Brain, Vol. 125, No. 2, 252-263,
February 1, 2002
© 2002 Oxford University Press
Calcium-activated potassium channel SK1- and IK1-like immunoreactivity in injured human sensory neurones and its regulation by neurotrophic factors
1 Peripheral Neuropathy Unit, Department of Neurology, Imperial College of Science, Technology and Medicine, Hammersmith Hospital and 2 Imperial Cancer Research Fund, Histopathology Unit, London, 3 GlaxoSmithKline, Medicines Research Centre, Stevenage and 4 Peripheral Nerve Injury Unit, Royal National Orthopaedic Hospital, Stanmore, UK
Correspondence to: Professor P. Anand, MD, Peripheral Neuropathy Unit, Imperial College School of Medicine, Area A, Ground Floor, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK E-mail: p.anand{at}ic.ac.uk
*These authors contributed equally to this work
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Calcium-activated potassium ion channels SK and IK (small and intermediate conductance, respectively) may be important in the pathophysiology of pain following nerve injury, as SK channels are known to impose a period of reduced excitability after each action potential by afterhyperpolarization. We studied the presence and changes of human SK1 (hSK1)- and hIK1-like immunoreactivity in control and injured human dorsal root ganglia (DRG) and peripheral nerves and their regulation by key neurotrophic factors in cultured rat sensory neurones. Using specific antibodies, hSK-1 and hIK-1-like immunoreactivity was detected in a majority of large and small/medium-sized cell bodies of human DRG. hSK1 immunoreactivity was decreased significantly in cell bodies of avulsed human DRG (n = 8, surgery delay 8 h to 12 months). There was a decrease in hIK1-like immunoreactivity predominantly in large cells acutely (<3 weeks after injury), but also in small/medium cells of chronic cases. Twenty-three injured peripheral nerves were studied (surgery delay 8 h to 12 months); in five of these, hIK1-like immunoreactivity was detected proximally but not distally to injury, whereas neurofilament staining confirmed the presence of nerve fibres in both regions. These five nerves, unlike the others, had all undergone Wallerian degeneration previously and the loss of hIK1-like immunoreactivity may therefore reflect reduced axonal transport of this ion channel across the injury site in regenerated fibres, as well as decreased expression in the cell body. In vitro studies of neonatal rat DRG neurones showed that nerve growth factor (NGF) significantly increased the percentage of hSK1-positive cells, whereas neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) failed to show a significant effect. NT-3 stimulated hIK1 expression, while NGF and GDNF were ineffective. As expected, NGF increased expression of the voltage-gated sodium channel SNS1/PN3 in this system. Decreased retrograde transport of these neurotrophic factors in injured sensory neurones may thus reduce expression of these ion channels and increase excitability. Blockade of IK1-like and other potassium channels by aminopyridines (4-AP and 3,4-DAP) may also explain the paraesthesiae induced by these medications. Selective potassium channel openers are likely to represent novel therapies for pain following nerve injury.
Keywords: calcium-activated potassium channel; hSK1; hIK1; hSK4; nerve injury
Abbreviations: AHP = afterhyperpolarization; BK = large conductance calcium-activated potassium channel; CHO = Chinese hamster ovary cell; DRG = dorsal root ganglion; EST = expression sequence tag; GDNF = glial cell line-derived neurotrophic factor; hIK1 = human intermediate conductance calcium-activated potassium channel type 1; hSK1 = human small conductance calcium-activated potassium channel type 1; hSK4 = human small conductance calcium-activated potassium channel type 4; IK = intermediate conductance calcium-activated potassium channel; NGF = nerve growth factor; NT-3 = neurotrophin3; PBS = phosphate-buffered saline; SDS = sodium dodecyl sulphate; SK = small conductance calcium-activated potassium channel; SNS/PN3 = sensory neurone-specific sodium channel
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Introduction |
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Calcium-activated potassium channels are gated by intracellular Ca2+ ions and their activity is responsible for part of the afterhyperpolarization (AHP) that follows a single action potential or a train of action potentials in neurones (for reviews, see Sah et al., 1996
; Vergara et al., 1998). This AHP serves a key role in spike frequency adaptation, setting interspike interval and generally suppressing membrane excitability. Calcium-activated potassium channels are also important in non-neuronal cells, such as the epithelia and visceral smooth muscle, where they regulate secretion and contractility (Vogalis et al., 1998; Devor et al., 1999). According to their single-channel conductance in symmetrical K+ solutions, calcium-activated potassium channels can be classified as BK, SK or IK [large (‘big’), small and intermediate conductance, respectively] (Latorre et al., 1989; Vergara et al., 1998). Each channel subtype has a distinct pharmacology: classical selective blockers of BK and some SK channels are iberiotoxin and apamin, respectively, whereas the antifungal agent clotrimazole selectively blocks IK channels (Garcia et al., 1997).
