Voltage-gated sodium channels initiate and propagate action potentials in excitable cells. The tetrodotoxin-resistant Na+ channel (Nav1.8/SNS) is expressed exclusively in damage-sensing neurons and ...plays an important role in pain pathways. Expression of functional Nav1. 8 in heterologous cells has proved problematic, even in the presence of known accessory β-subunits. This suggests that Nav1.8 requires regulatory proteins for transport or as a stabilizer to promote its functional expression on the plasma membrane in sensory neurons. We report the use of yeast 2-hybrid system and a rat dorsal root ganglion cDNA library to identify 28 different clones encoding proteins which interact with Nav1.8. Many clones are expressed at high levels in small diameter DRG neurons. Interacting proteins include enzymes, channels, motor proteins, and novel proteins. Co-mmunoprecipitation confirms that some of the proteins interact with, and may hence regulate, the properties of Nav1.8 in vivo. One of such clones is p11, annexin II light chain. We have reported that p11 is an essential co-factor for expression of functional Nav1.8 in sensory neurons (Nature 417(6889), 653-656). The aberrant current voltage relationship and relatively low levels of expression of Nav1.8 expressed with p11 in heterologous cells suggest that additional factors must play a role in promoting normal levels of expression of Nav1.8. The consequences of the interactions between Nav 1.8 and the other clones for the functional roles on Nav1.8 in the DRG are being assessed.
KV1.1 is a Shaker homologue K+ channel that contributes to the juxta-paranodal membrane conductance in myelinated axons, and is blocked by fampridine (4-aminopyridine), used to treat the symptoms of ...multiple sclerosis. The present experiments investigate KV1.1 function in primary sensory neurons and A-fibres, and help define its characteristics as a drug-target using sequence specific small-interfering RNAs (siRNAs). siRNA (71nM) was used to knock-down functional expression of KV1.1 in sensory neurons (>25μm in apparent diameter) in culture, and was also delivered intrathecally in vivo (9.3μg). K+ channel knock-down in sensory neurons was found to make the voltage-threshold for action potential generation significantly more negative than in control (p=0.02), led to the breakdown of accommodation and promoted spontaneous action potential firing. Exposure to dendrotoxin-K (DTX-K, 10–100nM) also selectively abolished K+ currents at negative potentials and made voltage-threshold more negative, consistent with KV1.1 controlling excitability close to the nominal resting potential of the neuron cell body, near −60mV. Introduction of one working siRNA sequence into the intrathecal space in vivo was associated with a small increase in the amplitude of the depolarising after-potential in sacral spinal roots (p<0.02), suggesting a reduction in the number of working K+ channels in internodal axon membrane. Our study provides evidence that KV1.1 contributes to the control of peripheral sensory nerve excitability, and suggests that its characteristics as a putative drug target can be assessed by siRNA transfection in primary sensory neurons in vitro and in vivo.
The voltage-gated sodium channel NaV1.8 is expressed exclusively in nociceptive sensory neurons and plays an important role in pain pathways. NaV1.8 cannot be functionally expressed in non-neuronal ...cells even in the presence of β-subunits. We have previously identified Pdzd2, a multi PDZ-domain protein, as a potential interactor for NaV1.8. Here we report that Pdzd2 binds directly to the intracellular loops of NaV1.8 and NaV1.7. The endogenous NaV1.8 current in sensory neurons is inhibited by antisense- and siRNA-mediated downregulation of Pdzd2. However, no marked change in pain behaviours is observed in Pdzd2-decificent mice. This may be due to compensatory upregulation of p11, another regulatory factor for NaV1.8, in dorsal root ganglia of Pdzd2-deficient mice. These findings reveal that Pdzd2 and p11 play collaborative roles in regulation of NaV1.8 expression in sensory neurons.
The voltage-gated sodium channel Na(V)1.8 is expressed exclusively in nociceptive sensory neurons and plays an important role in pain pathways. Na(V)1.8 cannot be functionally expressed in ...non-neuronal cells even in the presence of beta-subunits. We have previously identified Pdzd2, a multi PDZ-domain protein, as a potential interactor for Na(V)1.8. Here we report that Pdzd2 binds directly to the intracellular loops of Na(V)1.8 and Na(V)1.7. The endogenous Na(V)1.8 current in sensory neurons is inhibited by antisense- and siRNA-mediated downregulation of Pdzd2. However, no marked change in pain behaviours is observed in Pdzd2-decificent mice. This may be due to compensatory upregulation of p11, another regulatory factor for Na(V)1.8, in dorsal root ganglia of Pdzd2-deficient mice. These findings reveal that Pdzd2 and p11 play collaborative roles in regulation of Na(V)1.8 expression in sensory neurons.
The interaction of p11 (annexin II light chain) with the N‐terminal domain of Na
V
1.8, a tetrodotoxin‐resistant sodium channel, is essential for the functional expression of the channel. Here we ...show that p11 binds to Na
V
1.8 but not to sodium channel isoforms Na
V
1.2, 1.5, 1.7 or Na
V
1.9. The binding of amino acids 74–103 of Na
V
1.8 to p11 residues 33–78 occurs in a random coiled region flanked by two EF hand motifs whose crystal structure has been established. As Na
V
1.8 channel expression is associated with pain pathways, drugs that disrupt the Na
V
1.8–p11 interaction and down‐regulate channel expression may have analgesic activity.
