Growth Factor Receptor Tyrosine Kinases Acutely Regulate Neuronal Sodium Channels Through the Src Signaling Pathway

The Journal of Neuroscience, January 15, 1998, 18(2):590–600

Growth Factor Receptor Tyrosine Kinases Acutely Regulate Neuronal Sodium Channels through the Src Signaling Pathway

Michael D. Hilborn,1 Richard R. Vaillancourt,2 and Stanley G. Rane1 1Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, and 2Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721

Growth factor receptor tyrosine kinase (RTK)-activated signal- abrogate sodium channel inhibition in response to NGF and ing pathways are well established regulators of neuronal growth PDGF, respectively, showing that the intrinsic RTK activity of and development, but whether these signals provide mecha- these receptors is necessary for sodium channel inhibition. Use nisms for acute modulation of neuronal activity is just beginning of PDGF receptor mutants deficient for specific signaling activ- to be addressed. We show in pheochromocytoma (PC12) cells ities demonstrated that this inhibition is dependent on RTK that acute application of ligands for both endogenous RTKs interaction with Src but not with other RTK-associated signaling [trkA, basic FGF (bFGF) receptor, and epidermal growth factor molecules. Inhibition was also compromised in cells expressing (EGF) receptor] and ectopically expressed platelet-derived dominant-negative Ras. These results suggest a possible growth factor (PDGF) receptors rapidly inhibits whole-cell so- mechanism for acute physiological actions of RTKs, and they dium channel currents, coincident with a hyperpolarizing shift in indicate regulatory functions for Ras and Src that may comple- the voltage dependence of inactivation. Sodium channel inhi- ment the roles of these signaling proteins in long-term neuronal bition by trkA and PDGF receptors is mutually occlusive, sug- regulation. gestive of a common signal transduction mechanism. Further- Key words: PC12 cells; sodium channels; growth factor re- more, specific inhibitors for trkA and PDGF RTK activities ceptor tyrosine kinases; PDGF receptors; NGF; Ras; Src

Growth factors constitute a family of polypeptides essential for growth factors can modulate synaptic activity in hippocampal the establishment and maintenance of the mature nervous sys- neurons and developing Xenopus neuromuscular junctions (Ter- tem. Expressed throughout the CNS and PNS, growth factors act lau and Seifert, 1989; Abe et al., 1991; Kang and Schuman, 1995), by binding to their cognate receptor tyrosine kinases (RTKs), as well as modulate short-term potentiation and LTP (Lohof et which autophosphorylate and subsequently activate a complex of al., 1993; Stoop and Poo, 1996). These studies suggest that ion signaling proteins including the well characterized Ras and channel modulation may be an important component of acute mitogen-activated kinase (MAPK) pathway, phosphatidylinositol growth factor actions, and they are complemented by work show- 3-kinase (PI3-K), phospholipase C␥ (PLC␥), and the Src family of ing growth factors can acutely modulate the high-voltage- ϩ ϩ ϩ nonreceptor tyrosine kinases (for review, see Panayotou and activated Ca 2 channel and Ca 2 -activated K channels (Wil- Waterfield, 1993; Johnson and Vaillancourt, 1994; Kaplan and dering et al., 1995; Holm et al., 1997). However, the signaling Stephens, 1994; Szebereńyi and Erhardt, 1994; van der Geer et paths underlying these physiological events are undetermined, ϩ al., 1994). The variety of neuronal responses to growth factors, and although both NMDA receptors and the human Kv1.5 K including proliferation and migration, establishment and mainte- channel associate with the nonreceptor tyrosine kinase Src nance of synaptic connections, and survival of many mature (Holmes et al., 1996; Yu et al., 1997), it is not yet known whether neurons, ultimately depends on the activation of specific compo- this interaction underlies a growth factor-initiated action. Fur- nents comprising this signaling network (for review, see Korsch- thermore, studies suggesting that the activation of the MAPK ing, 1993; Snider, 1994; van der Geer et al., 1994; Bothwell, 1995; cascade is essential for events associated with long-term facilita- Lewin and Barde, 1996). tion in Aplysia neurons have not ascertained whether growth In addition to the wealth of information on long-term cellular factors are vital in initiating this response (Bailey et al., 1997; effects, there is increasing evidence that growth factors and their Martin et al., 1997). Thus, there is much to be determined signaling pathways play pivotal roles in the acute regulation of the concerning how growth factors physiologically modulate the ma- mature nervous system (for review, see Thoenen, 1995). In hip- ture nervous system and which signaling pathways are essential to pocampal neurons, depolarizing stimuli and stimulation protocols this modulation. that induce long-term potentiation (LTP) increase neurotrophin Past efforts to examine the signaling mechanisms that medi- mRNA levels (Lu et al., 1991; Patterson et al., 1992). In turn, ate the effects of chronic growth factor exposure in both the PNS and CNS have used the well characterized rat pheochro- Received Aug. 28, 1997; revised Oct. 23, 1997; accepted Oct. 24, 1997. mocytoma (PC12) model neuronal cell line (for review, see This work was supported by a grant from the Whitehall Foundation (S.G.R.), Grant DK 08897 (R.R.V.), and a Predoctoral Fellowship from the American Heart Chao, 1992; Keegan and Halegoua, 1993; Marshall, 1995). Association, Indiana Affiliate (M.D.H.). We are grateful to Dr. S. Rossie for critical Application of NGF causes PC12 cells to differentiate into a review of this manuscript. neuronal-like phenotype characterized by numerous morpho- Correspondence should be addressed to Dr. Stanley G. Rane, Department of Biological Sciences, Lilly Hall, West Lafayette, IN 47907. logical and physiological changes, including the persistent ac- Copyright © 1998 Society for Neuroscience 0270-6474/98/180590-11$05.00/0 tivation of the Ras and MAPK pathway, cessation of cellular Hilborn et al. • Growth Factor RTK Regulation of Neuronal Naϩ Channels J. Neurosci., January 15, 1998, 18(2):590–600 591