BK and SK channels are widely distributed in both excitable and non-excitable cells (Gribkoff et al., 1997). The voltage-dependent human BK channel gene was first cloned from brain (Tseng-Crank et al., 1996) and genes encoding three voltage-independent SK subtypes were also isolated from brain [hSK1, hSK2 and hSK3 (h = human)] (Köhler et al., 1996; Chandy et al., 1998; Desai et al., 2000). BK and SK sequence data were then used to clone a further channel, human IK (hIK1 or hSK4), from placenta (Joiner et al., 1997), pancreas (Ishii et al., 1997) and T lymphocytes (Khanna et al., 1999). The human IK channel (hIK) is closely related to but distinct from the SK family of channels. IK channels are present in various tissues, including blood cells (Grygorczyk, 1983), lymphocytes (Grissmer et al., 1993; Rader et al., 1996), epithelial cells (MacVinish et al., 1998), smooth muscle (Vogalis et al., 1998) and keratinocytes (Koegel and Alzheimer, 2001). Jensen et al. (1998) further characterized hIK by the use of an expression sequence tag (EST) database; RNA analysis revealed widespread tissue expression, with the highest levels located in the salivary gland, placenta, trachea and lung. Although mRNA for IK has not been demonstrated previously in neurones, there are several reports of neuronal IK-like conductances, e.g. in visceral neurones (Hay and Kunze, 1994) and magnocellular neurones of the rat supraoptic nucleus (Greffrath et al., 1998).
Mutations in SK and IK channels may underlie a wide range of disorders (Litt et al., 1999). SK3 channels have been implicated in myotonic muscular dystrophy (Behrens et al., 1994) and IK channels in Diamond–Blackfan anaemia (Ghanshani et al., 1998). Since calcium-activated potassium channels play a crucial role in governing neuronal excitability, their presence and distribution in the PNS could contribute to the pathophysiology of pain and paraesthesiae after nerve injury. Calcium-dependent potassium currents and AHPs have been observed previously in dorsal root ganglion (DRG) neurones, notably in a subset of slowly conducting putative nociceptors (Akins and McClesky, 1993; Gold et al., 1996; Villiere and McLachlan, 1996; Amir and Devor, 1997). Interestingly, the AHP is inhibited by inflammatory mediators, such as prostaglandin E2 (Fowler et al., 1985; Gold et al., 1996; Cordoba-Rodriguez et al., 1999), and this may be a key mechanism by which prostanoids sensitize nociceptors. Following nerve injury, there are a number of mechanisms and sites involved in the pathophysiology of sensory nerves (Janig et al., 1996; Devor and Seltzer, 1999; Michaelis et al., 2000). Calcium-activated potassium channels have already been heavily implicated in ectopic spontaneous discharges of injured rat DRG neurones (Amir and Devor, 1997; Xing and Hu, 1999) and in a model of neuropathic pain (Honma et al., 1999). These channels may thus represent novel therapeutic targets for pain relief.
