Splice Variants of Sodium Channels

16 May

A further level of complexity in the expression of sodium channels is created by the existence of splice variants  of sodium  channel  α-subunits  that  may be regulated during development and by regulators of splicing choice in the adult. Because fly genetics is so advanced, more information is available about

Drosophila splice choice sodium channel variants and their functional roles than the vertebrate equivalents. However, it seems reasonable to suppose that alternative  splicing and RNA editing and transport may also have roles in regulating mammalian  sodium channel function. In Drosophila, a mutation in a dsRNA helicase led to a lowering of expression of Para-encoded  sodium channels. Reenan et al. (2000) showed that this was due to a failure to edit the Para transcript with adenosine to inosine substitutions, which apparently required the helicase for secondary structure modification of the mRNA tran- script. At least three positions in the Para transcript  are known to be edited (Hanrahan  et al. 2000) by adenosine deaminase to give A to I substitutions, and these events are developmentally regulated.

The editing process requires a complementary  sequence of intronic  RNA

to form secondary structure with the edited sequence in a similar manner to that demonstrated for mammalian glutamate receptors. Other editing events in cockroach sodium channels and Drosophila para have been correlated with dramatic functional changes. Liu et al. (2004) have shown that a U to C edit- ing event resulting  in a phenylalanine  to serine modification  can produce a sodium channel with persistent  TTX-sensitive properties  rather  similar to currents  identified in mammalian  CNS neurons,  raising the possibility that

similar events could occur in mammals. Song et al. (2004) have catalogued fur- ther editing events that have functional consequences in terms of thresholds of activation and are developmentally regulated in the cockroach. Tan et al. (2002) have also found that alternatively spliced transcripts  can have distinct pharmacological profiles as well as altered gating characteristics. They found alternative exons encoding transmembrane segments in DIII of a cockroach sodium  channel, which had conserved splice sites across evolution in fish, flies, mice and men. The alternatively spliced forms were found in different tissues. One form with a premature stop codon occurred only in the peripheral nervous system whilst the two other functional forms differed in their sensi-

tivity to pyrethroid  insecticides such as δ-methrin. Remarkably, fetal mouse brain also contains transcripts of the SCN8A gene (Nav1.6) that contains a stop codon at the same site as the fly genes predicting the production of two domain

truncated sodium channel transcripts (Plummer et al. 1997). The role of these transcripts is unknown.

In mammals, mutually exclusive exon usage also occurs. The type III channel

exists as an embryonic or adult spliced form with different exons that code for the S3 and S4 segments in DI of the rat channel. Despite the fact that the two exons both encode 29 amino acids, only a single amino acid residue is altered in these two alternative forms. Single amino acid changes may also occur through alternative  3   splice site selection. Kerr et al. (2004) have found  that  both Nav1.8 and Nav1.5—two TTX-resistant sodium channels found in peripheral neurons—both exist as alternative forms containing an additional glutamine residue within the cytoplasmic loop linking DII and DIII of these channels. As well as alternative exon usage or amino acids insertions, transcripts encoding exon repeats have been identified in DRG neurons. The presence of a transcript with a three-exon repeat encoding Nav1.8 is enhanced by treatment with NGF, suggesting that this neurotrophin may regulate trans-splicing events in these cells (Akopian et al. 1999). Once again the functional consequences of these conserved changes have yet to be established.

The regulation of splice choice in response to external signals is still little understood. Buchner et al. (2003), studying a modifier locus in different mouse lines that determines the lethality of a Nav1.6 splice site mutation, discovered that the efficiency of action of a splice factor determined the amount of func- tional channel produced and hence the lethality of the original mutation. Thus, on a C57Bl6 background little correctly spliced mRNA was produced, causing a lethal phenotype, whilst on a wild-type background, 10% of the transcripts were correctly spliced, leading to a viable if dystonic phenotype.