ϩ division, neurite extension, the induction of immediate early HEPES, pH 7.3. For recording voltage-gated Ca 2 channel currents, the and neuronal-specific genes, and an induction of electrical bath solution consisted of 135 mM TEA-Cl, 4 mM KCl, 10 mM BaCl2 ,1 mM MgCl ,5mM glucose, 10 mM HEPES, and 1 ␮M tetrodotoxin, pH excitability because of an increase in ion channel expression 2 7.3. In all cases, the patch pipette solution was 150 mM CsCl, 2 mM (Greene and Tischler, 1982). Via the analysis of PC12 cell lines MgATP, 0.5 mM GTP, 2 mM BAPTA, and 10 mM HEPES, pH 7.3. expressing mutant receptors or intracellular signaling mole- Whole-cell capacitive transients, elicited by 20 mV depolarizing steps, cules, the signaling mechanisms that mediate these long-term were compensated with the analog compensation circuitry of the patch- changes have been well characterized (Rausch et al., 1990; clamp amplifier. Whole-cell capacitance, a measure of membrane area, was read directly from the compensation control dial of the amplifier. Szebereńyi et al., 1990; Ginty et al., 1992; Traverse et al., 1992; We waited from 2 to 5 min after establishing whole-cell patch-clamp Fanger et al., 1993, 1997; Cavalie´ et al., 1994; Cowley et al., access before beginning experimental data collection. During this time, ϩ 1994; Loeb et al., 1994; Stephens et al., 1994; Vaillancourt et the Na I–V relationship was assessed by holding the cell at Ϫ90 mV and Ϫ al., 1995; Pollock and Rane, 1996; Hilborn et al., 1997). For by evoking command steps from 60 to 60 mV in 10 mV increments at 1.5–3 sec intervals. Two changes were observed when this protocol was example, analyses of PC12 cell lines expressing mutated repetitively administered. First, as has been noted in a number of studies ϩ platelet-derived growth factor (PDGF) receptors deficient for on voltage-gated currents, the Na I–V relationship shifted to the left by activating specific signaling pathways have determined that 5–10 mV during the first 30–90 sec of recording. We also observed that both Src and PLC␥ are required for growth factor-induced in addition to this shift, current amplitudes increased at any given voltage during the first 1–3 min of recording, and then they stabilized. Growth neurite extension (Vaillancourt et al., 1995). An analysis of ϩ factor inhibition of Na current was assessed at the command voltage these same cell lines, along with PC12 cells expressing the N17 giving maximal current (as shown by the I–V protocol) only after these ϩ Ras dominant-negative mutant, demonstrated that although spontaneous changes in Na current were complete. Any cell that ϩ growth factor-induced upregulation of brain type II/IIA Na showed continuous variations in current of Ͼ5% at this voltage was rejected. To test for significant differences in growth factor inhibition of channels is independent of Ras activation, the activation of Src ϩ Na current between experimental groups, we used a two-tailed, non- is important for establishing the full functional expression of paired, t test (at p Ͻ 0.05). All experiments were performed at 22–25°C. these channels (Fanger et al., 1993, 1997; Pollock and Rane, Growth factor and drug application. Growth factors dissolved in the 1996). bath solution were acutely applied to cells via pressure ejection from In this report, we use this system to show that growth factors can blunt-tipped, fire-polished micropipettes positioned ϳ5–10 ␮m from the ϩ acutely inhibit voltage-gated Na currents in undifferentiated and cell. For RTK and Src inhibitor studies, tyrphostin AG9, tyrphostin AG879, and PP1 were dissolved in 70% ethanol, 70% methanol, and differentiated PC12 cells and that this inhibition is dependent on DMSO, respectively, before dilution to their appropriate concentrations RTK coupling to Src and Ras. This inhibition does not share the in intracellular solution (for details of inhibitor specificity, see Bilder et ϩ mechanistic properties of Na current inhibition induced by sig- al., 1991; Levitzki and Gilon, 1991; Ohmichi et al., 1993; Levitzki and naling molecules such as the cAMP-dependent protein kinase Gazit, 1995; Hanke et al., 1996). After whole-cell access, the intracellular solution containing the inhibitor was allowed to diffuse into the cell for (PKA) or protein kinase C (PKC), further suggesting that this ϳ3–5 min before recording. For all experiments, inclusion of inhibitors ϩ modulation relies on intracellular mechanisms distinguishable did not affect basal Na current densities. Furthermore, recordings with from pathways such as those driven by receptors of the seven intracellular solutions containing only ethanol, methanol, or DMSO did ϩ transmembrane spanning region superfamily. Thus we show that not alter basal Na channel current densities or acute responses to signals mediated by growth factors and their RTKs are capable of growth factors. regulating an ion channel fundamental to neuronal excitation and that this regulation occurs through Src and Ras. Although Src is RESULTS ؉ extensively expressed in the mature PNS and CNS and contributes Initial characterization of growth factor-induced Na to long-term growth factor effects, its physiological function has channel current inhibition in PC12 cells expressing remained elusive. Our results suggest that acute regulation of wild-type PDGF receptors ϩ neuronal Na channels may be one such function. In PC12 cells, cellular differentiation is mediated by NGF binding to its cognate RTK. The wtPDGF receptor, when expressed in MATERIALS AND METHODS PC12 cells, mediates neuronal differentiation in a manner char- Tissue culture. Stock PC12 cell cultures were grown on rat tail collagen- acteristic of NGF; both growth factors induce the persistent coated Petri dishes and maintained in DMEM. For cells ectopically activation of the Ras and MAPK pathway and of PLC␥, induce ϩ expressing N17 Ras (for details of cell line generation, see Szebereńyi et the expression of the early gene c-fos, and upregulate Na chan- al., 1990), the media were supplemented with 5% fetal calf serum and nels and other neuronal-specific mRNAs (Heasley and Johnson, 10% horse serum. For PC12 cells expressing either the human form of the ␤PDGF [wild-type PDGF (wtPDGF)] receptor or mutant PDGF 1992; Fanger et al., 1993, 1997). Furthermore, observations with receptor (for details of cell line generation, see Vaillancourt et al., 1995), mutant PDGF receptors demonstrate