We have studied, for the first time, the presence and distribution of hSK1- and hIK1-like immunoreactivity in injured human DRG and peripheral nerves and their regulation by key target-derived neurotrophic factors—nerve growth factor (NGF), neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF)—in cultured neonatal rat DRG neurones.
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Antibody generation and validation
Stable lines of CHO-K1 (Chinese hamster ovary-K1) and HEK293 cells were produced as follows. CHO-K1 cells were maintained in MEM-
medium with Glutamax (Life Technologies, Paisley, UK) with 10% foetal calf serum and 1 x non-essential amino acids (Life Technologies). HEK (human embryonic kidney cells) 293 cells were maintained in Dulbecco-modified essential medium (Life Technologies) with 10% foetal calf serum, 1 x glutamine and 1 x non-essential amino acids (Life Technologies). Full-length human cDNA coding regions of SK1, SK2, SK3 and IK1 were isolated from EST clones and total brain cDNA (our unpublished data) and cloned into pCin mammalian expression vector (Rees et al., 1996) under the control of the CMV (cytomegalovirus) promoter, and with G418 resistance. CHO stable cell lines were isolated in the presence of 0.5 mg/ml G418. HEK293 stable cell lines were isolated in the presence of 0.8 mg/ml G418. Clones with the highest channel activity were selected and demonstrated to express the corresponding channel by RT–PCR (reverse transcription–polymerase chain reaction) and immunoblotting. Antibodies were raised in rabbits. L155 was raised against an hSK1 C-terminal peptide (CSSPYRWTPVAPSDYG), M6 against an hSK1 N-terminal peptide (PGPRAAYSEPN PYTQC) and M20 against an hIK1 N-terminal peptide (GGDLVLGLGALRRRKC). Immunoglobulins were affinity-purified using the corresponding immobilized peptides.
Immunoblotting was used to profile the antibodies. Cells were lysed in 50 mM Tris–HCl, pH 7.6, containing 2 mM EDTA, 150 mM NaCl and 1% NP40. Samples were then boiled for 10 min in buffer containing 0.25 M Tris–HCl, 10% glycerol, 5% sodium dodecyl sulphate (SDS), 0.05% bromophenol blue and 0.1 M dithiothreitol. Total cell protein extract (10 µg) was loaded in each well on 4–20% gradient Tris–glycine gels (Novex GmbH, Frankfurt/Main, Germany) for electrophoresis. Resolved proteins were then electroblotted onto nitrocellulose membranes. Remaining protein binding sites were blocked with phosphate-buffered saline (PBS) plus 5% fat-free milk powder for >1 h and incubated in 1 µg/ml immunoglobulins in blocking solution at 4°C overnight. Following a washing step (4 x 15 min in PBS plus 0.1% Tween 20), immunoreactive bands were localized using horseradish peroxidase-conjugated goat anti-rabbit IgG (Dako, Ely, UK) and SuperSignal reagents (Pierce now Perbio, Tattenhall, UK).
Human tissues
Cervical DRG whose roots had been avulsed from the spinal cord following traumatic injury to the brachial plexus were collected from eight male patients (age range 20–37 years). The delay between injury and collection of tissue at operation ranged from 8 h to 12 months (of these cases, five were regarded as acute, i.e. <3 weeks after injury). Control cervical DRG were obtained from five male and three female patients (age range 43–81 years) with an autopsy delay of <24 h.
Non-injured control peripheral nerves were obtained from five patients (age range 27–67 years) immediately after amputation of a limb for non-neurological indications. Injured nerves, trimmed proximal and distal to the injury site, were collected from 11 patients (age range 22–47 years) with brachial plexus injury and from 12 patients (age range 20–57 years) with a more distal limb peripheral nerve injury. The delay between injury and surgical treatment ranged from 8 h to >1 year (of these cases, 18 were regarded as acute, i.e. less than 3 weeks after injury).
All tissues were removed as a necessary part of surgical repair and not for research purposes. The patients gave informed consent for this study, which had approval from the Regional Ethics Committee of the Royal National Orthopaedic Hospital, Stanmore, UK.
