ERK1/2 mitogen-activated protein kinase phosphorylates sodium channel Na(v)1.7 and alters its gating properties.

Na(v)1.7 sodium channels can amplify weak stimuli in neurons and act as threshold channels for firing action potentials. Neurotrophic factors and pro-nociceptive cytokines that are released during development and under pathological conditions activate mitogen-activated protein kinases (MAPKs). Previous studies have shown that MAPKs can transduce developmental or pathological signals by regulating transcription factors that initiate a gene expression response, a long-term effect, and directly modulate neuronal ion channels including sodium channels, thus acutely regulating dorsal root ganglion (DRG) neuron excitability. For example, neurotrophic growth factor activates (phosphorylates) ERK1/2 MAPK (pERK1/2) in DRG neurons, an effect that has been implicated in injury-induced hyperalgesia. However, the acute effects of pERK1/2 on sodium channels are not known. We have shown previously that activated p38 MAPK (pp38) directly phosphorylates Na(v)1.6 and Na(v)1.8 sodium channels and regulates their current densities without altering their gating properties. We now report that acute inhibition of pERK1/2 regulates resting membrane potential and firing properties of DRG neurons. We also show that pERK1 phosphorylates specific residues within L1 of Na(v)1.7, inhibition of pERK1/2 causes a depolarizing shift of activation and fast inactivation of Na(v)1.7 without altering current density, and mutation of these L1 phosphoacceptor sites abrogates the effect of pERK1/2 on this channel. Together, these data are consistent with direct phosphorylation and modulation of Na(v)1.7 by pERK1/2, which unlike the modulation of Na(v)1.6 and Na(v)1.8 by pp38, regulates gating properties of this channel but not its current density and contributes to the effects of MAPKs on DRG neuron excitability.

Neuropathic pain memory is maintained by Rac1-regulated dendritic spine remodeling after spinal cord injury.

Localized increases in synaptic strength constitute a synaptic basis for learning and memory in the CNS and may also contribute to the maintenance of neuropathic pain after spinal cord injury (SCI) through the de novo formation or elaboration of postsynaptic dendritic structures. To determine whether SCI-induced dendritic spine remodeling contributes to neuronal hyperexcitability and neuropathic pain, we analyzed spine morphometry, localization, and functional influence in dorsal horn (DH) neurons in adult rats 1 month after sham surgery, contusion SCI, and SCI treated with a selective inhibitor of Rac1 activation, NSC23766. After SCI, DH neurons located in lamina IV-V exhibited increased spine density, redistributed spines, and mature spines compared with control neurons, which was associated with enhancement of EPSCs in computer simulations and hyperexcitable responsiveness to innocuous and noxious peripheral stimuli in unit recordings in vivo. SCI animals also exhibited symptoms of tactile allodynia and thermal hyperalgesia. Inhibition of the small GTP-binding protein Rac1 ameliorated post-SCI changes in spine morphology, attenuated injury-induced hyperexcitability of wide-dynamic range neurons, and progressively increased pain thresholds over a 3 d period. This suggests that Rac1 is an important intracellular signaling molecule involved in a spinal dendritic spine pathology associated with chronic neuropathic pain after SCI. Our report provides robust evidence for a novel conceptual bridge between learning and memory on the one hand, and neuropathic pain on the other.

Fibroblast growth factor homologous factor 2 (FGF-13) associates with Nav1.7 in DRG neurons and alters its current properties in an isoform-dependent manner.

Fibroblast Growth Factor Homologous Factors (FHF) constitute a subfamily of FGF proteins with four prototypes (FHF1-4; also known as FGF11-14). FHF proteins have been shown to bind directly to the membrane-proximal segment of the C-terminus in voltage-gated sodium channels (Nav), and regulate current density, availability, and frequency-dependent inhibition of sodium currents. Members of the FHF2 subfamily, FHF2A and FHF2B, differ in the length and sequence of their N-termini, and, importantly, differentially regulate Nav1.6 gating properties. Using immunohistochemistry, we show that FHF2 isoforms are expressed in adult dorsal root ganglion (DRG) neurons where they co-localize with Nav1.6 and Nav1.7. FHF2A and FHF2B show differential localization in neuronal compartments in DRG neurons, and levels of expression of FHF2 factors are down-regulated following sciatic nerve axotomy. Because Nav1.7 in nociceptors plays a critical role in pain, we reasoned that its interaction with FHF2 isoforms might regulate its current properties. Using whole-cell patch clamp in heterologous expression systems, we show that the expression of FHF2A in HEK293 cell line stably expressing Nav1.7 channels causes no change in activation, whereas FHF2B depolarizes activation. Both FHF2 isoforms depolarize fast-inactivation. Additionally, FHF2A causes an accumulation of inactivated channels at all frequencies tested due to a slowing of recovery from inactivation, whereas FHF2B has little effect on these properties of Nav1.7. Measurements of the Nav1.7 current in DRG neurons in which FHF2 levels are knocked down confirmed the effects of FHF2A on repriming, and FHF2B on activation, however FHF2A and B did not have an effect on fast inactivation. Our data demonstrates that FHF2 does indeed regulate the current properties of Nav1.7 and does so in an isoform and cell-specific manner.

