Intermediate-conductance Ca2+-activated K+ channel is expressed in C2C12 myoblasts and is downregulated during myogenesis

Bernard Fioretti, Tiziana Pietrangelo, Luigi Catacuzzeno, Fabio Franciolini


We report here the expression in C2C12 myoblasts of the intermediate-conductance Ca2+-activated K+ (IKCa) channel. The IKCa current, recorded under perforated-patch configuration, had a transient time course when activated by ionomycin (0.5 μM; peak current density 26.2 ± 3.7 pA/pF; n = 10), but ionomycin (0.5 μM) + 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (100 μM) evoked a stable outward current (28.4 ± 8.2 pA/pF; n = 11). The current was fully inhibited by charybdotoxin (200 nM), clotrimazole (2 μM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (300 μM), but not by tetraethylammonium (1 mM) or d-tubocurarine (300 μM). Congruent with the IKCa channel, elevation of intracellular Ca2+ in inside-out patches resulted in the activation of a voltage-insensitive K+ channel with weak inward rectification, a unitary conductance of 38 ± 6 pS (at negative voltages), and an IC50 for Ca2+ of 530 nM. The IKCa channel was activated metabotropically by external application of ATP (100 μM), an intracellular Ca2+ mobilizer. Under current-clamp conditions, ATP application resulted in a membrane hyperpolarization of ∼35 mV. The IKCa current downregulated during myogenesis, ceasing to be detectable 4 days after the myoblasts were placed in differentiating medium. Downregulation was prevented by the myogenic suppressor agent basic FGF (bFGF). We also found that block of the IKCa channel by charybdotoxin did not inhibit bFGF-sustained myoblast proliferation. These observations show that in C2C12 myoblasts the IKCa channel expression correlates inversely with differentiation, yet it does not appear to have a role in myoblast proliferation.

  • ATP
  • cell proliferation

myogenesis is a highly regulated process during which proliferating myoblasts withdraw from the cell cycle and fuse to form an ordered array of large, multinucleated muscle fibers that express specific proteins (34). C2C12 myoblasts (2, 37) represent a myogenic cell line that has been widely used as an in vitro model of myogenesis for a variety of biochemical and physiopathological studies (19, 38). When cultured with a high serum concentration, C2C12 myoblasts remain in the myoblast state while continuing to proliferate. On serum withdrawal, these cells arrest proliferation and begin differentiating. Ion channels are centrally involved in this process, with several types being either upregulated or downregulated during the complex steps leading to differentiated muscle fibers (1). Proliferating C2C12 myoblasts express an ATP-induced K+ (KATP) current, and a swelling-activated Cl current (ICl,sw) (14, 33). During myogenesis the KATP current and ICl,sw disappear and other currents appear, such as the TTX-sensitive Na+ current, the delayed-rectifier K+ (DRK) current, the inward-rectifier K+ (Kir) current, and the L-type Ca2+ (CaL) currents (14, 15, 33, 35). In this study we report the expression of the intermediate-conductance Ca2+-activated K+ (IKCa) channel in C2C12 myoblasts and its downregulation during myogenesis.

Previous studies have reported three distinct classes of Ca2+-activated K+ channels (3, 32). Large-conductance Ca2+-activated K+ (BKCa) channels are gated by the concerted action of internal Ca2+ and membrane potential and have a unitary conductance from 100 to 220 pS. In contrast, small-conductance Ca2+-activated K+ (SKCa) and IKCa channels, solely gated by internal Ca2+, have a unitary conductance of 2–20 and 20–80 pS, respectively (3, 10, 11, 32). The IKCa channels (KCa3.1; Ref. 7) have a distinct pharmacological profile, being blocked by the scorpion venom toxin charybdotoxin (CTX) and the antifungal imidazole compound clotrimazole (CTL) but resistant to tetraethylammonium (TEA) and d-tubocurarine (d-TC), effective blockers of BKCa channels and of two of the three subtypes (SK2 and SK3) of SKCa channels, respectively (3, 32). These biophysical and pharmacological properties were used to identify the IKCa current in C2C12 myoblasts. The IKCa channel was previously shown to be expressed in the myogenic mesodermal stem cell line C3H10T1/2 expressing the myogenic regulatory factor 4 (MRF4) (C3H10T1/2-MRF4), required for complete myogenic differentiation (24, 25). In this cell line, the upregulation of IKCa channels sustained by basic FGF (bFGF) was shown to prevent myogenic differentiation while maintaining proliferation (24–26). The results presented here show that the IKCa channel is abundantly expressed in C2C12 myoblasts and is downregulated during myogenesis. Unlike the C3H10T1/2-MRF4 cell line, the IKCa channel in C2C12 myoblasts was not required for proliferation. The IKCa channel may thus represent a marker for undifferentiated C2C12 myoblasts.


Cell culture.

