Collapsin response mediator proteins (CRMPs) mediate signal transduction of neurite outgrowth and axonal guidance during neuronal development. Voltage-gated Ca2+ channels and interacting proteins are essential in neuronal signaling and synaptic transmission during this period. We recently identified the presynaptic N-type voltage-gated Ca2+ channel (Cav2.2) as a CRMP-2-interacting partner. Here, we investigated the effects of a functional association of CRMP-2 with Cav2.2 in sensory neurons. Cav2.2 colocalized with CRMP-2 at immature synapses and growth cones, in mature synapses and in cell bodies of dorsal root ganglion (DRG) neurons. Co-immunoprecipitation experiments showed that CRMP-2 associates with Cav2.2 from DRG lysates. Overexpression of CRMP-2 fused to enhanced green fluorescent protein (EGFP) in DRG neurons, via nucleofection, resulted in a significant increase in Cav2.2 current density compared with cells expressing EGFP. CRMP-2 manipulation changed the surface levels of Cav2.2. Because CRMP-2 is localized to synaptophysin-positive puncta in dense DRG cultures, we tested whether this CRMP-2-mediated alteration of Ca2+ currents culminated in changes in synaptic transmission. Following a brief high-K+-induced stimulation, these puncta became loaded with FM4-64 dye. In EGFP and neurons expressing CRMP-2–EGFP, similar densities of FM-loaded puncta were observed. Finally, CRMP-2 overexpression in DRG increased release of the immunoreactive neurotransmitter calcitonin gene-related peptide (iCGRP) by ∼70%, whereas siRNA targeting CRMP-2 significantly reduced release of iCGRP by ∼54% compared with control cultures. These findings support a novel role for CRMP-2 in the regulation of N-type Ca2+ channels and in transmitter release.
Potassium channels play a critical role in regulating many aspects of action potential (AP) firing. To establish the contribution of the voltage-dependent potassium channel Kv1.1 in regulating excitability, we used the selective blocker dendrotoxin-K (DTX-K) and small interfering RNA (siRNA) targeted to Kv1.1 to determine their effects on AP firing in small-diameter capsaicin-sensitive sensory neurons. A 5-min exposure to 10 nM DTX-K suppressed the total potassium current (I(K)) measured at +40 mV by about 33%. DTX-K produced a twofold increase in the number of APs evoked by a ramp of depolarizing current. Associated with increased firing was a decrease in firing threshold and rheobase. DTX-K did not alter the resting membrane potential or the AP duration. A 48-h treatment with siRNA targeted to Kv1.1 reduced the expression of this channel protein by about 60% as measured in Western blots. After treatment with siRNA, I(K) was no longer sensitive to DTX-K, indicating a loss of functional protein. Similarly, after siRNA treatment exposure to DTX-K had no effect on the number of evoked APs, firing threshold, or rheobase. However, after siRNA treatment, the firing threshold had values similar to those obtained after acute exposure to DTX-K, suggesting that the loss of Kv1.1 plays a critical role in setting this parameter of excitability. These results demonstrate that Kv1.1 plays an important role in limiting AP firing and that siRNA may be a useful approach to establish the role of specific ion channels in the absence of selective antagonists.