Neurotransmitter signaling in postnatal neurogenesis: The first leg.

Like the liver or other peripheral organs, two regions of the adult brain possess the ability of self-renewal through a process called neurogenesis. This raises tremendous hope for repairing the damaged brain, and it has stimulated research on identifying signals controlling neurogenesis. Neurogenesis involves several stages from fate determination to synaptic integration via proliferation, migration, and maturation. While fate determination primarily depends on a genetic signature, other stages are controlled by the interplay between genes and microenvironmental signals. Here, we propose that neurotransmitters are master regulators of the different stages of neurogenesis. In favor of this idea, a description of selective neurotransmitter signaling and their functions in the largest neurogenic zone, the subventricular zone (SVZ), is provided. In particular, we emphasize the interactions between neuroblasts and astrocyte-like cells that release gamma-aminobutyric acid (GABA) and glutamate, respectively. However, we also raise several limitations to our knowledge on neurotransmitters in neurogenesis. The function of neurotransmitters in vivo remains largely unexplored. Neurotransmitter signaling has been viewed as uniform, which dramatically contrasts with the cellular and molecular mosaic nature of the SVZ. How neurotransmitters are integrated with other well-conserved molecules, such as sonic hedgehog, is poorly understood. In an effort to reconcile these differences, we discuss how specificity of neurotransmitter functions can be provided through their multitude of receptors and intracellular pathways in different cell types and their possible interactions with sonic hedgehog.

Expression, localization and functions in acrosome reaction and sperm motility of Ca(V)3.1 and Ca(V)3.2 channels in sperm cells: an evaluation from Ca(V)3.1 and Ca(V)3.2 deficient mice.

In spermatozoa, voltage-dependent calcium channels (VDCC) have been involved in different cellular functions like acrosome reaction (AR) and sperm motility. Multiple types of VDCC are present and their relative contribution is still a matter of debate. Based mostly on pharmacological studies, low-voltage-activated calcium channels (LVA-CC), responsible of the inward current in spermatocytes, were described as essential for AR in sperm. The development of Ca(V)3.1 or Ca(V)3.2 null mice provided the opportunity to evaluate the involvement of such LVA-CC in AR and sperm motility, independently of pharmacological tools. The inward current was fully abolished in spermatogenic cells from Ca(V)3.2 deficient mice. This current is thus only due to Ca(V)3.2 channels. We showed that Ca(V)3.2 channels were maintained in sperm by Western-blot and immunohistochemistry experiments. Calcium imaging experiments revealed that calcium influx in response to KCl was reduced in Ca(V)3.2 null sperm in comparison to control cells, demonstrating that Ca(V)3.2 channels were functional. On the other hand, no difference was noticed in calcium signaling induced by zona pellucida. Moreover, neither biochemical nor functional experiments, suggested the presence of Ca(V)3.1 channels in sperm. Despite the Ca(V)3.2 channels contribution in KCl-induced calcium influx, the reproduction parameters remained intact in Ca(V)3.2 deficient mice. These data demonstrate that in sperm, besides Ca(V)3.2 channels, other types of VDCC are activated during the voltage-dependent calcium influx of AR, these channels likely belonging to high-voltage activated Ca(2+) channels family. The conclusion is that voltage-dependent calcium influx during AR is due to the opening of redundant families of calcium channels.

Junctate, an inositol 1,4,5-triphosphate receptor associated protein, is present in rodent sperm and binds TRPC2 and TRPC5 but not TRPC1 channels.

The acrosome reaction, the first step of the fertilization, is induced by calcium influx through Canonical Transient Receptor Potential channels (TRPC). The molecular nature of TRPC involved is still a debated question. In mouse, TRPC2 plays the most important role and is responsible for the calcium plateau. However, TRPC1 and TRPC5 are also localized in the acrosomal crescent of the sperm head and may participate in calcium signaling, especially in TRPC2-deficient mice. Activation of TRPC channels is an unresolved question in germ and somatic cells as well. In particular, in sperm, little is known concerning the molecular events leading to TRPC2 activation. From the discovery of IP3R binding domains on TRPC2, it has been suggested that TRPC channel activation may be due to a conformational coupling between IP3R and TRPC channels. Moreover, recent data demonstrate that junctate, an IP3R associated protein, participates also in the gating of some TRPC. In this study, we demonstrate that junctate is expressed in sperm and co-localizes with the IP3R in the acrosomal crescent of the anterior head of rodent sperm. Consistent with its specific localization, we show by pull-down experiments that junctate interacts with TRPC2 and TRPC5 but not with TRPC1. We focused on the interaction between TRPC2 and junctate, and we show that the N-terminus of junctate interacts with the C-terminus of TRPC2, both in vitro and in a heterologous expression system. We show that junctate binds to TRPC2 independently of the calcium concentration and that the junctate binding site does not overlap with the common IP3R/calmodulin binding sites. TRPC2 gating is downstream phospholipase C activation, which is a key and necessary step during the acrosome reaction. TRPC2 may then be activated directly by diacylglycerol (DAG), as in neurons of the vomeronasal organ. In the present study, we investigated whether DAG could promote the acrosome reaction. We found that 100 microM OAG, a permeant DAG analogue, was unable to trigger the acrosome reaction. Altogether, these results provide a new hypothesis concerning sperm TRPC2 gating: TRPC2 activation may be due to modifications of its interaction with both junctate and IP3R, induced by depletion of calcium from the acrosomal vesicle.