that both Src and PLC␥ are the media were supplemented with 2.5% fetal calf serum (Sigma, St. important in regulating morphological differentiation (Vaillan- Louis, MO) and PDGF-deficient 12.5% plasma-derived horse serum court et al., 1995). Thus, wtPDGF and mutant PDGF receptors (Sigma) to obviate differentiation. All cell lines were kept under G418 selection and maintained in 25 U/ml penicillin and 25 ␮g/ml streptomy- serve as reasonable models for neurotrophin receptors and the cin. In preparation for electrophysiology, cells were seeded onto 35 mm signaling pathways underlying their actions. collagen-coated dishes and allowed to grow for1dinstandard culture We first confirmed the robustness of signaling in PC12 cells medium. Then, fresh medium with either NGF (100 ng/ml), PDGF (20 ectopically expressing the wtPDGF receptor by showing that ng/ml), or basic FGF (bFGF) (50 ng/ml) was applied. Medium and chronic application of PDGF (20 ng/ml) could induce both mor- growth factor were replaced every other day. Electrophysiology. Conventional patch-clamp techniques and phological and physiological differentiation. In agreement with computer-assisted data acquisition and analysis (Pulse and PulseFit soft- previous observations (Heasley and Johnson, 1992; Vaillancourt ware; Instrutech Corporation, Great Neck, NY) (Pollock and Rane, et al., 1995), the morphological differentiation elicited by chronic ϩ 2ϩ 1996) were used to study whole-cell Na and Ca channel currents. All application of PDGF for a week, specifically neurite extension, current records were leak subtracted with a standard P/N procedure and filtered at 5 kHz before storage on disk for off-line analysis. For record- was comparable with that in cells treated with NGF (100 ng/ml) ϩ ing Na currents, the bath solution was 138 mM NaCl, 9 mM KCl, 1 mM for the same period of time (data not show). To determine ␮ CaCl2 ,1mM MgCl2 ,10mM TEA-Cl, 200 M CdCl2 , and 10 mM whether morphological differentiation was accompanied by ion 592 J. Neurosci., January 15, 1998, 18(2):590–600 Hilborn et al. • Growth Factor RTK Regulation of Neuronal Naϩ Channels channel upregulation, we used whole-cell patch-clamp electro- (100 ng/ml) or PDGF (20 ng/ml). As seen in differentiated cells, ϩ physiology to examine whether chronic application of PDGF to NGF elicited a decrease in Na current levels (40.6 Ϯ 4.3%; n ϭ PC12 cells expressing wtPDGF receptors could increase func- 18) with an onset of 96 Ϯ 13 sec (n ϭ 15) and a rapid partial ϩ ϩ tional Na channel expression. Peak Na currents of undiffer- recovery to 90.0 Ϯ 4.8% (n ϭ 7) of the total current observed entiated and differentiated PC12 cells were normalized to whole- before NGF application. Likewise, PDGF induced a decrease in ϩ cell capacitance (as an indirect measure of cell size) and used as Na current (42.4 Ϯ 3.4%; n ϭ 12) with an onset of 154 Ϯ 17 sec ϩ an estimation of the Na channel current density in individual (n ϭ 12) which was partially reversible to 80.4 Ϯ 6.1% (n ϭ 4) of cells. In accordance with previous observations (Fanger et al., the total current observed before PDGF application. These re- 1995, 1997), PDGF elicited an approximately threefold increase sults suggest that the signaling pathways required for growth ϩ ϩ ϩ in the functional expression of Na channels, elevating Na factor-induced Na current inhibition were present in both un- current densities from 32.7 Ϯ 5.9 pA/pF (n ϭ 14) in undifferen- differentiated and differentiated cells expressing wtPDGF recep- tiated cells to 90.3 Ϯ 8.4 pA/pF (n ϭ 17) in differentiated cells, tors and thus are not dependent on physiological events necessary ϩ which paralleled an increase in Na current densities observed in for neuronal differentiation. In subsequent studies, however, we cells that had been differentiated with NGF (85.9 Ϯ 12.5 pA/pF; limited our experiments to the acute regulation involved in dif- ϩ n ϭ 22). As an additional control, we compared Ca 2 current ferentiated PC12 cells because the presence of several markers densities in both undifferentiated cells and cells differentiated specific for neuronal differentiation was necessary to ensure that ϩ with PDGF and found that PDGF elicited an increase in Ca 2 the effects of mutant PDGF receptors were specific to the inhib- current densities comparable with increases seen in positive con- itory response (see below). trols treated with NGF (data not shown). These results, combined To test whether the acute effects of growth factors were depen- with previous observations from other laboratories (Heasley and dent on the activation of their respective RTKs, we took advan- Johnson, 1992; Fanger et al., 1995, 1997; Vaillancourt et al., tage of a family of protein tyrosine kinase inhibitors, the tyr- 1995), demonstrated that PC12 cells differentiated with PDGF phostins (for review, see Levitzki and Gilon, 1991; Levitzki and are both morphologically and physiologically comparable with Gazit, 1995), specifically tyrphostin AG9, which inhibits PDGF PC12 cells differentiated with NGF and thus serve as an excellent receptor autophosphorylation and the ability of the receptor to model for examining the signaling mechanisms underlying the phosphorylate intracellular signaling molecules (Bilder et al., physiology of fully differentiated neurons. Furthermore, the sim- 1991), and AG879, which prevents the activation of the NGF ilarities in chronic effects of wtPDGF and NGF receptor activa- receptor and neurite extension in PC12 cells (Ohmichi et al., tion suggest similarities in their signaling mechanisms, making 1993). For our experiments, individual cells were loaded with the the wtPDGF receptor a valuable surrogate for analyzing neuro- appropriate inhibitor by including either AG9 or AG879 in the trophin receptor signaling. intracellular recording solution and by allowing the solution to ϩ ϩ To analyze the possibility that NGF could acutely regulate Na diffuse into the cell for 3–5 min before recording Na current channels, we differentiated PC12 cells with PDGF (20 ng/ml) over responses to growth factor application (Fig. 2). We found that ϩ a period of 4–6 d during which Na current densities are up- inclusion of AG9 (10 ␮M) in the intracellular solution dramati- ϩ regulated. Cells differentiated with PDGF were stepped to volt- cally reduced PDGF inhibition of Na currents (10.4 