Recent papers have suggested roles for sodium channels in regulating synap- tic efficacy, as well as functions  in immune  system cells. Macrophages and microglia express Nav1.6, a channel that is broadly expressed in the nervous system and which is functionally compromised in the naturally occurring med mutant  that leads to dystonia. Interestingly, macrophage function is also in- hibited in these animals. When microglia or macrophages are activated, Nav1.6

expression is up-regulated,  and this event seems to be important  in terms of phagocytic activity, as the uptake of latex beads is partially blocked in med macrophages, or in normal macrophages treated with TTX. This suggests that voltage-gated sodium channels play an important functional role in immune system cell function. These kinds of unsuspected roles for voltage-gated chan- nels may give rise to significant problems in attempts to use selective blockers as analgesic in chronic pain states.

Calcium Channels

Voltage-gated calcium channels comprise a single α-subunit and show strong structural homology with sodium channels, but the accessory subunits associ- ated with these channel are much more complex. The functional calcium chan- nel complexes contain four proteins: α1 (170 kDa), α2 (150 kDa), β (52 kDa), δ (17–25 kDa) and γ (32 kDa). Four α2δ subunit genes have now been cloned.

Both the message and protein  for the α2δ-1 subunit  is highly expressed in

sensory neurons but is also found almost ubiquitously in other tissues (Gong et al. 2001). All α2δ subunits have a predicted N-terminal signal sequence, in- dicating that the N-terminus is extracellular, with an intracellular C-terminus and potential transmembrane region. There are up to 14 conserved cysteines throughout the α2δ-1, 2 and 3 sequences, six of which are within δ, providing

additional evidence that α2 and δ are disulphide-bonded. Following the iden-

tification of α2δ subunits as components of skeletal muscle calcium channels, they have also been shown to be associated with neuronal  N- and P/Q-type channels. The α2δ-1 subunit has been shown to bind to extracellular regions

including DIII on the Cav1.2 subunit (Felix et al. 1997).

Voltage-Gated Calcium Channels

High voltage-activated and low voltage-activated calcium channels are now known to comprise a number of cloned α-subunits (Peres-Reyes 2003). Cav2.2, Cav2.3 and Cav3.2 are now known to play an important  role in pain sensation and are interesting analgesic drug targets.

The evidence of an important specialised role for certain voltage-gated cal- cium channels in the pathogenesis of neuropathic  pain is strong. A variety of drugs targeted at calcium channel subtypes are effective analgesics, and mouse null mutants of N-type Cav2.2 calcium channels show dramatic diminution  in neuropathic pain behaviour in response to both mechanical and thermal stim- uli. Opioid peptides are known to inhibit the action of N-type calcium channels through post-translational mechanisms. In addition, two highly effective anal-

gesic drugs used in neuropathic  pain  conditions  selectively target  calcium channel subtypes. The conotoxin ziconotide blocks Cav2.2 α-subunits, and the widely prescribed drug gabapentin binds with high affinity to α2δ subunits of calcium channels.

Gabapentin binds to high-affinity sites in the brain, and the target bind- ing site has been identified  as the α2δ-1 subunit.  Transient  transfection  of cells with α2δ-1 increased the number of gabapentin binding sites (Gee et al.

1996). Subsequently, gabapentin has been found to bind to two isoforms of α2δ

subunits (the α2δ-1 and α2δ-2 isoforms, but not α2δ-3 or α2δ-4) (Gee et al.

1996; Gong et al. 2001). The effects of gabapentin on native calcium currents

are controversial,  with some authors  reporting  small inhibitions  of calcium currents in different cell types. Gabapentin could interfere with α2δ binding to the α1 subunit, thus destabilizing the heteromeric complex. Interestingly, α2δ1 up-regulation  in neuropathic  pain correlates well with gabapentin sensitivity (Luo et al. 2002), suggesting that the α2δ-1 isoform is the most likely site of action of gabapentin.