The murine skeletal myoblast cell line C2C12 (at passages 15–30) was obtained from the American Type Culture Collection (ATCC, Rockville, MD; CRL 1772). C2C12 proliferating myoblasts, plated at 2,000 cells/cm2, were maintained in a humidified atmosphere (CO2 5%-air 95%, 37°C) and passaged by standard trypsinization every 3 days (27) in a growth medium (GM) consisting of DMEM supplemented with 20% fetal bovine serum, 4 mM l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (GIBCO BRL, Gaithersburg, MD). For differentiation-committed cells, cells were plated at 6,000/cm2 directly in differentiation medium (DM) consisting of DMEM supplemented with 2% horse serum (HS), 4 mM l-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin (GIBCO). The experiments were carried out both on proliferating myoblasts at days 1–4 after being plated in either GM or bFGF (EuroClone) and on differentiation-committed cells at days 1–4 after plating in DM.


Whole cell (perforated), cell-attached, and inside-out patch-clamp configurations were used for electrophysiological recordings on C2C12 myoblasts. Electrode resistance was 3–5 MΩ for whole cell experiments and 5–10 MΩ for cell-attached and inside-out experiments. Single-channel and whole cell currents were amplified with a List EPC-7 amplifier (List Medical, Darmstadt, Germany), and digitized with a 12-bit analog-to-digital converter (TL-1, DMA interface; Axon Instruments, Foster City, CA). The pCLAMP software package (version 7.0; Axon Instruments) was used. For online data collection, macroscopic and single-channel currents were filtered at 5 and 0.5 kHz and sampled at 20 and 200 μs/point, respectively. Membrane capacitance (Cm) measurements were made by using the Membrane Test routine of the pCLAMP software. Whole cell currents were routinely expressed as current densities (pA/pF) calculated with respect to the measured cell Cm. In cell-attached experiments the membrane potential (Vm,patch) sensed by the patch results from the sum of the applied (pipette) potential and the cell membrane potential (Vm,patch = Vpipette + Vm,cell). During the ATP response, Vm,cell was estimated to reach and remain stable for 5–20 s at about −60 mV (cf. Fig. 4, D and E). The Na+-to-K+ permeability ratio (PNa/PK) for single IKCa channels, bathed in 150 mM internal Na+ and 150 mM external K+ solutions, was assessed with the Goldman-Hodgkin-Huxley (GHK) relationship PNa/PK = exp(RT/ErevF), where Erev is the single-channel current reversal potential and R, T, and F are the gas constant, temperature, and Faraday constant, respectively (8). Experiments were carried out at room temperature (RT; 18–22°C), and the resulting data are presented as means ± SE.

Solutions and drugs.

Macroscopic currents were recorded with the perforated-patch method. With this configuration the bathing physiological salt solution (PSS) contained (in mM) 106.5 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 5 MOPS, 20 glucose, and 30 Na-gluconate at pH 7.25, and the pipette solution contained (in mM) 57.5 K2SO4, 55 KCl, 5 MgCl2, and 10 MOPS at pH 7.2. Electrical access to the cytoplasm was achieved by adding amphotericin B (200 μM) to the pipette solution. Access resistances in the range of 10–20 MΩ were achieved within 10 min after seal formation. In cell-attached recordings the bathing solution contained (in mM) 106.5 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 5 MOPS, 20 glucose, and 30 Na-gluconate at pH 7.25, and the pipette solution contained (in mM) 150 KCl, 2 CaCl2, 2 MgCl2, 5 MOPS, and 10 glucose at pH 7.4. In inside-out single-channel recordings the bathing solution contained (in mM) 150 KCl (NaCl), 1 EGTA-K (EGTA-Na), 5 MOPS, and 1 MgCl2, pH 7.2 at the indicated free Ca2+ concentration, and the pipette solution contained (in mM) 150 KCl, 2 CaCl2, 2 MgCl2, 5 MOPS, and 10 glucose, pH 7.4. All chemicals used were of analytical grade. DMSO, TEA, d-TC, and CTL were purchased from Sigma (St. Louis, MO). CTX was purchased from Alomone Labs (Jerusalem, Israel). We routinely used CTL because of its greater selectivity toward IKCa channels compared with other Ca2+-activated K+ channels. In practice, however, the experiments were usually repeated with CTX. In contrast, in the proliferation assay we only used CTX, because preliminary experiments showed that CTL had toxic actions. Ionomycin, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) were purchased from Tocris Cookson (Bristol, UK). CTL, NPPB, DCEBIO, ionomycin, and amphotericin B were similarly dissolved in stock solutions with DMSO to concentrations of 20, 100, 100, 1, and 50 mM, respectively. Pharmacological agents were dissolved daily in the appropriate solution at the concentrations stated and were bath applied by gravity-fed superfusion at a flow rate of 2 ml/min, with complete solution exchange within the recording chamber in ∼1 min. The maximal DMSO concentration in the recording solution was ∼1%. ATP was applied with a Picospritzer II (General Valve, Fairfield, NJ) via a glass pipette with tip diameter about twice as large as that used for whole cell recording. The glass pipette was normally placed within 40 μm of the patched cell to accelerate the increase in drug concentration at the cell, and to allow brief application times to minimize desensitization of the ATP response or IKCa channel rundown.

Cell proliferation assays.