Ϯ 4.8%; ϩ ages that induced peak Na current amplitudes (typically be- n ϭ 13), whereas the acute effects of NGF remained unaffected tween Ϫ10 and 0 mV) and then were subjected to acute (38.9 Ϯ 5.9%; n ϭ 9). The converse was true in differentiated cells ϩ application of NGF (100 ng/ml). Figure 1, A and C, shows that loaded with AG897 (25 ␮M). The inhibition of Na current ϩ NGF caused a rapid decrease in Na current amplitude (36.5 Ϯ caused by NGF (13.6 Ϯ 4.6%; n ϭ 11) was significantly reduced, 3.0%; n ϭ 13) that was partially recoverable to 80.0 Ϯ 4.0% (n ϭ whereas the inhibition caused by PDGF (29.2 Ϯ 2.7%; n ϭ 11) 6) of the total current observed before NGF application. NGF- was only slightly affected when compared with the inhibition induced inhibition was not associated with any change in the observed in control cells. These results strongly suggest that ϩ voltage dependence or time course of the currents. Because growth factors inhibit Na channels via the activation of their ϩ future experiments would entail analyzing Na channel currents cognate RTKs. in PC12 cells expressing mutant receptors, it was essential to Finally, because there is significant conservation of signaling ϩ determine whether PDGF could acutely modulate Na currents mechanisms among the growth factor RTKs, especially in early in a manner similar to NGF. Figure 1, B and C, shows that events (Chao, 1992; Marshall, 1995), we asked whether other ϩ application of PDGF (20 ng/ml) to cells differentiated with NGF growth factors were capable of acutely modulating Na channels ϩ (100 ng/ml) also caused an acute decrease in Na current ampli- (Fig. 3). In addition to the NGF RTK trkA, PC12 cells express tudes (54.0 Ϯ 3.8%; n ϭ 21) that was partially reversible to 71.5 Ϯ RTKs for epidermal growth factor (EGF) and bFGF. Therefore, 7.3% (n ϭ 12) of the total current observed before PDGF appli- either EGF (100 ng/ml) or bFGF (50 ng/ml) was acutely applied cation. Again, this was not the result of a shift in the voltage to cells that had been differentiated with NGF. Like PDGF and ϩ dependence of activation or current kinetics. A comparison of NGF, acute application of EGF inhibited Na currents by 44.4 Ϯ cells acutely treated with NGF and PDGF showed that, in gen- 3.6% (n ϭ 7) with a time to maximum inhibition of ϳ50–75 sec ϩ eral, the time to maximal PDGF-induced Na current inhibition and a recovery to 80.2 Ϯ 6.5% (n ϭ 6) of the total current (160 Ϯ 15 sec; n ϭ 21) was slow relative to the NGF time course observed before growth factor application. Likewise, bFGF ini- ϩ (82 Ϯ 11 sec; n ϭ 12). Furthermore, the magnitude of inhibition tiated a 38.7 Ϯ 3.1% (n ϭ 8) decrease in Na current with a time observed in cells acutely treated with PDGF was generally greater to maximum inhibition of ϳ15–30 sec and a recovery to 86.5 Ϯ than was the inhibition caused by NGF. 3.9% of the total current observed before growth factor applica- We also examined whether the acute responses to NGF and tion. Combined, these results demonstrate that the acute effects ϩ PDGF were the result of signaling mechanisms specific to the of NGF and PDGF on Na channels are shared by other growth differentiated phenotype. Undifferentiated cells expressing factors and that the magnitude of current inhibition and recovery wtPDGF receptors were exposed to acute application of NGF is remarkably preserved throughout the growth factor receptor Hilborn et al. • Growth Factor RTK Regulation of Neuronal Naϩ Channels J. Neurosci., January 15, 1998, 18(2):590–600 593

Figure 1. Growth factors inhibit ϩ Na currents. A, Representative time course of NGF-induced inhibition of ϩ whole-cell Na currents in PC12 cells expressing wtPDGF receptors. PC12 cells were differentiated with 20 ng/ml PDGF for a period of 4–6 d. Differ- entiated cells were then held at Ϫ90 mV and given sequential 10 mV steps from Ϫ60 to 60 mV in 1.5 sec intervals to determine the voltage of peak current amplitude. Command steps to induce peak current amplitudes (open diamonds) were then given every 5 sec before, during, and after acute application of 100 ng/ml NGF (horizontal bar); *selected current traces shown in the inset. B, Representative time course of 20 ng/ml PDGF-induced in- ϩ hibition of whole-cell Na currents in PC12 cells differentiated by chronic treatment with 100 ng/ml NGF. Elec- trophysiological protocols are identical to those described in A; * indicates selected current traces that are shown in the inset. C, Cumulative data for ϩ the inhibition and recovery of Na currents in response to acute application of either 100 ng/ml NGF or 20 ϩ ng/ml PDGF. Whole-cell Na currents recorded during or after growth factor treatment were normalized to whole-cell currents recorded before growth factor applications. Error bars represent the mean Ϯ SEM of nor- ϩ malized Na currents for the indicated growth factor; †The current after recovery from growth factor inhibition is statistically signiﬁcant from the fully inhibited current. The values of n for experimental groups are, from left to right, 13, 6, 21, and 12. family, although the time to reach maximum inhibition was de- tude was not the consequence of a shift in the voltage dependence pendent on the individual growth factor. The results again sug- of channel activation. For differentiated PC12 cells held at Ϫ90 ϩ gest that Na current inhibition is most likely mediated via a mV, the voltage threshold for activation of both control and ϩ signaling pathway that is conserved among growth factor RTKs. growth factor-inhibited whole-cell Na currents is approximately Ϫ50 mV, and the currents reach a maximum amplitude between Growth factors affect steady-state inactivation but not Ϫ steady-state activation of Na ؉ channels 10 and 0 mV (Fig. 4A). Analysis of the steady-state inactivation of control cells and cells acutely treated with growth factors, Similar to the acute effects observed with NGF and PDGF on ϩ ϩ however, demonstrated that acute application of NGF and PDGF PC12 Na current, phosphorylation of rat brain Na channels by ϩ shifted the voltage dependence of inactivation by Ϫ10.0 Ϯ 1.3 mV either PKA or PKC results in a reduction of Na current ampli- (n ϭ 10) and Ϫ8.5 Ϯ 0.8 mV (n ϭ 6), respectively (Fig. 4B). This tude; however, changes in kinetic behavior differ among individ- suggests that the acute inhibition caused by NGF and PDGF