Differential regulation of potassium currents by FGF-1 and FGF-2 in embryonic Xenopus laevis myocytes

Authors


Corresponding author A. E. Spruce: Department of Pharmacology, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. Email: a.e.spruce@bham.ac.uk

Abstract

  • 1Fibroblast growth factors (FGFs) are involved in the regulation of many aspects of muscle development. This study investigated their role in regulating voltage-dependent K+ currents in differentiating Xenopus laevis myocytes. Both FGF-1 and FGF-2 are expressed by developing muscle cells, so their actions were compared. Experiments were performed on cultured myocytes isolated from stage 15 embryos.
  • 2Long-term exposure of the embryonic myocytes to FGF-1 downregulated inward rectifier K+ current (IK(IR)) density as well as both sustained and inactivating voltage-dependent outward K+ currents (IK,S and IK,I, respectively) and their densities. In contrast, FGF-2 upregulated these currents, although, because of an increase in capacitance caused by FGF-2, current density did not change with this factor.
  • 3The regulation of IK(IR) by FGF-1 was prevented by the cytoplasmic tyrosine kinase inhibitor herbimycin A, but that of IK,S and IK,I was unaffected, indicating that FGF-1 achieves its regulatory effects on electrical development via separate signalling pathways. The receptor tyrosine kinase inhibitor genistein in isolation suppressed K+ currents, but this may have occurred through a channel-blocking mechanism.
  • 4In many cells, IK,S was found to be composed of two components with differing voltage dependencies of activation. The FGFs brought about an alteration in the amount of total IK,S by equal effects on each component. Conversely, herbimycin A increased the proportion of low voltage-activated current without affecting total current amplitude. Therefore, we suggest that a single species of channel whose voltage dependence is shifted by tyrosine phosphorylation generates IK,S.
  • 5In summary, FGF-1 and FGF-2 exert opposite effects on voltage-dependent K+ currents in embryonic myocytes and, furthermore, FGF-1 achieves its effects on different K+ currents via separate second messenger pathways.

Fibroblast growth factors (FGFs) inhibit muscle differentiation and promote cell proliferation during myogenesis (for review, see Olson, 1992). Both FGF-1 and FGF-2, which are endogenously produced by skeletal myoblasts, are able to elicit these effects (Hannon et al. 1996). Release from this repression and consequent activation of the terminal differentiation programme arises from a decline in the expression of FGFs and their receptors (Moore et al. 1991; Itoh et al. 1996). Nevertheless, the influence of FGFs continues even in differentiated muscle, in which they are still expressed (Joseph-Silverstein et al. 1989). The biological effects of the FGFs alter substantially in differentiating muscle cells, in that they act to promote development. At the developing amphibian neuromuscular junction, FGF-2 is able to induce the clustering of acetylcholine receptors (AChRs; for review, see Hall & Sanes, 1993) as well as possibly influencing the metabolic stability of the embryonic subtype of AChR (Dai & Peng, 1992). More ubiquitous effects of FGF on muscle electrical development are suggested by demonstrations of modulation of voltage-dependent ionic currents by FGF-2 in other cell types: neonatal cardiac myocytes (Guo et al. 1995) and embryonic hippocampal neurones (Shitaka et al. 1996).

FGFs mediate their effects via binding to receptor tyrosine kinases, and four distinct receptor genes have been cloned (Jaye et al. 1992). A variety of signalling pathways may transduce the growth factor signal into its effects. Thus, ligand-dependent activation of the tyrosine kinase domain on the intracellular portion of the receptor has been shown to result in activation of phospholipase C (Jaye et al. 1992) and the mitogen-activated protein kinase (MAP kinase) cascade (Milasincic et al. 1996). Alternatively, FGF signalling may occur via internalization of the growth factor-receptor complex and translocation to the nucleus (for review, see Mason, 1994). Therefore, there is the potential for different FGFs to initiate separate biological responses in a particular cell type.