Functional interaction between mouse spermatogenic LVA and thapsigargin-modulated calcium channels.

The acrosome reaction in mouse is triggered by a long-lasting calcium signaling produced by a chain of openings of several calcium channels, a low-voltage-activated (LVA) calcium channel, an inositol trisphosphate receptor (IP(3)R), and the store-operated calcium channel TRP2. Since mature sperm cells are refractory to patch clamp experiments, we study the functional interactions among those sperm calcium channels in spermatogenic cells. We have studied the role of cytosolic calcium in voltage-dependent facilitation of low voltage-activated calcium channels. Calcium concentration was modified through the inclusion of the calcium buffers, EGTA and BAPTA, in the recording pipette solution, and by addition of calcium modulators like thapsigargin and the calcium ionophore A23187. We demonstrate that lowering calcium concentration below resting level allows to evidence a voltage-dependent facilitation. We also show that LVA calcium channels present strong voltage-dependent inhibition by thapsigargin. This effect is independent of cytosolic calcium elevation secondary to calcium store depletion and to the activation of TRP channels. Our data evidence an interesting functional relationship, in this cell type, between LVA channels and proteins whose activity is related to calcium filling state of the endoplasmic reticulum (presumably TRP channels and inositol triphosphate receptor). These relationships may contribute to the regulation of calcium signaling during acrosome reaction of mature sperm cell.

Biophysical and pharmacological characterization of spermatogenic T-type calcium current in mice lacking the CaV3.1 (alpha1G) calcium channel: CaV3.2 (alpha1H) is the main functional calcium channel in wild-type spermatogenic cells.

Mammalian acrosome reaction (AR) requires successive activation of three different types of calcium channels (T-type channels, Inositol-3-phosphate (InsP3) receptors, and TRPC2 channels). All the calcium signaling is under the control of the activation of the first-one, a T-type calcium channel. The molecular characterization of the T-type calcium channel is still a matter of debate, previous reports showing the presence of transcripts for Ca(V)3.1 and Ca(V)3.2 subunits. Using mice deficient for Ca(V)3.1 subunit, we show that the T-type current density in spermatogenic cells is not reduced in deficient mice versus control mice. We characterized the biophysical and pharmacological properties of T-type current in spermatogenic cells from Ca(V)3.1 deficient mice. Biophysical and pharmacological properties of spermatogenic T-type current from wild-type and Ca(V)3.1 deficient mice demonstrate that Ca(V)3.3 does not contribute to T-type current. Moreover, nickel and amiloride inhibit T-type currents in deficient and wild-type mice with similar potencies. These results demonstrate that T-type currents in spermatogenic cells is due to Ca(V)3.2 subunit and that Ca(V)3.1 contributes to a very negligible extent to the T-type currents. Thus, the deficient Ca(V)3.1 mouse model allows the characterization of native Ca(V)3.2 currents in spermatogenic cells. Spermatogenic Ca(V)3.2 currents present specific feature in comparison to the cloned Ca(V)3.2 current so far. More particularly, the time-dependence of recovery from short-term inactivation of native spermatogenic Ca(V)3.2 is close to 100 millisecond, a value expected for Ca(V)3.1 current.

Repositioning of charged I-II loop amino acid residues within the electric field by beta subunit as a novel working hypothesis for the control of fast P/Q calcium channel inactivation.

We have investigated the contribution of the Ca(v)beta subunits to the process of inactivation dependent of the I-II loop of Ca(v)alpha(2.1). Two amino acid residues located in the alpha1 interaction domain (AID) of the I-II loop of Ca(v)alpha(2.1) (Arg(387) and Glu(388)) have been directly implicated in voltage-dependent inactivation of this channel. Various point mutations of these residues disrupt the interaction between the I-II loop and the III-IV loop, and thereby modify the inactivation properties of the channel by accelerating its kinetics and shifting the steady-state inactivation curve towards hyperpolarized potentials. A similar disruption is produced by Ca(v)beta(4) subunit association with the I-II loop. Moreover, in the presence of Ca(v)beta(4) subunit, introducing negatively charged residues at positions 387 or 388 slows inactivation kinetics down, whereas introducing positive charges has the opposite effect. The shift of the steady-state inactivation curve is also amino acid charge-dependent. In contrast, mutation of Arg(387) or Glu(388) does not alter the differential regulation of the different Ca(v)beta isoforms on inactivation. These results suggest that the expression of Ca(v)beta(4) alters the contribution of charged residues at positions 387 and 388 to inactivation. We discuss these results with regard to the actual hypotheses on the mechanisms of calcium channel inactivation. We introduce the working concept that Ca(v)beta-subunits produce a conformational repositioning of charged AID residues within the electric field.