is a ual inhibitory agents, suggesting different mechanisms of action. ϩ direct result of a negative shift in the voltage dependence of Na For example, application of puriﬁed PKA to excised membrane ϩ channel inactivation. The similarity in NGF and PDGF effects on patches reduces peak Na current amplitudes without any effect ϩ the Na current steady-state inactivation relationship also sug- on time course or the voltage dependence of activation or inac- ϩ gests that both receptors share a common mode of signaling in tivation (Li et al., 1992), whereas phosphorylation of Na chan- ϩ modulating Na current. nels by PKC slows inactivation in addition to reducing peak current amplitude (Numann et al., 1994). Analysis of acute effects of growth factors on PC12 Current–voltage relationships for morphologically differenti- cells expressing mutant PDGF receptors ated PC12 cells acutely treated with either NGF or PDGF indi- The use of exogenously expressed PDGF receptors in PC12 cells cated that the growth factor-induced reduction in current ampli- assumes that the physiological responses induced by these recep- 594 J. Neurosci., January 15, 1998, 18(2):590–600 Hilborn et al. • Growth Factor RTK Regulation of Neuronal Naϩ Channels

ϩ Figure 2. Inhibition of Na currents by NGF (100 ng/ml) or PDGF (20 ng/ ml) is reduced by inhibitors specific for the wtPDGF and trkA receptors. A, Representative time courses of NGF- and PDGF-induced inhibition of ϩ whole-cell Na currents in differentiated cells loaded with either 25 ␮M AG879 (open diamonds)or10␮M AG9 (closed diamonds). Differentiation protocols and electrophysiological protocols for determining voltages of peak current amplitudes are identical to those described in Figure 1. After achieving whole-cell access, the intracellular solution containing the indicated inhibitor was allowed to diffuse into the cell for a period of 3–5 min. ϩ Peak Na current amplitudes were subsequently recorded before and during acute application of the indicated growth factor (horizontal bar). For comparison, currents were normalized to the total whole-cell current recorded before growth factor application. B, Representative current traces selected from A. Traces depict total whole-cell current and the total current remaining after the indicated growth factor in the presence of the indicated inhibitor reached its maximum effect. C, Cumu- lative data from A. Error bars represent ϩ mean Ϯ SEM of peak Na currents normalized to the total current recorded before indicated growth factor application; † indicates a statistically significant difference from cells that were not subjected to RTK inhibitors. The values of n for experimental groups are, from left to right, 13, 11, 9, 10, 11, and 13. tors are mediated by signaling cascades shared by endogenous Use of PC12 cells expressing mutant PDGF receptors proved RTKs that mediate similar, physiological events. One method of advantageous in identifying the signaling molecules necessary for ϩ testing this possibility is to determine whether the application of neurite extension and for the chronic upregulation of Na chan- one growth factor can occlude, or “mask,” the response of the nels during PC12 cell neuronal differentiation (Vaillancourt et al., second growth factor. We used this approach to determine 1995; Fanger et al., 1997). The tyrosine phosphorylation sites of ϩ whether NGF and PDGF modulate Na channel currents via the receptor are well characterized, and point mutations of these similar, if not identical, signaling pathways. NGF was acutely residues result in the inability of the receptor to associate with applied to cells (Fig. 5A). As expected, NGF induced an inhibi- and activate specific signaling molecules. PLC␥ associates with ϩ tion of Na current (29.2 Ϯ 2.4%; n ϭ 13), but subsequent residues 1009 and 1021, PI3-K associates with residues 740 and application of PDGF after NGF reached its maximum effect 751, the nonreceptor tyrosine phosphatase Syp associates with ϩ caused only minor inhibition of Na current (10.5 Ϯ 3.0%; n ϭ tyrosine 1009, and the GTPase-activating protein (GAP) associ- 13). Similarly, acute application of PDGF to differentiated cells ates with the tyrosine at position 771. The tyrosine at position 716 ϩ (Fig. 5B) caused an inhibition of Na current (30.4 Ϯ 3.5%; n ϭ enhances binding with Grb2 and is thought to be involved in Ras ϩ 10), whereas the inhibition of Na current caused by subsequent activation, although phenylalanine substitutions of this particular NGF treatment (7.7 Ϯ 1.6%; n ϭ 10) was dramatically reduced tyrosine have been unable to inhibit Ras activation to any great relative to inhibition in response to NGF alone. In conjunction extent (for review, see Claesson-Welsh, 1994; van der Geer et al., with previous observations that signals from PDGF receptors 1994). Therefore, to elucidate the signaling pathways underlying ϩ serve as models for NGF-induced signaling in wild-type PC12 the PDGF-induced acute Na current inhibition that we ob- cells, our results strongly favor a common signaling pathway for served, we took advantage of the following paradigm: cells ex- ϩ growth factor-mediated Na channel modulation. pressing exogenous mutant PDGF receptors were morphologi- Hilborn et al. • Growth Factor RTK Regulation of Neuronal Naϩ Channels J. Neurosci., January 15, 1998, 18(2):590–600 595

Figure 3. Activation of either EGF or ϩ bFGF receptors inhibits Na current. A, B, Results are shown from two PDGF receptor-expressing PC12 cells that had been differentiated by chronic ϩ NGF treatment. Peak Na current amplitudes (open diamonds) are plotted as a function of time, showing inhibition in response to EGF (A) or bFGF (B) application (horizontal bars). Currents were evoked every 5 sec; *Selection of raw data traces shown in the insets. cally differentiated with NGF; then, during recording sessions, the 581, F5/579, and F5/581 mutant receptors. However, we found ϩ ϩ ability of PDGF to acutely inhibit Na currents was analyzed that the ability of PDGF to inhibit Na current was reduced in (Fig. 6A,B). cells expressing the F579/581, F5/579, or F5/581 receptors. In ϩ The F5 mutant PDGF receptor is characterized by five these cells PDGF inhibited Na currents by only 20.2 Ϯ 3.6% tyrosine-to-phenylalanine substitutions at residues 740, 751, 771, (n ϭ 20), 17.0 Ϯ 3.0% (n ϭ 14), or 6.7 Ϯ 