We investigated the ability of FGF-1 and FGF-2 to modulate the amount of voltage-dependent K+ currents present in embryonic Xenopus myocytes. Surprisingly, these factors exerted almost completely opposite effects on K+ currents and, moreover, the effects of FGF-1 on delayed rectifier and inward rectifier K+ currents appear to be mediated via separate biochemical pathways.

METHODS

Preparation

Xenopus laevis embryos were generated as described previously (Spruce & Moody, 1992). Briefly, mature oocytes were extruded into 1 × MBS (composition (mm): 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO3)2, 0.41 CaCl2, and 10 Hepes, pH 7.4 with NaOH) from females injected 12–18 h earlier with 1000 i.u. human chorionic gonadotrophin, and fertilized with sperm solution (in 1 × MBS) prepared from an excised testis. Adult male frogs were anaesthetized by immersion in 0.2 % MS-222 (3-aminobenzoic acid ethyl ester; Sigma) and killed, following testes removal, by cervical section before they could recover from the anaesthetic. Within 1–2 h after fertilization, the jelly coat was removed from the embryos using 2 % cysteine solution (pH 7.8). Embryos were rinsed in 0.1 × MBS and then left in this solution to develop at 16–20°C.

Embryonic dissections were carried out at stages 15–19. The neural plate region of the embryo (including underlying mesodermal tissue) was excised and placed into Danilchik's medium (composition (mm): 53 NaCl, 15 NaHCO3, 4.5 potassium gluconate, 1 MgSO4, 1 CaCl2, and 27 sodium isethionate, pH 8.3 with Na2CO3) containing 1 mg ml−1 papain. After 10 min the different tissue types were easily separated and the mesodermal tissue (the developing somitic muscle) on either side of the notocord was isolated and placed into Ca2+-Mg2+-free solution (composition (mm): 52.8 NaCl, 0.7 KCl, 0.4 EDTA, and 4.6 Tris, pH 7.5 with HCl). After 20 min, when the presumptive muscle cells had dissociated, cells were aspirated using a pulled Pasteur pipette and plated onto tissue culture plastic (Falcon 3001) in Danilchik's medium and left overnight at room temperature (18–22°C). This was the control condition. For the experimental conditions, growth factors and tyrosine kinase inhibitors (all from Calbiochem-Novabiochem (UK) Ltd) were added to the myocyte cultures for overnight incubation, as follows: FGF-1, 10 ng ml−1; FGF-2, 10 ng ml−1; herbimycin A, 1 μm; genistein, 50 μm; FGF-1 + herbimycin A, 10 ng ml−1 and 1 μm, respectively. Immediately before recordings were made, all cultures were washed with fresh Danilchik's solution.

Electrical recording

Whole-cell patch clamp recordings were made from the cultured myocytes using an internal (pipette) solution containing (mm): 90 potassium aspartate, 10 KCl, 10 NaCl, 2 MgCl2, 2 EGTA, 3 glucose, 2 theophylline, 2 Na2ATP, 0.1 cAMP, and 10 Hepes, pH 7.4 with KOH. The seal was made in Danilchik's medium, and then external recording solution (composition (mm): 120 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, and 10 Hepes, pH 7.4 with NaOH) was perfused onto the cell via an electrode placed to within 100 μm of the cell.

Ionic currents were measured using a List Electronic L/M PC amplifier, filtered at 3 kHz (6-pole Bessel filter), and acquired at 2 kHz for inward rectifier K+ current and 20 kHz for outward K+ current using pCLAMP software (Axon Instruments). The scaled leak current (measured in response to 10 mV steps between −48 and −68 mV) was subtracted from all traces. Quantitative comparisons of current amplitude were made as follows: for ‘sustained’ outward K+ current (IK,S), the average current at +32 mV (after correction for liquid junction potential) at 77.5–80 ms after the voltage step; for inactivating outward K+ current (IK,I), the maximum current at 32 mV averaged over a period of 150 μs minus IK,S; and for inward rectifier K+ current (IK(IR)), the current at 8 ms after the voltage step. Current density was measured by dividing total cell current by cell capacitance, measured from the dial of the patch clamp amplifier. The accuracy of this reading was validated in a number of cells by comparison with a method described previously employing a triangular voltage waveform (Simoncini & Moody, 1991).