Immunohistochemistry
Human tissues were removed at surgery and immediately snap-frozen in liquid nitrogen. Tissues were first embedded in optimum cutting temperature medium (Tissue Tek, supplied by Bayer, Newbury, UK) then sectioned at 8–10 µm using a cryostat (Bright Instrument, Huntingdon, UK). Sections were thaw-mounted on to glass slides precoated with poly-L-lysine. After sectioning, tissues were post-fixed in fresh paraformaldehyde fixative (4% in PBS) for 30 min and treated with 0.3% hydrogen peroxide (in industrial methylated spirit) for a further 30 min to eliminate endogenous peroxidase activity. Sections were washed in PBS three times between each incubation. Cultured neonatal DRG were fixed after 48 h with 2% paraformaldehyde for 30 min and put into peroxide. Primary antibodies (Table 1) were applied to two tissue sections from each patient (two LabTek wells per cell preparation), diluted in normal goat serum, and left incubating overnight. In addition, further sections of each tissue in LabTek wells (Nalge-Nunc, Naperville, USA) were incubated with normal rabbit serum and PBS as negative controls. A further two sections from each tissue in LabTek wells were incubated with a positive antibody control [anti-neurofilament (Sigma, Poole, UK)]. The following day, sections/wells were washed three times in fresh PBS and secondary antibody was applied (biotinylated goat anti-rabbit) for 60 min. After a further PBS wash, visualization of specific antibody binding was performed by a standard immunoperoxidase method with nickel enhancement (Vector Elite ABC method using Vectastain; Vector Laboratories, Burlingame, Calif., USA) as described by Shu et al. (1988). Tissue sections were counterstained in 1% w/v aqueous neutral red and photographed with an Olympus photomicroscope. Cell cultures were not counterstained, but double staining was performed for all ion-channel antibodies using neurofilament antibody in Tris-buffered saline, visualized by a standard alkaline phosphatase method (Vector Laboratories). Specificity of the hSK1, hIK1 and sensory neurone-specific sodium channel (SNS/PN3) antibodies was tested by preincubating the primary antibodies with an excess of corresponding peptides.
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Neuronal cell size distribution for human DRG sections was defined using the criteria described by Holford et al. (1994
) as small/medium (<50 µm) or large (>50 µm) diameter. All cell bodies with a visible nucleus were measured before counting the number of immunopositive and -negative neurones (categorized according to cell size) by visual examination under a light microscope equipped with a calibrated eyepiece graticule. Cells were considered positive for hSK1/hIK1 if they showed dense and diffuse cytoplasmic staining that was clearly above the background level. An average of 85 DRG neurones per section was counted. Cultured rat neurones were classified as small (<17 µm), medium (17–25 µm) and large (>25 µm) according to Bowie et al. (1994). An average of 290 cells per well were counted with 10 wells per growth factor and antibody. Cells were counted blind by two independent observers, who achieved an overlap of 89.8%. As it was impossible to develop all wells in the same run, an internal control was included in each run and analysed for staining intensity (minimum 20 cells out of two wells per developing bath). Inter-run variability (coefficient of variation) was 6.2%.
Rat DRG in vitro studies
Neonatal Wistar rats (age 3–5 days) were killed and DRG from all spinal levels obtained and pooled. DRG were collected in Ham’s F12 Nutrient Mix (Gibco, now Life Technologies) and spin-washed three times in fresh Ham’s F12. DRG were then transferred into a 1.5 ml Eppendorf tube and immersed in 1 ml of papain solution (Sigma) at 10 U/ml for 50 min at 37°C under 8% CO2. The digested tissue was spun and the excess papain removed, and the tissue was then triturated in trypsin inhibitor solution (Sigma). Complete BSF2 medium (Ham’s F12, bovine serum albumin, apotransferrin, progesterone, insulin, sodium selenite, putrescine, penicillin, streptomycin and 2% heat-inactivated foetal calf serum) was added to a volume of 1 ml and cells were counted using a haemocytometer. The extract was diluted to a concentration of 4000–5000 cells/ml using complete BSF2 medium, and growth factors were added at the following concentrations: NGF 100 ng/ml; NT-3 50 and 500 ng/ml; GDNF 50 and 500 ng/ml. These concentrations were chosen as they have been reported to produce morphological effects in cultured neonatal rat sensory neurones (Gavazzi et al., 1999). Cells were then plated at 250 µl per well of a LabTek plate or 500 µl per well of a four-well plate, precoated with poly-L-lysine and laminin (both Sigma). Cells were grown for 48 h at 37°C under 8% CO2.