3.0% (n ϭ 15), 1009, and 1021. Biochemical analyses of this receptor in PC12 respectively. This suggested that PDGF receptors with mutations ϩ cells and other cell lines suggest that it is unable to associate with in their Src binding domains were unable to mediate Na inhi- or activate PLC␥, Syp, PI3-K, or GAP (Valius and Kazlauskas et bition in comparison with their wild-type counterparts, despite al., 1993; Vaillancourt et al., 1995). Despite the inability of this their ability to initiate morphological differentiation (with the receptor to activate these signaling pathways, PC12 cells express- exception of the F5/579 receptor), activate the Ras and MAPK ing the F5 receptor respond to PDGF with the extension of pathway, and induce both c-fos and transin mRNA in a PDGF- ϩ neurites and with the induction of mRNA for c-fos,Na chan- dependent manner (Vaillancourt et al., 1995; Fanger et al., 1997). nels, and the neural-specific metalloprotease transin (Vaillan- To confirm further that the decrease in PDGF-induced inhibition court et al., 1995; Fanger et al., 1997). As expected, our cell lines, was the result of loss of Src activation, we loaded cells expressing when chronically treated with NGF, upregulated the functional the wtPDGF receptor with the Src kinase inhibitor PP1 (Hanke et ϩ expression of Na channels, elevating current densities to 92.6 Ϯ al., 1996) by including the inhibitor in the intracellular recording 8.8 pA/pF (n ϭ 22). Furthermore, acute application of PDGF solution and allowing the solution to diffuse into the cells for 3–5 ϩ elicited a 39.8 Ϯ 3.1% (n ϭ 20) decrease in Na channel currents, min after establishment of whole-cell access and before PDGF suggesting that PLC␥, Syp, PI3-K, and GAP have little effect on application. Addition of PP1 (1 ␮M), which inhibits Src and other ϩ growth factor-mediated Na current inhibition. members of the Src family of kinases, including Fyn and Lyk, had ϩ Because of the presence of tyrosine residues 579 and 581 little to no effect on Na current densities in cells that had been located at the juxtamembrane region, the F5 PDGF receptor still differentiated with either NGF [139.1 Ϯ 13.1 pA/pF (n ϭ 18) retains its ability to activate members of the Src family nonrecep- compared with 115.6 Ϯ 11.4 pA/pF (n ϭ 18) in cells without PP1] tor tyrosine kinases. We next analyzed two PDGF mutants con- or PDGF [103.2 Ϯ 17.5 pA/pF (n ϭ 14) compared with 107.8 Ϯ taining the same five-point substitution motif characteristic of the 14.7 pA/pF (n ϭ 14) in cells without PP1]. However, PP1 effec- ϩ F5 receptor but possessing additional tyrosine-to-phenylalanine tively reduced Na current inhibition by both NGF (15.1 Ϯ 3.4%; substitutions at either residue 579 (F5/579 receptor) or 581 (F5/ n ϭ 14) and PDGF (17.2 Ϯ 3.0%; n ϭ 21), arguing further that 581 receptor) and a third receptor with substitutions at residues the Src family of nonreceptor tyrosine kinases plays a role in ϩ 579 and 581 (F579/581 receptor) with the remainder of the growth factor-induced Na current inhibition. intracellular region intact. Despite similarities in substitution motifs, the ability of each receptor to associate with Src and to Analysis of acute effects of growth factors in PC12 induce Src phosphorylation differ, with PC12 cells expressing the cells expressing a dominant-negative mutant form F579/581 or F5/579 receptor showing no Src association or phos- of Ras phorylation, whereas the F5/581 receptor shows modest Src asso- In PC12 cells, activation of the Ras and MAPK pathway is ciation and phosphorylation (Vaillancourt et al., 1995). To test essential for a variety of morphological and physiological events, the ability of these mutant receptors to mediate PDGF-induced including neurite extension (Szebereńyi et al., 1990; Traverse et ϩ Na current inhibition, we differentiated cells expressing these al., 1992; Cowley et al., 1994; Loeb et al., 1994; Stephens et al., ϩ receptors via chronic treatment with NGF. In agreement with 1994) and the upregulation of Ca 2 channels (Pollock and Rane, previous observations (Fanger et al., 1997), NGF induced mor- 1996). Furthermore, there is evidence that signals mediated by ϩ phological differentiation and upregulated Na channel currents, Src are required for the activation of Ras, suggesting that Ras increasing current densities to 46.6 Ϯ 5.7 pA/pF (n ϭ 19), 78.3 Ϯ might lie downstream of Src in certain RTK signaling cascades 12.1 pA/pF (n ϭ 14), and 44.6 Ϯ 7.4 pA/pF (n ϭ 13) in morpho- (Kremer et al., 1991; Rusanescu et al., 1995). The long-term ϩ logically differentiated cells expressing, respectively, the F579/ upregulation of Na channels during PC12 cell differentiation, 596 J. Neurosci., January 15, 1998, 18(2):590–600 Hilborn et al. • Growth Factor RTK Regulation of Neuronal Naϩ Channels

Figure 4. Current–voltage and steady- ϩ state inactivation plots for Na currents inhibited by growth factor receptor activation. A, Results are shown from two wtPDGF receptor-expressing cells that had been differentiated by chronic application of 20 ng/ml PDGF (left)or100 ng/ml NGF (right). Cells were held at Ϫ90 mV, and command steps from Ϫ60 to 60 mV in 10 mV intervals were given every 5 sec before growth factor application (closed diamonds) and after the acute application of the indicated growth factor had reached its maximum effect (open diamonds). B, Results are shown from two wtPDGF receptor-expressing cells that had been differentiated by chronic PDGF (left) or NGF (right) treatment. Cells were sequentially held at each of the indicated potentials for 3 sec before a command step to Ϫ10 mV to ϩ evoke Na current. The protocol was then repeated in the presence of acutely applied NGF (100 ng/ml) or PDGF (20 ng/ml). Peak current amplitudes were normalized to the current obtained from the Ϫ110 mV hold. Fitted curves are of ϭ ϩ Ϫ the form: I/Imax 1/1 exp(Vhold V1/2 /k), where Vhold is the holding voltage from which the command step was evoked, V1/2 is the voltage corresponding to half-inactivation of the current, and k is the slope constant. The mean Ϯ SEM shift in V1/2 in response to NGF was 10.0 Ϯ 1.3 mV (n ϭ 10 cells). The Ϯ mean SEM shift in V1/2 in response to PDGF was 8.5 Ϯ 0.8 mV (n ϭ 6 cells). however, is completely independent of Ras (Fanger et al., 1993), tiation, it is unlikely that the decrease in inhibition is the