C2C12 myoblasts were seeded at 3,000 cells/cm and held for 5 h in a synchronization medium containing low-glucose DMEM supplemented with 2% HS, plus 4 mM l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (DM). As assessed by cytofluorimetric analysis, under this condition the cell cycle distribution was: G0/G1 82 ± 7% and G2/S 15 ± 5% (n = 2). Myoblasts were then stimulated with 200 nM CTX, 20 ng/ml bFGF, or 20 ng/ml bFGF in combination with either 200 nM CTX or the src kinase inhibitor PP-2 at 50 μM. PP-2 was dissolved in DMSO. As control, we verified that DMSO, applied to myoblasts at the same concentration used in our experiments, did not significantly interfere with C2C12 growth. CTX and PP-2 were added 2–5 min and 15 min, respectively, before bFGF addition. Cell proliferation was assessed by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) incorporation assays. The MTT assay was carried out by incubating the cells for 3 h with 5 mg/ml MTT and solubilizing the tetrazolium salts precipitated on the cells with 200 μl of DMSO for 30 min (23). The absorbance was read on a Titertek Multiscan Microelisa reader (Flow Laboratories). We considered 16 wells for treatment and repeated the experiments twice. BrdU incorporation assay was performed by treating the cells with 50 μM BrdU (Sigma). Coverslips were then washed with PBS, and the cells were fixed with 95% ethanol for 5 min at RT. Cells were then treated with 2 M HCl for 1 min at RT, washed with PBS, and maintained for 1 h in 10% normal goat serum (NGS) plus 0.2% Triton X-100 in PBS. The cells were then incubated with 1:100 monoclonal mouse anti-BrdU (DAKO) in 10% NGS in PBS for 90 min at 37°C. Cells were then subjected to three washes with PBS for 5 min each at RT and incubation with 1:200 anti-mouse secondary antibody (mouse immunoglobulin biotin, DAKO no. E0354) in PBS for 40 min at RT. After three washes with PBS for 5 min each at RT, we amplified the signal by incubating with AB complex for 45 min (StreptABComplex/HRP, no. K0377, DAKO). After 2 washes with PBS, we revealed the staining with diaminobenzidine and H2O2, per kit directions. The pictures of the cells were acquired at ×200 (Leica DMIL) and recorded on a host computer. From the pictures (10 random fields on 3 distinct coverslips for each treatment) we calculated the mean and SE of BrdU-immunoreactive (IR) cells. The experiments were done in duplicate. Finally, cell cycle distribution was assessed by washing with PBS and incubating for 30 min at 37°C at a density of 106 cells per milliliter of solution containing 250 mg of Na-citrate, 5 mg/ml RNase, 750 μl of Nonidet P-40, and 16.5 mg of propidium iodide dissolved in 200 ml of deionized H2O. After a wash in PBS, 106 cells resuspended in 1 ml of PBS were scanned with a Beckman Epics-XL Coulter cytofluorimeter interfaced to a PC. The experiments were done in duplicate.

Immunocytochemistry for myosin heavy chain proteins.

C2C12 myoblasts were seeded on glass coverslips at 6,000 cells/cm in DM (DMEM with 2% HS, l-glutamine, and antibiotics as described in Cell culture) and allowed to differentiate for 1–4 days. The cells were washed twice with PBS and fixed with 95% ethanol for 5 min at RT. After two washes with PBS, the cells were treated with 2% fetal calf serum in PBS for 30 min at 37°C. The samples were then incubated with 1:50 monoclonal mouse anti-myosin heavy chain antibody (MF20, Hybridoma Bank) in PBS for 90 min at 37°C, washed three times with PBS, and incubated with 1:200 anti-mouse secondary antibody (mouse immunoglobulin biotin, DAKO no. E0354) in PBS for 40 min at RT. After three further washes with PBS (5 min each at RT) we amplified the signal, and the staining was revealed as described in the StreptABComplex/HRP instructions (DAKO no. K0377; see also Cell proliferation assays). The pictures of the cells were acquired and stored as described above. The percentage (mean ± SE) of MF20-IR cells was calculated from the stored pictures by inspecting 10 random fields on three distinct coverslips. The experiments were done in duplicate. Statistical analysis of the data was carried out with Student's paired t-test derived from Prism 2.0 software (GraphPad Software, San Diego, CA).


Intracellular Ca2+ elevation activates IKCa current in C2C12 myoblasts.

Macroscopic and unitary Ca2+-activated K+ currents were recorded from C2C12 myoblasts on application of the Ca2+-mobilizing agent ionomycin (Fig. 1). On myoblasts in the perforated-patch configuration, bathed in PSS and with no applied potential, ionomycin (0.5 μM) evoked transient outward currents with mean peak current density of 26.2 ± 3.7 pA/pF (n = 10; Fig. 1, A and B). Measurements of the reversal potential of the ionomycin-induced current were used to assess its selectivity. Voltage ramps from −150 to +150 mV were applied during the ionomycin-induced response, and the reversal potential of the current was determined as the voltage at which the current traces under control conditions and in the presence of ionomycin intersected (Fig. 1A, inset). The mean reversal potential was −87 ± 4 mV (n = 3), a value close to the K+ equilibrium potential for our recording conditions (EK = −90 mV, calculated from the Nernst relationship). Ionomycin application shifted the zero-current potential from −26 ± 5 to −63 ± 9 mV (n = 3). These results indicate that C2C12 myoblasts possess a Ca2+-activated K+ current.

Fig. 1.