Recordings were made about 1 day after stage 15 (t= 0 h; Nieuwkoop & Faber, 1967). For each condition, the recording times and capacitance values for the K+ current recordings were as follows: control, 25.8 ± 0.8 h and 50.8 ± 2.1 pF (n= 25); FGF-1, 24.1 ± 0.4 h and 50.9 ± 2.7 pF (n= 15); FGF-2, 30.4 ± 0.5 h and 69.1 ± 4.1 pF (n= 22); herbimycin A, 28.6 ± 0.5 h and 57.6 ± 3.0 pF (n= 10); FGF-1 + herbimycin A, 23.4 ± 0.5 h and 69.0 ± 3.3 pF (n= 10); genistein, 24.7 ± 1.5 h and 38.3 ± 2.2 pF (n= 11).

The voltage dependence of current activation was analysed from conductance-voltage plots. For IK,S, conductance was calculated using the theoretical value for the equilibrium potential for K+ (EK; −91.9 mV). Normalized conductance (gK)-voltage plots were fitted using a least-squares procedure (SigmaPlot; Jandel Scientific, Erkrath, Germany) to either single or double Boltzmann relations:

display math(1)
display math(2)

where V½ is the voltage at half-maximal conductance and c is a factor which describes the slope of each relation (c=kT/zF; where z, F and T have their usual meanings and k is the Boltzmann constant). The L and H subscripts refer to low and high voltage-activated current, respectively, and fL is the fractional amount of low voltage-activated current. The cut-off between V½ ranges for low and high voltage-activated currents was defined as −25 mV to ensure complete separation of the ranges. Equation (2) was used only when the fit generated using this equation was substantially better than that to eqn (1) as judged by visual inspection. Often, however, when the fit to eqn (1) was good, there was no choice as the fitting procedure used for eqn (2) aborted.

For IK(IR), conductance (gK(IR)) was calculated using an estimated current reversal potential of −53 mV. A significant Na+ permeability of IK(IR) in Xenopus myocytes leading to a substantial deviation from EK at low K+ concentrations has been demonstrated previously (Spruce & Moody, 1992). Normalized gK(IR)-voltage plots were fitted to:

display math(3)

All potentials were corrected for the liquid junction potential. Between potassium aspartate and either Danilchik's medium or external recording solution it was −8 and −9 mV, respectively (Spruce & Moody, 1992). Means are expressed ± standard error of the mean. For statistical analysis involving multiple comparisons, analysis of variance was used followed by Dunnet's post hoc test.

RESULTS

Previous studies have described the appearance of voltage-dependent ionic currents in aneural cultures of Xenopus laevis embryonic myocytes isolated from early/mid-neurula stage embryos (Spruce & Moody, 1992; Linsdell & Moody, 1995). In the present study, we have investigated the effects of long-term exposure of the myocytes to FGF-1 and FGF-2 on the functional expression of voltage-activated outward K+ current (IK) and inward rectifier K+ current (IK(IR)). Myocytes were isolated from embryos between stages 15 and 19 and left overnight in Danilchik's solution to which one of the following chemicals or chemical combinations were added: 10 ng ml−1 FGF-1; 10 ng ml−1 FGF-2; 10 ng ml−1 FGF-1 + 1 μm herbimycin A (Herb A; tyrosine kinase inhibitor); 1 μm herbimycin A; 50 μm genistein (tyrosine kinase inhibitor). Immediately before recordings commenced, these chemicals were removed by washing out the culture dish using Danilchik's solution. Control recordings were made from cells cultured in the absence of these factors. The timing of all recordings is given relative to the time at which the embryos reached stage 15, and the mean recording times are quite similar for each condition (approximately 24 h; see Methods).

