Elsevier

Cell Calcium

Volume 43, Issue 6, June 2008, Pages 562-575
Cell Calcium

Growth factors differentially regulate neuronal Cav channels via ERK-dependent signalling

https://doi.org/10.1016/j.ceca.2007.10.001Get rights and content

Abstract

Voltage-gated calcium channels (Cav) are tonically up-regulated via Ras/extracellular signal-regulated kinase (ERK) signalling in sensory neurones. However, the mechanisms underlying the specificity of cellular response to this pathway remain unclear. Neurotrophic factors are attractive candidates to be involved in this process as they are key regulators of ERK signalling and have important roles in neuronal survival, development and plasticity. Here, we report that in rat dorsal root ganglion neurones, endogenous nerve growth factor (NGF), glial derived neurotrophic factor (GDNF) and epidermal growth factor (EGF) are all involved in tonic ERK-dependent up-regulation of Cav channels. Chronic (overnight) deprivation of growth factors inhibits total Cav current according to developmental changes in expression of the cell surface receptors for NGF, GDNF and EGF. Whilst EGF specifically regulates transcriptional expression of Cavs, NGF and GDNF also acutely modulate Cav channels within a rapid (∼10 min) time-frame. These acute effects likely involve changes in the biophysical properties of Cavs, including altered channel gating rather than changes in surface expression. Furthermore, NGF, GDNF and EGF differentially regulate specific populations of Cavs. Thus, ERK-dependent regulation of Cav activity provides an elegant and extremely flexible system with which to tailor calcium influx to discrete functional demands.

Introduction

Voltage-gated calcium channels (Cav) play a fundamental role in regulating cellular activity by coupling changes in membrane potential to the influx of calcium, a pivotal second messenger involved in diverse cellular processes such as muscle contraction, neurotransmitter release and gene expression [1]. Multiple Cav subtypes (L-, N-, P/Q-, R- and T-type) exist that differ in their biophysical properties and distributions both within and between cells/tissues, thereby allowing cells to tailor calcium influx to discrete functional demands [1]. Furthermore, Cavs are multi-subunit proteins composed of a pore-forming α1 subunit and auxiliary β, α2δ and possibly γ subunits [1]. With at least ten α1, four β and four α2δ subunit genes so far identified, many with multiple splice variants, there is enormous structural and functional diversity of Cavs [2]. Aberrant Cav function is implicated in numerous conditions including cardiac arrhythmias, epilepsy, migraine and neuropathic pain. Consequently, a definition of the mechanisms that regulate calcium influx through Cavs is crucial to cell biology and medicine alike.

In addition to their intrinsic voltage-dependent regulation, Cav channels are also tightly regulated via a number of intracellular signalling pathways that include heterotrimeric G proteins and protein kinases, notably protein kinases A and C (PKA, PKC) and calcium-calmodulin kinase II (CaMKII) [1], [3], [4]. More recently, signalling via the small G protein, Ras and its downstream kinases, ERK1/2 (extracellular signal-regulated kinase or p44/p42 MAPK), has also been identified as a key pathway in regulation of neuronal Cavs [5], [6], [7]. ERK-dependent enhancement of Cav currents is most often reported to involve gene expression-mediated synthesis of new channels [8], [9]. However, in sensory dorsal root ganglion neurones (DRGs), we have previously shown that acute suppression of Ras/ERK activity induces rapid inhibition of Cav current by up to 40% within 5–10 min [6], [10]. These effects are consistent with post-translational modulation of channels at the plasma membrane, and/or, changes in surface expression [4], [11], [12]. Furthermore, we recently reported that recombinant N-type Cav channels (Cav2.2) are directly phosphorylated by ERK and that this process underlies ERK-dependent enhancement of Cav2.2 current [13]. Since multiple Cav subtypes possess putative ERK phosphorylation sites, it is possible that numerous calcium-dependent cellular processes could be directly regulated via ERK phosphorylation [13], [14]. DRGs express all Cav subtypes (L-, N-, P/Q-, R- and T-type) and importantly, ERK-dependent up-regulation of whole cell Cav current in these cells is reported to involve all high voltage-activated (HVA) channels (L-, N-, P/Q- and R-type) to a similar extent [10]. This raises important questions as to how differential regulation of Cavs and hence specificity of cellular response to calcium entry through these channels might be achieved via this pathway.