Chi XX, Nicol GD. The sphingosine 1-phosphate receptor, S1PR 1 , plays a prominent but not exclusive role in enhancing the excitability of sensory neurons. J Neurophysiol 104: 2741-2748. First published September 15, 2010 doi:10.1152/jn.00709.2010. Sphingosine 1-phosphate (S1P) through its interaction with a family of G protein-coupled receptors (S1PR) is proving to have a significant impact on the activation of a variety of cell types, most notably those cells mediating the inflammatory response. Previously, we showed that S1P enhanced the excitability of small diameter sensory neurons, and mRNA for S1PR 1-4 was expressed in sensory neurons. These initial findings did not determine which S1PR subtype(s) mediated the increased excitability. Here, we report that exposure to the selective S1PR 1 agonist, SEW2871, produced a significant increase in excitability of some, but not all, sensory neurons. To further examine the role of S1PR 1 , neurons were treated with siRNA targeted to S1PR 1 . siRNA reduced S1PR 1 protein expression by 75% and blocked the sensitization produced by SEW2871, although some neurons remained responsive to subsequent exposure to S1P. Treatment with scramble siRNA did not alter S1PR 1 expression. Recordings from siRNA-and scramble-treated neurons suggested three distinct populations based on their sensitivities to SEW2871 and S1P. Approximately 50% of the neurons exhibited a significant increase in excitability after exposure to SEW2871 and subsequent S1P produced no additional increase; ϳ25% were not affected by SEW2871 but S1P significantly increased excitability; and ϳ25% of the neurons were not sensitized by either SEW2871 or S1P. RT-PCR measurements obtained from single neurons showed that 50% of the small diameter neurons expressed the mRNA for S1PR 1 . These results indicate that S1PR 1 plays a prominent, although not exclusive, role in mediating the enhancement of excitability produced by S1P.
Neurotrophin-mediated signalling cascades can be initiated by activation of either the p75 neurotrophin receptor (p75 NTR ) or the more selective tyrosine kinase receptors. Previously, we demonstrated that nerve growth factor (NGF) increased the excitability of sensory neurons through activation of p75 NTR to liberate sphingosine 1-phosphate. If neurotrophins can modulate the excitability of small diameter sensory neurons through activation of p75 NTR , then brain-derived neurotrophic factor (BDNF) should produce the same sensitizing action as did NGF. In this report, we show that focally applied BDNF increases the number of action potentials (APs) evoked by a ramp of depolarizing current by reducing the rheobase without altering the firing threshold. This increased excitability results, in part, from the capacity of BDNF to enhance a tetrodotoxin-resistant sodium current (TTX-R I Na ) and to suppress a delayed rectifier-like potassium current (I K ). The idea that BDNF acts via p75 NTR is supported by the following observations. The sensitizing action of BDNF is prevented by pretreatment with a blocking antibody to p75NTR or an inhibitor of sphingosine kinase (dimethylsphingosine), but not by inhibitors of tyrosine kinase receptors (K252a or AG879). Furthermore, using single-cell RT-PCR, neurons that were sensitized by BDNF expressed the mRNA for p75 NTR but not TrkB. These results demonstrate that neurotrophins can modulate the excitability of small diameter capsaicin-sensitive sensory neurons through the activation of p75 NTR and its downstream sphingomyelin signalling cascade. Neurotrophins released upon activation of a variety of immuno-competent cells may be important mediators that give rise to the enhanced neuronal sensitivity associated with the inflammatory response.
Little is known about the neuronal voltage-gated sodium channels (NaVs) that control neurotransmission in the parasympathetic nervous system. We evaluated the expression of the a subunits of each of the nine NaVs in human, guinea pig, and mouse airway parasympathetic ganglia. We combined this information with a pharmacological analysis of selective NaV blockers on parasympathetic contractions of isolated airway smooth muscle. As would be expected from previous studies, tetrodotoxin potently blocked the parasympathetic responses in the airways of each species. Gene expression analysis showed that that NaV 1.7 was virtually the only tetrodotoxin-sensitive NaV1 gene expressed in guinea pig and human airway parasympathetic ganglia, where mouse ganglia expressed NaV1.1, 1.3, and 1.7. Using selective pharmacological blockers supported the gene expression results, showing that blocking NaV1.7 alone can abolish the responses in guinea pig and human bronchi, but not in mouse airways. To block the responses in mouse airways requires that NaV1.7 along with NaV1.1 and/or NaV1.3 is blocked. These results may suggest novel indications for NaV1.7-blocking drugs, in which there is an overactive parasympathetic drive, such as in asthma. The data also raise the potential concern of antiparasympathetic side effects for systemic NaV1.7 blockers.
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