Effects of fibroblast growth factors

FGF-1 and FGF-2 were found, surprisingly, to have largely opposite effects on K+ currents (Figs 1, 2 and 3). Thus, FGF-1 caused suppression of IK and IK(IR), whereas FGF-2 enhanced both of these currents. The extent and the significance of these modulatory effects on the maximum recorded currents and their densities are shown in Fig. 2. Figure 3 indicates the significance of the differences in current amplitude at each voltage.

Figure 1.

Regulation of voltage-dependent K+ currents by FGFs and a tyrosine kinase inhibitor in Xenopus myocytes

Myocytes were cultured overnight in the presence of the indicated chemicals and, for each condition, two recordings are shown which were obtained after washout of the chemicals. Recordings on the left were obtained by step depolarizations lasting for 80 ms from −98 mV in 10 mV increments to between −78 and +32 mV, and those on the right by step hyperpolarizations lasting for 900 ms from −38 mV in 10 mV increments to −138 mV.

Figure 2.

Effect of FGFs and a tyrosine kinase inhibitor on maximum current amplitude and density

A, IK,S; B, IK,I; C, IK(IR). For each current type, the top graph plots the amplitude data and the bottom graph illustrates current densities. The labels for the bars in the bottom graphs also apply to the top graphs. The bars plot means ±s.e.m. (n= 8–19). Asterisks indicate significant differences compared with control (*P < 0.05, **P < 0.01).

Figure 3.

Effect of growth factors and tyrosine kinase inhibitors on current-voltage relations

A, delayed rectifier K+ current measured at the end of the 80 ms step (IK,S). B, inward rectifier K+ current (IK(IR)) measured 8 ms after onset of the voltage step. Each point represents mean ±s.e.m. (n= 8–19). Aa and Ba, effects of growth factors (control, •; FGF-1, ○; FGF-2, □). Statistically significant differences from control are indicated by asterisks (*P < 0.05, **P < 0.01). Ab and Bb, effects of tyrosine kinase inhibitors (control data from Aa and Ba, continuous lines; herbimycin A, ▵; FGF-1 + herbimycin A, ▴; genistein, ⋄). For IK,S, the combination of herbimycin A and FGF-1 led to significant reductions in current (*P < 0.05). For IK(IR), genistein significantly inhibited current (*P < 0.05).

For IK, many recordings showed a prominent inactivating component (IK,I) as well as a sustained current (IK,S; Fig. 1). When IK is first detected in embryonic myocytes soon after stage 15, it is slowly activating, although a rapidly activating, inactivating current component appears after about 8 h (Spruce & Moody, 1992; Linsdell & Moody, 1995). The transient current does not show properties typical of A-current (Moody-Corbett & Gilbert, 1992). In qualitative terms, both IK,I and IK,S were affected similarly by each growth factor, i.e. enhancement by FGF-2 and suppression by FGF-1, although the effects were only significant for IK,S (Fig. 2A and B, top graphs). In order to predict the effects of the growth factors on myocyte excitability, it was important to determine the current density (obtained by dividing the maximum current by cell capacitance). Cell size increases dramatically during development and the growth factors may modulate this process. However, FGF-1 was not found to affect cell capacitance (see Methods), thereby leading to a significant reduction in the density of IK,S and, in fact, that of IK,I also (Fig. 2A and B, bottom). In contrast, a concomitant increase in cell capacitance brought about by FGF-2 (P < 0.01, Dunnet's test) resulted in a non-significant difference in the density of IK,S compared with control (Fig. 2A, bottom).

For IK(IR), FGF-1 induced a reduction in the amplitude of the current, although this does not quite achieve significance (Fig. 2C, top). Nevertheless, the difference in the densities of IK(IR) between FGF-1-treated and control myocytes is significant (Fig. 2C, bottom). FGF-2, on the other hand, induced a significant increase in IK(IR) (Fig. 2C, top). Again, however, as with IK,S, the density of IK(IR) differs little from control (Fig. 2C, bottom) because of the FGF-2-induced increase in cell size. Sodium current was not blocked in these experiments to allow preliminary analysis of the effects of growth factors on this current. The amplitude and density of the sodium current were not modulated, however (data not shown).