Ionomycin-activated K+ currents in C2C12 myoblasts. A: representative experiment in perforated-patch configuration, showing the transient outward current activated after external application of ionomycin (0.5 μM). The bath solution perfusing the myoblast was physiological salt solution (PSS), the pipette solution contained 170 mM K+ as main salt, and the holding potential was 0 mV. Inset: current recordings in response to voltage ramps from −150 to +150 mV, under control conditions (CTRL) and 20 and 200 s after the addition of ionomycin (iono) to the bathing solution. B: mean peak current (26.2 ± 3.7 pA/pF; n = 10) induced by ionomycin (0.5 μM) application to myoblasts in perforated-patch configuration under control conditions. C: representative cell-attached recording showing the effect of bath application of ionomycin (0.5 μM) on single-channel activity. The bath solution was PSS, the pipette solution contained 150 mM K+ as main salt, and the holding potential was 0 mV. Bottom: time expansions of short segments of the single-channel recording shown at top, under control conditions (left) and after ionomycin application (right).

The time course of the ionomycin-evoked outward current was transient, reverting to near-control values within 100–200 s (Fig. 1A). A transient time course was previously reported for the macroscopic IKCa current, this behavior being ascribed to a Ca2+-dependent rundown process of the IKCa channel (9, 18). However, because intracellular Ca2+ concentration ([Ca2+]i) cannot be controlled in the perforated-patch configuration of our experiments, the transient time course of the ionomycin-activated current may also reflect a transient change of submembrane [Ca2+].

The effect of ionomycin-induced [Ca2+]i increase was also investigated at the single-channel level, in the cell-attached configuration, with 150 mM K+ solution in the pipette. No single-channel activity could be detected in the absence of ionomycin (control conditions) at the resting membrane potential (0 mV applied potential; Fig. 1C). Ionomycin (0.5 μM) activated a single-channel activity in four of the five patches examined, the mean unitary current being −2.24 ± 0.14 pA (at 0 mV applied potential; n = 4). With a mean zero-current potential of −63 mV, as obtained in the presence of ionomycin (see above), a single-channel conductance of ∼35 pS results. In accordance with the macroscopic current, the ionomycin-activated unitary current response was transient, with single-channel activity ceasing after 50–250 s, suggesting that the macroscopic current is underpinned by this unitary activity (Fig. 1C). The results from Fig. 1, namely Ca2+ dependence, K+ selectivity, and small unitary current, provide compelling evidence to identify the ionomycin-activated current as sustained by either the SKCa or the IKCa channel.

Coapplication of the SKCa/IKCa channel activator DCEBIO (30) together with ionomycin activated a stable K+ current that displayed pharmacological properties congruent with the IKCa current (Fig. 2). A typical experiment illustrating the effects of coapplication of DCEBIO (100 μM) and ionomycin (0.5 μM) to a C2C12 myoblast in the perforated-patch configuration is presented in Fig. 2A. Individual data points in Fig. 2A represent the current assessed at 0 mV with the ramp protocol stimulation under control conditions and after application of DCEBIO + ionomycin and CTL (2 μM; in the continuous presence of DCEBIO + ionomycin; see Fig. 2B, inset). The average current obtained from 11 such experiments was 28.4 ± 8.2 pA/pF at 0 mV (n = 11). Figure 2B illustrates the results of the pharmacological test, showing that the DCEBIO + ionomycin-activated K+ current was insensitive to either TEA (1 mM) or d-TC (300 μM), indicating that neither SK2/SK3 nor BKCa channels underlie this current. In contrast, the DCEBIO + ionomycin-activated K+ current was markedly inhibited by CTX (200 nM), CTL (2 μM), and the newly identified IKCa channel antagonist NPPB (300 μM; see Ref. 4), suggesting that it was sustained by IKCa channels. Figure 2C shows that the ionomycin-activated current block by both CTL and CTX was dose dependent. Fit of the data with the Hill relationship gave an IC50 of 23.7 nM for CTX and 631 nM for CTL (Fig. 2C). The ionomycin-activated K+ current displayed the same pharmacological properties as the DCEBIO + ionomycin-activated K+ current (data not shown), indicating that combined DCEBIO + ionomycin activates the same current activated by ionomycin alone. No significant change of the holding current (measured at 0 mV) was recorded after 10-min CTX incubation [holding current density was 2.5 ± 0.4 pA/pF in control myoblasts (n = 10) and 3.3 ± 0.8 pA/pF in the presence of 200 nM CTX (n = 5)]. These results suggest that the IKCa channels in C2C12 myoblasts have a low basal activity under our recording conditions.

Fig. 2.