In this regard, growth factors such as the neurotrophin, nerve growth factor (NGF), are particularly attractive candidates to be involved in fine-tuning calcium entry through Cavs. In DRG neurones, NGF, neurotrophin-3 and brain-derived neurotrophic factor are important regulators of neuronal survival, development and plasticity. Many of their actions are mediated through the activation of Ras and subsequently ERK, via binding to and activation of, their respective Trk-family receptor tyrosine kinases (RTKs), TrkA, TrkB, TrkC [15], [16]. In addition, differential effects of these neurotrophins on long-term (hours–days) transcriptional regulation of Cavs has been reported in a variety of neuronal cell types and in DRGs, rapid (minutes) up-regulation of Cav current via NGF/TrkA/Ras signalling has also been shown [6], [8]. The small-medium diameter C-fibre DRG neurones that are predominantly nociceptive, are dependent on NGF for their survival at early postnatal stages [17], [18]. However, during early post-natal development, a sub-population of these cells progressively lose dependence on NGF for survival and become dependent on glial-derived neurotrophic factor (GDNF) [17], [18]. GDNF is important in proliferation, survival and regeneration of sensory nerves following injury and has been shown in other neuronal cell types to regulate ion channels including Cavs [19], [20], [21], [22]. GDNF exerts its actions via a multi-component receptor complex composed of Ret tyrosine kinase and the GDNF-family-receptor α1 (GFRα1) which binds the ligand and thus, confers specificity for GDNF signalling over other GDNF-family ligands [19]. GDNF can activate Ras/ERK signalling via Ret tyrosine kinase [23]. Also expressed in DRG neurones is epidermal growth factor receptor (EGFR), which in common with other RTKs, activates Ras/ERK via its intrinsic tyrosine kinase activity [24]. Although the role of EGF-mediated stimulation of ERK in DRGs is at present unclear, EGF is known to have neurotrophic actions in the developing and mature central nervous system (CNS) and has been reported to regulate transcriptional expression of Cavs in a variety of cell types [25], [26], [27], [28], [29]. Differential effects of neurotrophins, EGF and GDNF on transcriptional regulation of Cavs and other ion channels have been reported in a variety of neuronal systems [8], [9], [21], [28], [29]. Thus, in DRG neurones, differential signalling via growth factors may also regulate ERK-dependent calcium entry through Cavs, thereby promoting specificity of cellular response to Cav activity. In the present study we have investigated this possibility by studying the effects specifically of NGF, GDNF and EGF, primarily on short-term ERK-dependent regulation of whole cell Cav current in rat DRG neurones. Briefly, our data demonstrate that all three growth factors tonically enhance HVA Cav currents in these cells. However, specific growth factors differentially regulate calcium entry through discrete populations of Cavs. Further differentiation of cellular response to Cav activity is observed according to developmental changes in growth factor receptor expression and differences in the mechanisms underlying current enhancement (transcriptional versus post-translational). Thus, we provide strong evidence that growth factor-dependent regulation of multiple Cav channels via ERK signalling provides a physiologically important means by which numerous calcium-dependent cellular processes can be dynamically regulated in space and time.

Section snippets

Cell culture

Rat dorsal root ganglion neurones from either 0- to 2- or 7- to 9-day old (P0–2 or P7–9) Sprague–Dawley rat pups were cultured essentially as described by Fitzgerald and Dolphin [6]. Briefly, dorsal root ganglia (DRGs) were isolated from rat pups that had been killed by cervical dislocation. Ganglia were then incubated with collagenase (1.25 μg ml−1; Sigma–Aldrich, Gillingham, Dorset, UK) for 13 min followed by trypsin (2.5 μg ml−1; Sigma–Aldrich) for 6 min in DMEM/F12 media (Invitrogen, Paisley, UK)

Are NGF, GDNF and EGF involved in tonic up-regulation of Cav channels in DRG neurones?

Our previous work in DRG neurones has shown that stimulation of Ras/ERK signalling tonically up-regulates calcium entry through HVA Cav channels and that this process is at least partially mediated via NGF/RTK activation [6], [10]. However, other converging or parallel pathways, involving GDNF, EGF and/or Src, may also be important [6]. Preliminary experiments were, therefore, conducted to determine whether GDNF and EGF, in addition to NGF, tonically up-regulate Cav currents in DRGs. Cells were

Discussion

A definition of the mechanisms by which extra-cellular signalling cues control voltage-gated ion channel function is a fundamental issue in excitable cell signalling. We have previously identified a novel post-translational pathway by which multiple HVA Cav channels in DRG neurones are tonically up-regulated via Ras/ERK signalling [6], [10]. However, the cellular mechanisms responsible for regulating individual Cavs and hence specificity of cellular response, remain unclear. Neurotrophic

Acknowledgements

We thank Mr. R.E. Paddon for technical support and the Wellcome Trust for financial support (project grant 069393 to E.M.F.)

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