Effects of tyrosine kinase inhibitors

The broad-spectrum receptor tyrosine kinase inhibitor genistein (50 μm) was tested alone first. Reductions in both IK,S and IK(IR) (non-significant and significant, respectively) were found (Fig. 3Ab and Bb, current records not shown), perhaps suggesting that K+ current in the aneural myocytes is constitutively upregulated by a tyrosine kinase-dependent pathway. However, some studies have suggested that genistein directly blocks ion channels (see Discussion). Therefore, even though the cultures were washed with genistein-free solution prior to recording, and despite the observation that capacitance was significantly reduced in genistein-treated cultures (38.3 ± 2.2 pF, n= 10, P < 0.05 using Dunnet's test), which may indicate an effect related to its action on biochemical signalling pathways, it would have been too confusing to interpret the results using combinations of growth factors with genistein, and no further studies were made using this compound.

The effect of herbimycin A (1 μm), an inhibitor of cytoplasmic tyrosine kinases (Fukazawa et al. 1991) was also tested (Figs 1, 2, and 3Ab and Bb). It had no effect on K+ currents when applied alone. Interestingly, however, co-application of herbimycin A with FGF-1 reversed the suppression of IK(IR) produced by FGF-1 alone but had no effect on the suppression of IK,S and IK,I by FGF-1. This would imply that activation of a src-like protein tyrosine kinase is involved in the suppression of IK(IR) by FGF-1, but regulation of the amount of IK is controlled by a different pathway (although it is possible that a different cytoplasmic tyrosine kinase with a lower sensitivity to herbimycin A is involved in the regulation of IK).

Analysis of conductance-voltage relations reveals separate current subtypes underlying IK,S

Chord conductances were calculated from IK,S values, normalized, plotted against voltage and fitted to Boltzmann curves (see Methods). For each myocyte treatment condition, many conductance relations from individual cells were fitted quite poorly by single Boltzmann curves according to visual inspection, particularly over the region of more negative voltages. At these voltages, substantial increases in conductance were often observed, suggesting the activation of a separate, low voltage-activated (LVA) current subtype. Indeed, these relations were much better fitted by the sum of two Boltzmann curves. Figure 4A-C shows, for control, FGF-1-exposed and FGF-2-exposed myocytes, examples of individual relations from two cells indicating the apparent presence of high voltage-activated (HVA) current alone in one cell (fitted by a single Boltzmann curve), and both HVA and LVA currents in the other cell (better fitted by the sum of two Boltzmann curves). Therefore, on the basis of this analysis and for these conditions, all myocytes (except one in each of control and FGF-2-exposed cells) contained HVA IK,S and many also contained LVA IK,S. Values of V½ and the slope factor (c) were obtained from the fits (see Methods) for each presumed current subtype, as well as the fractional contribution of LVA IK,S to the total current (fL). The mean values of these parameters for each condition are given in Table 1. It is clear that neither growth factor had a significant effect on the voltage dependence nor on the relative amount of the currents underlying IK,S. Moreover, similar proportions of cells were found to contain LVA IK,S (59 % in control; 62 % in FGF-1; 71 % in FGF-2). Therefore, modulation of the amount of total IK,S by FGF-1 and FGF-2 is apparently achieved by approximately equal effects on LVA and HVA currents.

Figure 4.

Voltage dependence of IK,S current: evidence for subtypes

Each graph plots individual normalized chord conductance-voltage relations. A, control; B, FGF-1; C, FGF-2; D, herbimycin A (▵) and FGF-1 + herbimycin A (▴). For A, B and C, two relations are shown for each condition. One is well fitted by a single Boltzmann curve (dashed lines - see eqn (1) in Methods) and we would denote these cells as containing only high voltage-activated current. The other has been fitted to both single (dashed lines) and the sum of two Boltzmann curves (continuous lines - eqn (2)). Since the latter fit is better, we assume that these cells contain low as well as high voltage-activated current. In D, only one relation is shown for each condition (both fitted by the sum of two Boltzmann relations). Mean values for the Boltzmann parameters are given in Table 1.