Pharmacological profile of the Ca2+-activated K+ current in C2C12 myoblasts. A: representative experiment in perforated-patch configuration showing the time course of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO) + ionomycin (100 μM, 0.5 μM) current activation and its block by clotrimazole (CTL; 2 μM). Individual data points were determined at 0 mV, with the voltage ramp protocol illustrated in B, inset. The bath solution perfusing the myoblast was PSS, the pipette solution contained 170 mM K+ as main salt, and the holding potential was 0 mV. B: normalized residual current of the DCEBIO + ionomycin (100 μM, 0.5 μM)-activated current on perforated-patch myoblasts upon application of d-tubocurarine (d-TC; 300 μM), tetraethylammonium (TEA; 1 mM), charybdotoxin (CTX; 200 nM), CTL (2 μM), or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 300 μM). Inset: representative current recordings from a myoblast in the perforated-patch configuration in response to voltage ramps (from −100 to +100 mV, holding potential of 0 mV; 800-ms duration) under control conditions and after application of DCEBIO + ionomycin (100 μM, 0.5 μM) and CTL (2 μM) in the continuous presence of DCEBIO + ionomycin. The bath and pipette solutions and the holding potential were as in A. C: dose-response relationships of the DCEBIO + ionomycin-activated current block by CTX (n = 3) and CTL (n = 3). Solid lines represent the fit of the experimental data with the Hill relationship Iblocker/ICTRL = 1/[1 + ([blocker]/IC50)nH], where Iblocker is current in the presence of blocker, ICTRL is current under control condition, [blocker] is blocker concentration, and nH is the Hill coefficient. The best-fit parameters were: IC50 = 23.7 nM and nH = 1.0 for CTX and IC50 = 631 nM and nH = 1.4 for CTL.

Biophysical properties of IKCa channel in C2C12 myoblasts.

The biophysical properties of the IKCa channel were studied in inside-out patches under symmetrical 150 mM K+ conditions on activation with varying [Ca2+]i (0.1–10 μM). Under these conditions a single-channel activity similar in kinetics and conductance to that shown in Fig. 3A was often observed. The current-voltage (I-V) relationship constructed with a double Gaussian fit of the current amplitude histograms revealed a moderate inward rectification and a mean single-channel conductance of 38 ± 6 pS (n = 4), as assessed by linear fit of the I-V data at negative voltages (see Fig. 3B). When internal K+ was substituted for Na+, the single-channel current became inwardly directed at all voltages examined (Fig. 3B), suggesting a high selectivity for K+ vs. Na+ (PNa/PK < 0.043, as estimated from the GHK relationship). We also assessed the voltage dependence of channel activity. The channel open probability (Po) was assessed from the individual current traces by double Gaussian fits of corresponding amplitude histograms, and quantitative analysis was carried out by constructing a Po vs. voltage plot. As shown in Fig. 3B, inset, the channel Po did not vary appreciably for membrane potentials over the range of −100 to +80 mV, suggesting that this channel is not gated by voltage.

Fig. 3.

Properties of the intermediate-conductance Ca2+-activated K+ (IKCa) channel in C2C12 myoblasts. A: inside-out single-channel recordings obtained at the indicated voltages in symmetrical 150 mM K+ solutions and 10 μM intracellular Ca2+ concentration ([Ca2+]i). B: single-channel current-voltage relationship obtained from the patch shown in A under symmetrical 150 mM K+ (□) and after substitution of internal K+ with Na+ (○). Dashed line, representing linear fit of the 3 control data points obtained at the most negative voltages, gives a slope conductance (γ) of 35 pS for this channel. Inset: open probability (Po) vs. voltage for the patch shown in A. C: inside-out single-channel recordings obtained at −100 mV in symmetrical K+ conditions and at the indicated [Ca2+]i. D: IKCa channel Po vs. [Ca2+]i obtained from 3 experiments of the type shown in C. The solid line represents the best fit of the experimental data with a Hill relationship of the form Po = Po,max/[1 + (IC50/[Ca2+]i)nH]. The best-fit parameters were Po,max = 0.44 (A), IC50 = 530 nM, and nH = 2.59. E: temporal profile of the mean current (Im) and variance (σ2) values assessed from the inactivating portion of the current recording shown in Fig. 1A. Im and σ2 were estimated from 100-ms segments of the ionomycin-activated current traces, and the Im and σ2 of the current trace before addition of ionomycin were subtracted from the assessed values. F: σ2 vs. Im plot obtained from a perforated-patch recording at 0 mV of applied potential, with the IKCa current being stimulated by bath addition of 0.5 μM ionomycin (cf. Fig. 1). Solid line represents the best fit of the experimental data with the parabolic relationship σ2 = Im·iIm2/N, where i is the single-channel current and N is the total number of functional channels; i and N assessed for this cell were 0.12 pA and 10,728, respectively.

The Ca2+ dependence of the IKCa channel activity was assessed at the unitary level by varying the [Ca2+]i in inside-out patches. Single-channel recordings of an IKCa channel at varying [Ca2+]i are shown in Fig. 3C. The IKCa channel Po vs. [Ca2+]i data were well fitted by a Hill relationship with an IC50 of 530 nM and a Hill coefficient of 2.6, suggesting the presence of at least three Ca2+ binding sites on the channel protein (Fig. 3D). The plot shows that the IKCa channel Po was close to zero for [Ca2+]i ≤ 0.1 μM, and increased at higher [Ca2+]i, reaching a maximum value of 0.44 at [Ca2+]i greater than ∼3 μM (as estimated from the fitted curve).