The up-regulation  of α2-δ subunits  does not occur in all animal models of neuropathic  pain that result in allodynia. Luo et al. (2002) compared DRG and spinal cord α2δ-1 subunit levels and gabapentin sensitivity in allodynic rats  with mechanical  nerve injuries  (sciatic nerve chronic  constriction  in-

jury, spinal nerve transection,  or ligation), a metabolic disorder  (diabetes) or chemical neuropathy  (vincristine neurotoxicity). Allodynia occurred in all types of nerve injury investigated, but DRG and/or  spinal cord α2δ-1 sub- unit up-regulation  and gabapentin  sensitivity only co-existed in mechanical

and diabetic neuropathies.  This may partially explain why gabapentin  is in- effective in some neuropathic  pain  patients.  Recent studies  with knock-in mice have demonstrated that gabapentin  loses its effectiveness when it can

no longer bind to a mutated  form of α2δ-1, and the details of this work can be found in a patent application (Baron et al. 2005). Thus, the site of action of gabapentin/pregabalin seems resolved, but the mechanism of action is still


Further support for calcium channels as useful drug targets in neuropathic pain comes from an analysis of the characteristics of the Cav2.2-null mutant mouse generated by Saegusa et al. (2001). The same authors  (Saegusa et al.

2002) have compared  the Cav2.2- and 2.3-null mutants  in a variety of pain models. Despite the widespread expression of Cav2.2, it has proved possible to demonstrate major deficits in inflammatory and in particular neuropathic pain in this transgenic mouse using the Seltzer model. Thermal and mechanical thresholds were dramatically stabilized in this mutant mouse. The relationship between α2δ subunits and Cav2.2 has not been investigated in detail, so it is possible that gabapentin  has sites of action on calcium channels other than

Cav2.2—for example, T-type channels such as Cav3.2.

A role for Cav2.2 in chronic pain is consistent with a known analgesic role

for N-type calcium channel blockers. Ziconotide, a toxin derived from marine

snails, blocks Cav2.2 channels with high affinity and has been found to have analgesic actions in animal models and man. In a study of the anti-nociceptive properties  of ziconotide,  morphine  and  clonidine  in a rat  model of post- operative pain, heat hyperalgesia and mechanical allodynia were induced in the hind paw (Wang et al. 2000). Intrathecal ziconotide blocked established heat hyperalgesia in a dose-dependent manner  and caused a reversible blockade of established mechanical allodynia. Intrathecal  ziconotide was found to be more potent, longer acting, and more specific in its actions than intrathecal morphine in this model of post-surgical pain.

Brose (1997) found that intrathecal ziconotide provided complete pain relief with elimination of hyperaesthesia and allodynia in a dose dependent manner in a single patient suffering from intractable pain, although side-effects were experienced. This type of study has led to clinical use of ziconotide in the treatment of intractable pain in late stage cancer patients. Evidence that omega conotoxins also target P2X3 receptors (Lalo et al. 2001) suggests that ziconotide may act at a broader range of targets than first suspected.

Interestingly, a sensory neuron-specific  splice variant of Cav2.2 has been identified  in DRG neurons.  Lipscombe and  collaborators  (Bell et al. 2004) showed that the DRG-specific exon, e37a, is preferentially present in Cav2.2 mRNAs expressed in neurons  that contain nociceptive markers  TRPV1 and Nav1.8. Cell-specific inclusion of e37a correlated with the significantly larger N-type currents in nociceptive neurons, suggesting that splice-variant specific antagonists could have useful therapeutic effects. The situation in man has yet to be examined, however.

Cav3.2, a T-type calcium channel  has also recently been found  to play a role in pain. Antisense targeting Cav3.2 induced a knock-down of the Cav3.2 mRNA and protein expression as well as a large reduction of T-type calcium currents in nociceptive DRG neurons. Concomitantly, the antisense treatment resulted in major anti-nociceptive, anti-hyperalgesic and anti-allodynic effects, suggesting that Cav3.2 plays a major pronociceptive role in acute and chronic pain states. These antisense studies have also been confirmed by the generation and analysis of a knock-out mouse (Bourinet and Zamponi 2005).

Potassium Channels

Potassium  channels act effectively as brakes on neurotransmission and ex- citability, allowing the flow of potassium  ions out of the cell in response to a range of different stimuli. For example, some analgesic actions of opioids are mediated  by the activation  of calcium-dependent potassium  channels. Potassium channels have been subdivided into four major subsets based on their structure and mode of gating. Voltage-gated channels are extraordinarily diverse—there are 12 different families, Kv1–Kv12, containing different indi-

vidual members. However, all the voltage-gated channels comprise the classical six-transmembrane monomer that forms a tetrameric voltage-gated structure reminiscent of sodium and calcium channels (Fig. 3).