Table 1. Effects of growth factors and an inhibitor of cytoplasmic tyrosine kinases on the voltage dependence of IK,S subtypes and IK(IR)
  I K,S (LVA) I K,S (HVA) I K(IR)
  V 1/2 (mV) c (mV) f L V 1/2 (mV) c (mV) V 1/2 (mV) c (mV)
  1. Myocytes were cultured overnight in the indicated chemicals. The mean values of the Boltzmann parameters V1/2 and c for both LVA and HVA current subtypes, and also the fractional amount of LVA current (fL) were obtained from fits to eqns (1) and (2) for IK,S. Equation (3) was used to obtain V1/2 and c values for IK(IR).** Significantly different from control (P < 0.01, Dunnet's post hoc test).

Control−36.9±2.36.3±0.90.24±0.07−5.0±1.911.6±0.9−95.3±1.714.9±0.8
  n=10  n= 17 n= 16   n= 19  
FGF-1−36.3±2.84.2±0.40.21±0.07−0.4±2.110.9±0.8−93.1±3.311.1±1.0**
  n= 8   n= 13 n= 13   n= 9  
FGF-2−40.4±2.65.5±0.40.36±0.08−2.8±2.014.1±1.4−95.5±1.314.1±0.5
  n= 12   n= 17 n= 16   n= 13  
Herb A−39.3±2.56.1±0.80.76±0.09**1.0±3.610.7±1.7−101.2±2.012.6±0.6
  n= 7   n= 7 n= 5   n= 8  
FGF-1 + Herb A−38.8±2.53.5±0.20.20±0.080.1±2.111.3±1.0−102.2±1.912.4±0.4
  n= 7   n= 8 n= 8   n= 9  

Different results were obtained using herbimycin A, however. The conductance-voltage relation for herbimycin A in Fig. 4D suggests that the LVA subtype of IK,S was much more pronounced in these myocytes than in myocytes from all other conditions. Indeed, 100 % of cells contained LVA current and 71 % contained HVA current, and, as shown in Table 1, the increase in the fractional amount of LVA IK,S was highly significant. The voltage dependencies of both current subtypes were unaltered, however. Therefore, these data suggest that a constitutively active soluble protein tyrosine kinase suppresses LVA and upregulates HVA IK,S. Overall, total IK,S is unchanged by herbimycin A, however (see above; Figs 2A and 3Ab). This effect of herbimycin A has revealed an important mechanism for controlling the functional expression of K+ current.

In combination with FGF-1, the current modulation by herbimycin A reverted to the patterns seen in control myocytes as well as those treated with growth factor alone (Fig. 4D; Table 1). In particular, the fractional amount of LVA IK,S was unaltered. This was unexpected due to the lack of effect of FGF-1 on its own. Speculation as to the meaning of this result is made later.

Analysis of effects of growth factors on voltage dependence of IK(IR)

Individual normalized inward rectifier conductance (gK(IR)) vs. voltage relations were fitted well by single Boltzmann curves (mean relations are shown in Fig. 5). The mean values for the parameters of the fits (Table 1) demonstrated a lack of modulation of the voltage dependence of IK(IR) by FGF-1 and FGF-2 (the continuous lines in Fig. 5A and B are the fits to the control data), although the slope of the relation in FGF-1 was significantly steeper compared with control myocytes; however, the relevance of this effect is unclear. Herbimycin A, alone and in combination with FGF-1, appeared to shift the mean conductance-voltage relations (Fig. 5B; Table 1), but this effect was not significant.

Figure 5.