The number of functional IKCa channels on C2C12 myoblasts was estimated by applying noise analysis to the inactivation phase of the ionomycin-induced macroscopic currents similar to that shown in Fig. 1A. Variance (σ2) and mean current (Im) of the ionomycin-induced macroscopic current were assessed for each adjacent 100-ms interval (Fig. 3E). The resulting data were then fitted with the parabolic relationship σ2 = Im·iIm2/N, where i is single-channel current, which allowed an estimate of the total number of functional IKCa channels (N) of the cell (Fig. 3F). This analysis was performed on four cells, giving a mean number of functional IKCa channels per cell of 8,722 ± 1,132. Using the Cm assessed for these cells (22.5 ± 3.9 pF) and assuming a specific Cm of 1 μF/cm2 (8), we calculated a mean IKCa channel density of 3.9 ± 0.7 channels/μm2.

IKCa channel in C2C12 myoblasts responds to ATP.

We then tested whether the IKCa channel in this cell line could be activated by a physiological stimulus such as ATP, given that previous studies demonstrated that this agonist is capable of releasing Ca2+ from intracellular stores in C2C12 myoblasts (5, 17). A brief Picospritzer-driven application of ATP (3 s, 100 μM) on perforated myoblasts evoked, after a latency of several seconds (5–15 s), an outward current in 28 of the 55 cells tested. The current response took the form of either a single transient peak current (10 of 28; Fig. 4A, left) or current oscillations (15 of 28; Fig. 4A, right). The remaining three current responses could not be classified. With regard to the single transient peak current responses (which we deal with in this study), these could be evoked by successive applications of the agonist and displayed amplitude and time course similar to those of the first application (cf. Fig. 4B). To determine whether the ATP-induced transient outward current was a IKCa current, the agonist was applied while the myoblast was being superfused with the IKCa-specific inhibitor CTL (2 μM). Under these conditions, no response to ATP was observed (cf. Fig. 4B, third ATP application; n = 4). In two cells tested we found that pretreatment with CTX (200 nM) similarly prevented the ATP-induced response (data not shown). The ATP response was also absent after myoblast preincubation with the PLC inhibitor U-73122 (10 μM, 10 min; data not shown), indicating the involvement of inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores in the ATP-activated metabotropic cascade (13). Analysis of the ATP response at the single-channel level (cell attached) confirmed that the IKCa channel underlies this response. Cell-attached patches (with 150 mM K+ in the pipette, PSS in the bath, and no applied voltage) were subjected to brief Picospritzer-driven pulses of ATP (3 s, 100 μM). This protocol evoked transient, unitary inward currents after a delay of several seconds, which had a time course similar to that of the ATP-induced macroscopic currents shown in Fig. 4, A and B (cf. Fig. 4C). Reapplication of the agonist normally evoked a second transient unitary current episode similar to the first episode. In contrast, the response was absent when ATP was applied in the presence of the IKCa channel inhibitor CTL (2 μM; data not shown). The ATP-induced unitary current had an average amplitude (estimated from amplitude histograms) of ∼3.5 pA, as expected for IKCa channels at a Vm,patch of approximately −110 mV (i.e., Vm,patch = Vpipette + Vm,cell; see methods for details).

Fig. 4.

ATP activates the IKCa current and hyperpolarizes C2C12 myoblasts. A: representative current recordings in perforated-patch configuration showing the 2 types of current response [single transient peak (left) and oscillatory current response (right)] after a brief application of ATP (3 s, 100 μM). The bath solution was PSS, the pipette solution contained 170 mM K+ as main salt, and the holding potential was 0 mV. B: macroscopic current recording in perforated-patch configuration showing repeated current responses to brief applications of ATP (3 s, 100 μM). The first 2 ATP applications were in control conditions; the 3rd was after bath addition of the IKCa channel inhibitor CTL (2 μM). The bath and pipette solutions and the holding potential were as in A. C: cell-attached single-channel recording at −60 mV of applied potential showing the delayed unitary response to bath application of ATP (3 s, 100 μM). The bath solution was PSS, and the pipette solution contained 150 mM K+ as main salt. D: membrane potential recording obtained in perforated-patch configuration under current-clamp mode showing the hyperpolarizing response following a brief application of ATP (3 s, 100 μM) from a myoblast bathed in PSS. The bath and pipette solutions were as in A. E: mean membrane potential under control conditions (−20 ± 1 mV, n = 8) and after a brief application of ATP (3 s, 100 μM; −53 ± 12 mV, n = 8), following the experimental protocol described in D. Horizontal black bars in A–D represent ATP applications by Picospritzer.

We then evaluated the effect of ATP-induced IKCa channel activation on Vm. Brief applications of ATP (3 s, 100 μM) to perforated myoblasts under current-clamp recording conditions induced, after a short delay, a large, transient hyperpolarization (cf. Fig. 4D) that had a mean value of ∼35 mV (n = 8; Fig. 4E). The ATP-evoked hyperpolarizations closely matched the evoked transient currents we observed under voltage-clamp conditions, with regard to both the time course and latency of the response. The correspondence of the membrane voltage and current responses extended to the repeatability of the ATP-induced hyperpolarizations with successive applications (data not shown).

IKCa channel is downregulated during myogenesis.