The interaction of p11 (annexin II light chain) with the N-terminal domain of Na
V1.8, a tetrodotoxin-resistant sodium channel, is essential for the functional expression of the channel. Here we show ...that p11 binds to Na
V1.8 but not to sodium channel isoforms Na
V1.2, 1.5, 1.7 or Na
V1.9. The binding of amino acids 74–103 of Na
V1.8 to p11 residues 33–78 occurs in a random coiled region flanked by two EF hand motifs whose crystal structure has been established. As Na
V1.8 channel expression is associated with pain pathways, drugs that disrupt the Na
V1.8–p11 interaction and down-regulate channel expression may have analgesic activity.
We have examined the distribution of the sensory neuron‐specific Na
+
channel Na
v
1.8 (SNS/PN3) in nociceptive and non‐nociceptive dorsal root ganglion (DRG) neurons and whether its distribution is ...related to neuronal membrane properties. Na
v
1.8‐like immunoreactivity (Na
v
1.8‐LI) was examined with an affinity purified polyclonal antiserum (SNS11) in rat DRG neurons that were classified according to sensory receptive properties and by conduction velocity (CV) as C‐, Aδ‐ or Aα/β. A significantly higher proportion of nociceptive than low threshold mechanoreceptive (LTM) neurons showed Na
v
1.8‐LI, and nociceptive neurons had significantly more intense immunoreactivity in their somata than LTM neurons. Results showed that 89, 93 and 60 % of C‐, Aδ‐ and Aα/β‐fibre nociceptive units respectively and 88 % of C‐unresponsive units were positive. C‐unresponsive units had electrical membrane properties similar to C‐nociceptors and were considered to be nociceptive‐type neurons. Weak positive Na
v
1.8‐LI was also present in some LTM units including a C LTM, all Aδ LTM units (D hair), about 10 % of cutaneous LTM Aα/β‐units, but no muscle spindle afferent units. Na
v
1.8‐LI intensity was negatively correlated with soma size (all neurons) and with dorsal root CVs in A‐ but not C‐fibre neurons. Na
v
1.8‐LI intensity was positively correlated with action potential (AP) duration (both rise and fall time) in A‐fibre neurons and with AP rise time only in positive C‐fibre neurons. It was also positively correlated with AP overshoot in positive neurons. Thus high levels of Na
v
1.8 protein may contribute to the longer AP durations (especially in A‐fibre neurons) and larger AP overshoots that are typical of nociceptors.
Voltage-gated sodium channels initiate and propagate action potentials in excitable cells. The tetrodotoxin-resistant Na(+) channel (Na(V)1.8/SNS) is expressed in damage-sensing neurons (nociceptors) ...and plays an important role in pain pathways. Expression of high levels of functional Na(V)1.8 in heterologous cells has proved problematic, even in the presence of known sodium channel accessory beta-subunits. This suggests that other regulatory proteins are required for normal levels of Na(V)1.8 expression. Here we report the use of a yeast two-hybrid system and a rat dorsal root ganglion cDNA library to identify 28 different clones encoding proteins which interact with intracellular domains of Na(V)1.8. Many clones are expressed at high levels in small diameter DRG neurons as judged by in situ hybridization. Interacting proteins include cytoplasmic elements and linker proteins (e.g. beta-actin and moesin), enzymes (e.g. inositol polyphosphate 5-phosphatase and TAO2 thousand and one protein kinase), channels and membrane-associated proteins (voltage-dependent anion channel VDAC3V and tetraspanin), as well as motor proteins (dynein intermediate and light chain) and transcripts encoding previously undescribed proteins. Immunoprecipitation (pull-down) assays confirm that some of the proteins interact with, and may hence regulate, Na(V)1.8 in vivo.
Effects of various differentiating agents and DNA demethylating agents on the expression of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH), marker enzymes for cholinergic and ...adrenergic differentiation, respectively, were examined in N-18 neuroblastoma cells. Retinoic acid (RA) and a medium conditioned over C6-glioma cells (GCM), which have been shown to enhance the ChAT activity of PC12 cells, NG108-15 cells and fetal rat brain cells, did not induce ChAT activity of N-18 cells. Treatment of the cells with the DNA demethylating agents alone also did not affect ChAT activity. But after pretreatment of the cells with the DNA demethylating agents, ChAT activity of N-18 cells was greatly increased by either RA or GCM. TH activity of N-18 cells was enhanced by forskolin, an activator of adenylate cyclase. The pretreatment of the cells with the DNA demethylating agents greatly enhanced the induction of TH activity by forskolin. Levels of ChAT and TH messenger RNA were altered in accordance with changes in ChAT and TH activities. Possible mechanisms of the actions of the demethylating agents on cholinergic and adrenergic differentiation are discussed.
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