Voltage dependence of IK(IR)

Both graphs plot mean (±s.e.m.) data for normalized conductance-voltage relations. A, effect of FGFs (control, •; FGF-1, ○; FGF-2, □). A Boltzmann relation is fitted to the control data. For clarity, the fits to the FGF data are not included. B, effect of tyrosine kinase inhibitors (herbimycin A, ▵; FGF-1 + herbimycin A, ▴). The continuous line is the fit to the control data as shown in A.

DISCUSSION

Overnight treatment of terminally differentiating Xenopus laevis myocytes with either FGF-1 or FGF-2 resulted in the modulation of the amount of both IK(IR) and IK. Interestingly, FGF-1 suppressed the K+ currents, whereas they were enhanced by FGF-2. Specifically, FGF-1 significantly reduced the densities of the sustained and inactivating IK as well as the density of IK(IR). This is predicted to increase myocyte excitability. For FGF-2, the amplitudes of IK,S and IK(IR) were significantly enhanced but their densities were not because of a concomitant increase in cell size. Nevertheless, in isolation, this factor would promote muscle cell development during terminal differentiation, by inducing cell enlargement whilst maintaining cell electrical excitability. The effect of the combination of these factors is difficult to predict, however, and this needs to be tested. These effects of FGFs contrast with their actions at earlier stages of myogenesis, where they inhibit differentiation and promote proliferation (Hannon et al. 1996). The enhancement of ionic current by FGF-2 has been reported previously in many cells types (Puro & Mano, 1991; Guo et al. 1995; Zhong & Nurse, 1995; Shitaka et al. 1996), but the finding of opposite actions of two members of the same growth factor family on K+ current was unexpected. There are two main possible explanations for this.

Firstly, different FGF receptor subtypes are expressed by skeletal muscle (Stark et al. 1991; Templeton & Hauschka, 1992; Marcelle et al. 1995), and the large number of alternatively spliced variants are known to have unique (although overlapping) ligand-binding affinities (Ornitz et al. 1996). So, FGF-1 and FGF-2 may be activating different proportions of receptor subtypes, thereby activating separate second messenger pathways to different extents. The differences in the affinities of FGF-1 and FGF-2 may be further amplified because the assembly and activation of receptor hetero-oligomers (the active state of the receptor complex; Schlessinger & Ullrich, 1992) will depend on the distinct affinity profile of each component receptor (Jaye et al. 1992).

Secondly, the contrasting effects of FGF-1 and FGF-2 may arise as a result of internalization of the growth factor- receptor complex (Mason, 1994). After internalization, it has been shown that both FGFs can enter the nucleus and affect gene transcription (Wiedlocha et al. 1994). Perhaps each factor regulates expression of a different complement of genes.

Preliminary studies were carried out using herbimycin A, an inhibitor of cytoplasmic tyrosine kinases, to investigate the signalling pathways through which one of the growth factors, FGF-1, exerts its effects on muscle electrical development. Interestingly, the combination of herbimycin A and FGF-1 reversed the depression of IK(IR) by FGF-1, whereas the reduction in IK (both IK,S and IK,I) was unaffected. FGFs have previously been found to operate through the src family of cytoplasmic tyrosine kinases (Landgren et al. 1995). The modulation of IK(IR) by this pathway is unlikely to be endogenously active in the aneural myocytes as herbimycin A alone does not affect IK(IR) amplitude. Regulation of total IK by FGF-1 could occur through a number of alternative mechanisms including phospholipase C-γ (Mason, 1994), the MAP kinase cascade (Milasincic et al. 1996) as well as receptor internalization (see above).

Recently, a number of studies have implicated both receptor and cytoplasmic tyrosine kinase activity in modulating voltage-dependent K+ currents. Thus, tyrosine kinase activation suppresses Kv1.2 (Huang et al. 1993; Lev et al. 1995) and Kv1.5 (Holmes et al. 1996) currents. In addition, herbimycin A led to the induction of K+ currents in adenocarcinoma and fibroblast cell lines (Saad et al. 1994) and blocks the tyrosine phosphorylation and suppression of the activity of Kv1.3 channels in T-lymphocytes (Szabo et al. 1996). Finally, inhibition of the receptor tyrosine kinase prevents the upregulation of potassium currents in cardiac myocytes by FGF-2 (Guo et al. 1995).