In the engineered myogenic-like model C3H10T1/2-MRF4, it has been demonstrated that IKCa channels are downregulated during myogenesis and downregulation was prevented by the myogenesis-suppressing agents bFGF and transforming growth factor (24, 25). We have tested whether these findings apply to our myogenic model, i.e., whether myogenic progression in C2C12 myoblasts would likewise be paralleled by downregulation of the IKCa channel, and whether preventing myogenesis would prevent the IKCa channel downregulation. Differentiation of C2C12 myoblasts was induced by plating the cells in DM for 1–4 days. The induction of the myogenic program was confirmed by a marked increase in the percentage of IR myoblasts for the myosin heavy chain (Fig. 5C). In line with previous data, we found that IKCa current amplitude decreased during myogenesis induced by serum withdrawal (Fig. 5, A and B). Figure 5A illustrates representative DCEBIO + ionomycin-activated IKCa currents from C2C12 myoblasts kept in DM for either 1 or 4 days. As shown in Fig. 5B, the decrease in IKCa current usually started 24–48 h after myoblasts had been placed in DM, and after 4 days the IKCa current had decreased on average by ∼90%, compared with control myoblasts kept in GM. When bFGF was added to the DM to prevent myogenesis, downregulation was likewise removed, with the IKCa current remaining stable in amplitude at the levels found in myoblasts kept in GM (Fig. 5B). Previous reports showed that myoblasts placed in differentiation medium start expressing the DRK current, which is absent in myoblasts kept in growth medium (14). We monitored the DRK current in C2C12 myoblasts (kept in DM) over the time range in which the IKCa current was downregulated. We found that the DRK current, absent at day 1 in DM, normally appeared after 48–72 h, and at day 4 it reached a mean amplitude of 22.5 ± 8.8 pA/pF (Fig. 5C). Figure 5D illustrates representative DRK current families activated by depolarizing pulses, in DM at day 1 and day 4. Notably, no DRK current was ever observed in GM, regardless of the time for which myoblasts were kept in culture. The upregulation of the DRK current thus seems to correlate inversely with the downregulation of the IKCa current (cf. Fig. 5C, taken from the DM data of Fig. 5B).

Fig. 5.

IKCa current downregulation during myogenesis. A: representative experiments in perforated-patch configuration showing the time course of DCEBIO + ionomycin (100 μM, 0.5 μM) current activation and its block by CTL (2 μM) in cells held in DM for either 1 day (top) or 4 days (bottom). The bath solution perfusing the myoblast was PSS, the pipette solution contained 170 mM K+ as main salt, and the holding potential was 0 mV. B: IKCa current vs. days in culture from C2C12 myoblasts kept in differentiation medium (DM; n = 7), growth medium (GM; n = 7), or DM + basic FGF (bFGF, 4 days; n = 4). IKCa current amplitude was estimated as CTL-sensitive current from experiments similar to that shown in A. C: delayed-rectifier K+ (DRK) current density vs. days in DM. DRK current density was estimated as the current activated by a 100-ms-long depolarizing pulse to +40 mV, from a holding potential of −60 mV, divided by the membrane capacitance. Superimposed dotted line is the time course of the IKCa current vs. days in DM, from B. □, % of myoblasts immunoreactive (IR) to myosin heavy chain (MHC) proteins, at varying days in culture. D: representative families of voltage-activated K+ currents (DRK) from C2C12 myoblasts kept in DM for either 1 day (top) or 4 days (bottom). Myoblasts were bathed in PSS, and voltage-activated currents were evoked by depolarizing pulses from −40 to +60 mV in 20-mV steps, from a holding potential of −60 mV.

In C3H10T1/2 cells transfected with the muscle regulatory factor MRF4, and thereby capable of being induced toward a myoblastic lineage, the IKCa channel has been shown to be instrumental for promoting cell proliferation induced by bFGF (25). We therefore assessed whether the IKCa channel has a role on the proliferation of C2C12 myoblasts. Cells were maintained in either DM or DM + bFGF, and incubated for up to 48 h with saturating concentrations of the IKCa channel blocker CTX (200 nM). In both conditions used, CTX did not affect the percentage of viable cells with respect to control (no CTX), as assayed with the MTT method (Fig. 6A). Because of its toxicity to myoblasts on long exposure, CTL could not be used for these tests (data not shown). In contrast, treatment of C2C12 myoblasts with the src kinase inhibitor PP-2 (50 μM) was found to be effective in reducing cell proliferation under the same culture conditions (Fig. 6B), in line with previous studies (28). The lack of an antiproliferative effect of CTX on bFGF-induced C2C12 myoblast growth was confirmed by BrdU incorporation experiments (data not shown).

Fig. 6.

Effect of CTX and PP-2 on bFGF-induced proliferation. A: time course of cell growth rate [in arbitrary units, revealed as optical density (OD) with 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] in different media: DM, DM + CTX (200 nM), bFGF (20 ng/ml), and bFGF (20 ng/ml) + CTX (200 nM). The cells were kept for 5 h in the synchronization medium before a given medium was added. bFGF significantly stimulated cell growth compared with cells incubated in DM (bFGF vs. DM: P < 0.01 at 48 h). In contrast, CTX did not affect cell growth, regardless of the medium used. B: effect of the src kinase inhibitor PP-2 (50 μm) on cell growth (assessed as OD with MTT assay at 48 h and given as % of control) in bFGF or DM. Note that PP-2 (50 μm) inhibited by ∼50% the proliferation rate of cells both in bFGF and in DM.