result of although activation of Src seems to be important for establishing the inability of Ras to induce differentiation. Therefore, these ϩ fully functional Na channel expression (Fanger et al., 1997). results indicate a role for Ras in the acute effects of growth factors ϩ To determine whether the signaling mechanisms underlying on Na currents. ϩ acute regulation paralleled those responsible for long-term Na expression, we examined the ability of NGF to acutely regulate DISCUSSION ϩ Na channels in 17-2 cells, a PC12 cell line that overexpresses the Our results are the ﬁrst to ascertain that growth factors can ϩ N17 Ras dominant-negative mutant (Fig. 6C). In these cells, NGF acutely inhibit mammalian Na channels, which are fundamental and bFGF fail to induce morphological differentiation and the for the regulation of electrical excitability in neuronal cells. Ap- early-response genes c-fos, c-jun, and zif268 (Szebere´nyi et al., plication of growth factors to differentiated PC12 cells, which ϩ ϩ 1990), and are incapable of upregulating Ca 2 channels (Pollock express the PN-1 and brain type II/IIa Na channels (Mandel et ϩ and Rane, 1996). However, the upregulation of type II/IIA Na al., 1988; D’Arcangelo et al., 1993), resulted in a rapid, partially ϩ ϩ channel mRNA and functional Na channels is unaffected reversible inhibition of whole-cell Na current. The inhibition (Fanger et al., 1997). was dependent on the tyrosine kinase activity of the associated We chronically treated 17-2 cells with either NGF or bFGF (50 growth factor receptors, because RTK inhibitors reduced inhibi- ng/ml) for a period of 5–7 d. As expected, the cells failed to tion by at least two-thirds. These observations are consistent with ϩ extend neurites but expressed elevated levels of Na channels an increasing amount of evidence that in addition to their ability (data not shown). In cells that had been chronically treated with to upregulate ion channel expression during neuronal develop- ϩ bFGF, acute NGF application reduced Na current by only ment, growth factors and their signaling pathways may play an- 13.8 Ϯ 1.0% (n ϭ 10). Likewise, the ability of bFGF to inhibit other, fundamental role as acute regulators of ion channel activ- ϩ Na currents in cells chronically treated with NGF was also ity in both developing and mature nervous systems. Indeed, in rat impaired (15.9 Ϯ 1.0%; n ϭ 10) relative to our previous obser- brain neurons, both neurotrophin-3 (NT-3) and NGF are capable ϩ ϩ vations. Because we showed previously that growth factor- of activating Ca 2 -activated K channels (Holm et al., 1997), ϩ induced inhibition of Na currents does not depend on differen- which contribute to action potential repolarization and often Hilborn et al. • Growth Factor RTK Regulation of Neuronal Naϩ Channels J. Neurosci., January 15, 1998, 18(2):590–600 597

Figure 5. NGF- and PDGF-induced ϩ Na current inhibition are both mutually occlusive, suggesting a common ϩ signaling pathway. A,Na currents (open diamonds) were recorded from differentiated PC12 cells expressing the wtPDGF receptor. During whole- cell recording sessions, NGF (100 ng/ ml) was applied (upper bar). When the NGF response reached a maximum, PDGF (20 ng/ml) was then applied ϩ (lower bar). Inset, Representative Na current traces depicting total current, the current remaining after subsequent application of NGF, and the current remaining after application of ϩ PDGF. B,Na currents were recorded from differentiated PC12 cells expressing the wtPDGF receptor. During whole-cell recording sessions, PDGF (20 ng/ml) was applied (upper bar). When the PDGF response ϩ reached a maximum, NGF (100 ng/ml) was then applied (lower bar). Inset, Representative Na current traces depicting total current, the current remaining after subsequent application of PDGF, and the current remaining after the application of NGF. underlie the modulation of action potential frequency. Addition- Our results also support an increasing amount of evidence that ϩ ally, both NMDA receptors and the human Kv1.5 K channel activation of Src and/or Ras through RTKs is a critical compo- possess Src-specific SH3 binding domains, associate with Src, and nent of ion channel regulation in both developing and mature are modulated by this association (Holmes et al., 1996; Yu et al., nervous systems. For example, chronic treatment of 17-2 PC12 ϩ 1997), further arguing that signals activated by RTKs, particu- cells with NGF or bFGF fails to upregulate Ca 2 channels. larly Src, are intimately involved with ion channel regulation. In Likewise, PC12 cells morphologically differentiated by oncogenic ϩ agreement with this, our own observations with PDGF receptors Ras fail to show an increase in Ca 2 channel expression, suggest- deficient in activating the Src family of kinases demonstrate that ing that Ras is necessary but not sufficient for this upregulation ϩ Src is essential for growth factor-induced Na channel inhibition. (Pollock and Rane, 1996). PC12 cells induced to undergo mor- It should be noted, however, that neither the ␣ subunits of the phological differentiation by oncogenic Src, however, upregulate ϩ ϩ PN-1 and type II/IIa Na channels nor the ␤ subunits possess the Ca2 channels in a manner comparable with wild-type PC12 cells RPLPXXP SH3-binding domain motif indicative of direct Src differentiated by NGF or bFGF (Rausch et al., 1990). Further- ϩ association, suggesting that the influence of Src on Na channels more, our own observations with PC12 cells expressing PDGF is not direct and occurs via downstream effectors. receptors deficient in Src signaling suggest that Src may also be a It is interesting to note, then, that studies indicate that Src, Ras, necessary component in this upregulation (M. D. Hilborn and S. and MAP kinases act in sequence to initiate the induction of G. Rane, unpublished observations). Similar to the situation with ϩ ϩ early genes and morphological differentiation in PC12 cells (Kre- Ca2 channels, the upregulation of Na channel expression mer et al., 1991; D’Arcangelo and Halegoua, 1993; Rusanescu et during growth factor-induced PC12 cell differentiation seems to al., 1995). Because PDGF receptors deficient in activating the Ras be partially dependent on the activation of Src (Fanger et al., and MAPK pathway have yet to be successfully expressed, we 1997). However, this