Inhibition of the receptor tyrosine kinase by genistein would have been expected to block the effects of both FGF-1 and FGF-2. In fact, this compound led to modulation of current on its own. The results were generally the reverse of those produced by FGF-2 exposure, suggesting that the aneural myocytes endogenously produce and respond to FGF-2. Unfortunately, there is evidence to suggest that genistein is an ion channel blocker. Thus, genistein has been found to block IK in mammalian smooth muscle cells (Smirnov & Aaronson, 1995). Therefore, interpretation of the genistein results was in doubt and it was felt that there was no point in examining the effects of the combination of genistein with either FGF.

The results of this study suggest that two separate ion channel populations underlie the sustained outward K+ current (IK,S), each with a differing voltage dependence of activation. We presume that neither Ca2+-activated nor ATP-sensitive K+ currents contribute to IK,S, as the former current is not expressed in Xenopus myocytes at this developmental age (Spruce & Moody, 1992) and 2 mm ATP was included in the patch pipette. Both LVA and HVA current subtypes were affected approximately equally by FGF-1 and FGF-2, such that their relative amounts were unchanged in the presence of the growth factors. Therefore, it was surprising to find that herbimycin A on its own dramatically increased the proportion of the LVA current subtype. Since herbimycin A does not affect total IK, this result suggests that a constitutively active cytoplasmic tyrosine kinase (which is not activated by FGFs) may convert K+ channels to the HVA phenotype. Indeed, reduction in the voltage sensitivity of a K+ channel by phosphorylation has been described previously (Perozo & Bezanilla, 1990). During the development of control aneural Xenopus myocyte cultures, the voltage dependence of activation of IK shifts (Linsdell & Moody, 1995), i.e. in the first few hours after stage 15 the current activates at more depolarized voltages compared with myocytes at 24 h. This shift is opposite to that proposed to occur as a result of tyrosine kinase signalling. However, the developmental change may be only partially completed by 24 h (i.e. LVA current is still present) and what we see with herbimycin A may be its forced completion.

The most confusing result is that the combination of FGF-1 with herbimycin A does not alter the fractional amount of IK subtypes. Perhaps the conversion of the channels to the HVA phenotype is achieved not only via a cytoplasmic tyrosine kinase but also by a separate, FGF-activated pathway (possibly MAPK or PLC-γ)? Therefore, FGF can form HVA channels in the presence of herbimycin A but on its own it has no effect because the alternative pathway is normally maximally active.

An additional subtype of IK, the inactivating current (IK,I), is modulated by the FGFs in a manner similar to their effects on IK,S. Two classes of single K+ channels with marked differences in inactivation time course have been described in Xenopus myocytes (Ernsberger & Spitzer, 1995). The channel with the faster inactivation kinetics appears to correspond to IK,I in this study. Under oxidizing conditions, though, the rapidly inactivating channel converts to a slowly inactivating phenotype. Therefore, it is interesting to speculate that when both channels are slowly inactivating they generate the HVA and LVA components of IK,S.

The voltage dependence of IK(IR) was not significantly modulated by FGFs or herbimycin A, except for a relatively minor increase in slope brought about by FGF-1 exposure. Therefore, growth factors and tyrosine kinase activation exert little effect on the properties of inward rectifier channels, but substantially modulate the number of functional channels.

To summarize, FGF-1 will tend to promote electrical development and increase excitability by reducing the density of IK and IK(IR). Furthermore, although FGF-2 will not affect excitability, the increase in cell size together with K+ current amplitudes will promote differentiation. Finally, the results with tyrosine kinase inhibitors suggest complicated mechanisms underlie these effects, and, indeed, FGF-1 exerts its effects on electrical development via separate pathways.

Acknowledgements

This work was supported by The Wellcome Trust and the University of Birmingham.

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