Evidence has been obtained that potassium channel transcripts  are differ- entially regulated at the transcriptional level in animal models of neuropathic pain. Using RT-PCR, Ishikawa et al. (2002) found that, in a chronic constriction injury model of neuropathic  pain, Kv1.2, 1.4, 2.2, 4.2 and 4.3 mRNA levels in the ipsilateral DRG were reduced to 63%–73% of the contralateral sides of the same animal at 3 days and to 34%–63% at 7 days following CCI. In addition, Kv1.1 mRNA levels declined to about 72% of the contralateral  level at 7 days. No significant changes in Kv1.5, 1.6, 2.1, 3.1, 3.2, 3.5 and 4.1 mRNA levels were detectable in the ipsilateral DRG at either time. Interestingly, of the Kv channels present in DRG, Kv1.4 seems to be the main channel expressed in small-diameter sensory neurons, and the expression levels of this channel are much reduced in a Chung model of neuropathic  pain (Rasband et al. 1999).

The calcium-activated potassium  channel family comprises three subsets that have structural  similarities with the Kv channels. They have been classi- fied on the basis of their conductance into big, intermediate and small currents (BK, IK, SK). However, although voltage-dependent via some positive charges in the S4 domain, they are essentially opened by increases in cytoplasmic cal- cium concentrations through an interaction with a calmodulin binding domain found at the C-termini of these channels. They also differ from Kv channels in containing  an additional  N-terminal  transmembrane domain  that means the N-terminus  is extracellular, unlike voltage-gated channels. Fascinatingly, Xie et al. (2005) have found  that  splice choice for the BK channel  STREK exon is modulated  through  the actions of a CAM kinase response element, suggesting that  neuronal  excitability could be altered  both directly though the actions of calcium on potassium channels, as well as indirectly by chang-

ing the molecular  structure  of expressed  ion channels  through  alternative splicing.

Two-pore K channels (K2P channels) comprise a dimer of the two trans- membrane  regions that form the functional  pore with two P domains,  and form functional dimers that are gated by a variety of stimuli. This area has re- cently been reviewed by Kim (2005). Sixteen K2P channel genes are expressed in a range of tissues and have the properties  of leak K+ channels. They thus play a major role in setting resting membrane  potential  and regulating cell excitability. The stimuli that gate K2P channels are very diverse and include lipids, volatile anaesthetics, and heat, oxygen, acid and mechanical stimuli. Inward rectifier potassium channels retain just the two pore-forming subunits found in voltage-gated channels together with the potassium selective P loop and also form tetrameric structures that are, necessarily, voltage-independent. There is an enormous variety of these channels that have been classified into seven families. G protein-regulated potassium channels are gated by interac-

tions with G protein βγ subunits.

Recently a new set of K channels that are responsive to increased intracellular

levels of sodium have also been identified (Bhattacharjee and Kaczmarek 2005), but their role in setting pain thresholds has not been explored.

Passmore et al. (2003) have provided evidence that KCNQ potassium cur- rents  (responsible  for the M-current)  may also play a role in setting pain thresholds.  Retigabine potentiates  M-currents  and leads to a diminution  of nociceptive input into the dorsal horn of the spinal cord in both neuropathic and inflammatory pain models in the rat. Thus, despite the enormous complex- ity of potassium channels, there is some possibility of targeting pain thresholds through activating potassium channels expressed on damage-sensing sensory neurons.

Pacemaker Channels

Cyclic nucleotide-regulated hyperpolarisation-activated cation channels (HCN channels) play an important role in cardiac function and are expressed within sensory neurons. Chaplan and collaborators (2003) have described a novel role for HCN channels in touch-related  pain and spontaneous  neuronal discharge originating in the damaged DRG. Nerve injury markedly increased pacemaker currents  in large-diameter  DRG neurons  and resulted  in pacemaker-driven spontaneous action potentials in the ligated nerve. Pharmacological blockade of HCN activity using the specific inhibitor ZD7288 reversed hypersensitivity to light touch and decreased the firing frequency of ectopic discharges originating

in A-δ and  A-β fibres by 90% and  40%. Targeting these channels  appears problematic, however, given their other roles in the periphery.

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