Expression of IKCa channel in C2C12 myoblasts.

We report here the presence in C2C12 myoblasts of a Ca2+-activated K+ current whose pharmacological and biophysical properties are shown to be sustained by IKCa channels (3, 32). These can be summarized as follows: 1) single-channel conductance of 38 pS (at negative voltages) and moderate inward rectification in symmetrical 150 mM K+; 2) activation by intracellular Ca2+ with a IC50 of 530 nM; 3) voltage independence; 4) inhibition by CTX, CTL, and NPPB, but not by d-TC and TEA; and 5) sensitivity to the IKCa/SKCa channel activator DCEBIO. IKCa currents with properties similar to these have been found in other myogenic models including the mouse mesodermal stem cell line C3H10T1/2 (which under certain conditions can be induced toward myogenic differentation) and myogenic C3H10T1/2-MRF4 cells transfected with the muscle regulatory factor MRF4 (25).

IKCa channel is downregulated during myogenesis.

Ion channels are centrally involved in myogenesis, with several types being either upregulated or downregulated during differentiation. A major finding of this study is that the induction of differentiation by serum withdrawal results in downregulation of IKCa channel expression. In myoblasts placed in DM, the IKCa current decrease could be observed after 24–48 h, and by day 4 IKCa current had dropped below 10% of its value in control myoblasts (i.e., myoblasts kept in GM). When bFGF, a growth factor shown to prevent myogenesis, was added to the DM, the downregulation of the IKCa current was likewise prevented (Fig. 5B). These results, consistent with previous reports for the C3H10T1/2-MRF4 myogenic cell line (24), indicate that IKCa channel expression is modulated by a regulatory signaling pathway that also controls cell differentiation. This could be the bFGF receptor tyrosine kinase superfamily and its downstream signaling cascade, primarily involving the activation of the Ras/Raf/MEK/ERK pathway, as reported in another myogenic model (24).

During human myoblast differentiation a specific sequential expression of K+ currents [i.e., the ether-à-go-go (eag) and Kir currents] has been reported to be critical for myogenic progression and acquisition of cell excitability (1). Other K+ currents, including the DRK current, were shown to appear 24 h after C2C12 myoblasts had been placed in differentiation medium (14). We found that the DRK current, absent in myoblasts kept in GM, begins to appear after serum withdrawal, at about the same time the IKCa current starts to decrease. These results may indicate that the expressions of these two currents are sequentially ordered during myogenesis of the C2C12 cell line, although a causal link between these two currents has not been established. With regard to IKCa channel downregulation during myoblast differentiation, it is worthy of note that another current, ICl,sw, has been reported to disappear with a similar time course (33). The functional meaning, if any, of this simultaneous downregulation is likewise unknown. However, because these two currents (IKCa current and ICl,sw) are coexpressed in many cell models and coactivated in several important cellular processes including volume regulation (where their combined action mediates KCl efflux), their parallel downregulation may lead to inhibition of those processes that depend on cell volume changes, such as cell motility (21, 29, 36).

Functional role of IKCa channel in C2C12 myoblasts.

It has been shown that IKCa channel activity is essential for bFGF-sustained myoblast proliferation in the C3H10T1/2-MFR4 cell line (25). We therefore tested whether the IKCa current is also instrumental for sustaining cell proliferation in C2C12 myoblasts (6, 22, 25, 31). To this end, we used the IKCa channel inhibitor CTX (200 nM), which did not show any effect on the growth rate of C2C12 myoblasts (Fig. 6), indicating that the IKCa channel is not involved in this process. These observations allow us to conclude that the previously described modulatory role of IKCa channels on C3H10T1/2-MFR4 cell proliferation (25) cannot be generalized to all myoblastic cell lines.

The lack of a CTX-sensitive holding current under our resting conditions indicates that the IKCa channel does not contribute to the resting Vm. This conclusion is consistent with the relatively low Vm of this cell line (∼−20 mV, cf. Fig. 4E; also see Refs. 14, 33). We further showed that the IKCa current can be activated by a physiological agent such as ATP that leads to an increase in intracellular Ca2+ (5, 17). Our results indicate that the ATP response occurs via a PLC-mediated Ca2+ release from internal stores. The IKCa current activation induced by ATP occurred in intact myoblasts (perforated and cell-attached configurations) and resulted in a membrane hyperpolarization of ∼35 mV. As Vm plays an important modulatory role in a number of cellular responses, by linking changes in intracellular Ca2+ to Vm the IKCa channels may serve critical functions in several cellular processes of C2C12 myoblasts, including myogenesis (12, 16, 20).


This work was supported by Progetto di Ateneo from the University of Perugia and grants from Centro Universitario di Medicina Sportiva and Ministero dell’Istruzione, Universita’ e Ricerca, Università “G. d'Annunzio” di Chieti to S. Fulle.


We thank Stefania Fulle (University of Chieti) for helpful discussion and suggestions in planning the experiments on cell proliferation and Emilia Castigli (University of Perugia), Sandy Harper (University of Dundee), Paola Lorenzon, and Marina Sciancalepore (University of Trieste) for critical comments on the manuscript.


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