC-dependent down-regulation of IRK1 mRNA level appears to require de novo protein synthesis. DISCUSSION The expression of IRK in skeletal muscle seems to be subjected to developmental and neural regulations (20,26). To our knowledge, however, nothing is known about the mechanism underlying these phenomena. In this regard, the present work was undertaken to identify molecular mechanism(s) that might be involved in these regulations. One of the most important findings in the present studies is that calcium mediates muscle activity-IRK1 gene activation coupling through mRNA stabilization. In addition, tyrosine kinase-mediated signaling path-way is somehow involved in this regulation. In contrast, it appears that the increase of IRK1 mRNA induced by intracellular calcium does not seem to involve calmodulin-or cyclic nucleotide-dependent pathways since inhibitors or agonists of these cellular components had no effect. Instead, PKC-dependent pathway appears to decrease the level of IRK1 mRNA, and the PKC-mediated down-regulation of IRK1 expression is also modulated at the level of posttranscription.
Since depolarization triggered by neural activity at the neuromuscular junction results in skeletal muscle contraction, it seems possible that elements of the ECC pathway may be shared by the signaling pathway by which muscle activity is conveyed to regulate the IRK1 expression. Contraction of skeletal muscle has been shown to depend directly on sarcoplasmic reticulum calcium release (40). Therefore, calcium may act as a link between muscle activity and IRK1 gene activation. Consistent with this notion, activation of either L-type calcium channels or ryanodine receptors, the major elements of the ECC pathway, was found to increase the level of IRK1 mRNA (Fig. 3). In addition, the involvement of intracellular calcium was further confirmed by the observation that ionomycin induces the IRK1 expression in time-and dose-dependent manners (Fig. 4). Furthermore, the stimulatory effect of the increase in intracellular calcium on IRK1 expression is likely to mediate the tyrosine kinase pathway since a well known tyrosine kinase inhibitor genistein prevented the increase of IRK1 mRNA level by ionomycin (Fig. 5).
Intracellular calcium is an agonist of PKC, and hence elevated calcium levels either by influx or by release should lead to activation of the enzyme. Therefore, it was suspected that the intracellular calcium might increase the IRK1 expression through a PKC-dependent pathway. Nevertheless, treatment with TPA alone significantly reduced the level of IRK1 mRNA (Fig. 6). Yet, it is a well known fact that PKC also depends on intracellular calcium. It is thus no wonder that down-regulation of PKC with long term treatment of TPA or selective PKC inhibitor staurosporine further potentiates the ionomycin-mediated increase in IRK1 mRNA level (data not shown). It seems quite obvious that intracellular calcium can act on up-regulation of IRK1 expression by a mechanism that is distinct from the signaling pathway involving PKC, which instead is involved in down-regulation mechanism.
Stability of mRNA in eukaryotic cells, although not as widely and thoroughly studied as transcriptional control, is a regulated property that can determine the level of expression of a gene (41,42). Elevated intracellular calcium increases IRK1 mRNA level through the stabilization of mRNA as evidenced by the experiments for both nuclear run-on (Fig. 7) and actinomycin D pulse-chase (Fig. 8). PKC also seems to down-regulate IRK1 mRNA level by reducing the mRNA stability. Additionally, it is likely that labile destabilizing factors are involved in regulating IRK1 mRNA stability since inhibition of protein synthesis by cycloheximide is able to prevent inhibitory effect of TPA (Fig. 9). Although the precise mechanism for IRK1 mRNA stabilization and the involved cis elements are not characterized, it is clear that intracellular calcium and PKC oppositely regulate IRK1 expression by modulating the mRNA stability.
The mechanism involving calcium of muscle activity-dependent expression offers some suggestions concerning the developmental change in the IRK1 expression. During the differentiation of skeletal muscle, mononucleate myoblasts align along their bipolar axes and fuse to form multinucleate myotubes (43). David et al. (44) demonstrate that calcium entry is necessary for the onset of myoblast fusion. Therefore, increase in the IRK1 mRNA level concurrent with muscle differentiation is relevant to intracellular calcium increase that is a prerequisite for myoblast fusion. In addition, the increased expression of IRK1 in EDL contralateral to denervated muscle may be due to the increased cytoplasmic calcium caused by increased use of contralateral leg after denervation.
The functional implication for the up-regulation of the IRK1 expression by the increase in intracellular calcium is not obvious at present. One of the speculative roles of IRK1 is its involvement in a pathway that facilitates potassium ion reentry from potassium-loaded transverse tubules after each action potential. Potassium ions tend to accumulate in the lumen of the transverse tubules even under normal conditions, and this accumulation is exaggerated with the prolonged action potentials and thus tends to partially depolarize the muscle fibers and increase their excitability (45). Adrian and Peachey (46) calculate that a single action potential alters the luminal potassium concentration by about ϩ0.3 mM. Moreover, the extracellular potassium concentration elevated physiologically to 8 -9 mM in the vicinity of stimulated skeletal muscles, causing hyperkalemic periodic paralysis (47). The potassium accumulation in the lumen of the transverse tubules can only be dissipated relatively slowly by diffusion out of the mouth of the transverse tubules and by active pumping back into the myoplasm across the transverse tubule wall. Our present findings suggest that IRK1, which is up-regulated by muscle activity through the mechanism involving intracellular calcium, contributes to the uptake of accumulated luminal potassium, thereby preventing the hyperexcitability of stimulated muscle fibers.