upregulation is independent of Ras activa- turned to the 17-2 PC12 cell line, which overexpresses the tion (Fanger et al., 1993), an intriguing difference from our own dominant-negative mutant N17 Ras, to analyze the role of Ras in observations that Ras activation is necessary for acute regulation. ϩ Na current inhibition. Although we could not definitively con- This difference in signaling, in conjunction with signals generated clude that Src lies upstream of Ras, it is intriguing to note that the by sustained activation of RTKs, could account for the ability of ϩ block of growth factor-induced inhibition was comparable be- RTKs to regulate Na and other ion channels both acutely and tween cells loaded with RTK inhibitors and cell lines deficient for chronically. Further analysis of the role of Src, Ras, and their either Src or Ras signaling, which would be expected if these downstream effectors in acute ion channel regulation will perhaps molecules are activated in series. Still, it is possible that Ras and resolve these issues. ϩ Src inhibit Na channels via parallel, redundant pathways; thus Little is known about the receptor-mediated modulation of ϩ we cannot eliminate the possibility that activation of one signaling Na channels. Activation of G-protein-coupled pathways en- ϩ molecule can partially compensate for inactivity of the other. hanced brain Na channel activity in Chinese hamster ovary ϩ ϩ Furthermore, Na channel inhibition is not completely blocked (CHO) cells (Ma et al., 1994), and inhibition of cardiac Na in cell lines deficient in Src or Ras signaling, which suggests that channels by ␤-adrenergic agonists can also be mediated via pathways independent of Ras and Src could also be contributing G-proteins (Schubert et al., 1989). Stimulation of ␤2-adrenergic ϩ to acute Na channel regulation. However, the inability to com- receptors expressed in Xenopus oocytes can either enhance or ϩ pletely block inhibition is probably the result of a small amount of attenuate Na currents, depending on the levels of receptor residual Src and Ras activity, because growth factors applied to expression (Smith and Goldin, 1992, 1996), and both PKA and ϩ these cell lines are still capable of inducing Src- and Ras- PKC have been shown to modulate Na channel currents by dependent responses to a minor degree (Szebereńyi et al., 1990; directly phosphorylating key residues on the ␣-subunit of rat Kremer et al., 1991; D’Arcangelo and Halegoua, 1993; Vaillan- brain channels (Rossie and Catterall, 1987, 1989; Rossie et al., court et al., 1995; Fanger et al., 1997). 1987; West et al., 1991; Murphy and Catterall, 1992; for review, 598 J. Neurosci., January 15, 1998, 18(2):590–600 Hilborn et al. • Growth Factor RTK Regulation of Neuronal Naϩ Channels

Figure 6. Analysis of growth factor- ϩ induced inhibition of Na current in PC12 cells expressing mutant PDGF receptors or a dominant-negative Ras mutant. A, PC12 cells expressing either F5, F579/581, F5/579, or F5/ 581 mutant PDGF receptors were differentiated by chronic treatment ϩ with 100 ng/ml NGF. Peak Na current amplitudes (open diamonds) are plotted as a function of time, showing inhibition in response to 20 ng/ml PDGF (horizontal bars). Recording protocols are described in Figure 1; *Raw data traces selected for insets. B, Cumulative data for experiments described in A are shown. Whole-cell currents recorded during growth factor application were normalized to whole-cell currents recorded before growth factor application. Error bars represent the mean Ϯ SEM of nor- ϩ malized Na current amplitudes; †Statistically significant difference from PC12 cells expressing the wtP- DGF receptor. The values of n for each experimental group are, from left to right, 10, 20, 20, 15, and 14. C, Cumulative data for experiments with 17-2 PC12 cells, which express the dominant-negative mutant N17 Ras, are shown. Error bars represent the mean Ϯ SEM of normalized ϩ whole-cell Na currents after acute application of 100 ng/ml NGF (solid columns) or 50 ng/ml bFGF (shaded columns) reached its maximum effect in either 17-2 cells or PC12 cells expressing the wtPDGF receptor; †Sta- tistically significant difference from PC12 cells expressing the wtPDGF receptor. The values of n for each experimental group are, from left to right, 10, 8, 10, and 10. see Cukierman, 1996). Because NGF activates PLC␥ and PKA comparable with that observed with wild-type receptor, suggest- during differentiation and because activation of the PKA pathway ing that downstream effectors of PLC␥, including PKC, are not has been implicated in post-translational effects required for full, required for this response. Furthermore, PKC inhibition of rat ϩ ϩ functional Na channel expression during PC12 cell differentia- brain Na channels expressed in CHO cells is accompanied by a tion (Kalman et al., 1990; Ginty et al., 1992; D’Arcangelo et al., slowing of inactivation (Numann et al., 1994), and the attenuation ϩ 1993), both PKA and PKC might be considered candidates for of whole-cell Na currents is the consequence of a depolarizing ϩ mediating acute growth factor-induced Na channel inhibition. shift in the voltage dependence of channel activation (Dascal and ϩ However, attenuation of Na currents by PKA phosphorylation Lotan, 1991; Schreibmayer et al., 1991), both of which differ from occurs without a change in steady-state activation or inactivation the shift in steady-state inactivation that we observed. And al- ϩ properties of the channel (Gershon et al., 1992; Li et al., 1992, though a negative shift in the inactivation curve of Na channels 1993; Smith and Goldin, 1996), an observation that differs from in a mouse neuroblastoma cell line was observed in response to ϩ our own observation that growth factor-induced Na current some PKC activators (Godoy and Cukierman, 1994a,b), a inhibition is associated with a negative shift in steady-state inac- follow-up study demonstrated that the channel attenuation was a tivation. Additionally, the F5 mutant PDGF receptor, which is direct effect of the PKC activators (Renganathan et al., 1995). ϩ deficient in activating PLC␥, inhibited Na current to a level Overall, our studies complement an increasing amount of evi- Hilborn et al. • Growth Factor RTK Regulation of Neuronal Naϩ Channels J. 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