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Results |
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Antibody generation and validation
Site-directed anti-peptide antibodies L155 and M6 for hSK1 and M20 for hIK1 were characterized by immunoblotting with total cell extracts prepared from HEK293 and CHO stable cell lines expressing hSK1, hSK2, hSK3 and hIK1, and wild-type cell lines (Fig. 1). Both antibodies, but not preimmune serum, specifically reacted with the corresponding protein complexes of apparent molecular mass
240 kDa for hSK1 and 200 kDa for hIK1 in the appropriate cell lines. For all of the antibodies, the reaction was blocked by the corresponding peptide (data not shown). These findings were obtained in boiled and unheated extracts, which suggests that, like some other potassium channel complexes, these homotetramers are resistant to heat and SDS, as discussed below.
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Human DRG
In control post-mortem DRG (n = 4) (Fig. 2A), strong hSK1 immunostaining was observed. Ninety-five per cent of the large-diameter neurones (>50 µm) and 87% of the small- and medium-diameter neurones (<50 µm) were immunopositive. Only a few fibres were observed to be hSK1-positive. In acute avulsed DRG (n = 5) (Fig. B), i.e. those for which the delay between injury and surgery was <3 weeks, we saw a significant decrease in the number of large (53% positive, P = 0.0003) and small/medium cells (67% positive, P = 0.0004), when compared with controls. Chronic avulsed DRG (n = 3) (Fig. C) showed more hSK1-positive cells than DRG from acute states, but their numbers were still reduced when compared with control DRG, for large (66% positive, P = 0.002 compared with controls, P = 0.02 compared with acute avulsed) and small/medium neurones (83% positive, P = 0.002 compared with controls, P = 0.006 compared with acute avulsed) (Fig. 3A and B).
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hIK1-like immunoreactivity was detected in 87% of large-diameter and 94% of small- and medium-diameter neurones in control DRG (n = 5) (Fig. D). In the five acute avulsion cases studied, a significant decrease in numbers of large immunopositive cells was detected (74% positive, P = 0.0016), but small and medium cells showed no change (Fig. E). In chronic avulsed DRG, numbers of both large (63% positive, P = 0.0004) and small/medium cells (76% positive, P = 0.0078) had decreased significantly (Figs F and 3C and D).
Human nerves
hSK1-positive fibres were below the detection limit or were only weakly immunostained in control and injured nerves using the method described above, but their presence was clearly detected in control nerve freshly fixed by immersion in Zamboni’s medium (2% w/v formalin, 0.1 M phosphate, 15% v/v saturated picric acid) (Fig. 4A). The hSK1 immunoreactivity was successfully preabsorbed with immunizing peptide (Fig. B). Zamboni-fixed injured nerves were not available at the time of the study. hIK1-like immunoreactivity was found in control nerve (Fig. D), but also showed differential distribution in injured nerves. In five out of 23 injured nerves taken >3 weeks after injury, IK1-like immunostaining was detected proximal but not distal to the injury site (Fig. F). Immunostaining with neurofilament antibodies in the corresponding nerve segments is shown in Fig. C and Fig. E, respectively; abundant nerve fibres were observed proximal and distal to the injury site.
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Cultured neonatal rat DRG neurones
In culture medium without any added growth factor, 42% of all neurones showed immunoreactivity for hSK1 (Fig. 5). On addition of NGF at 100 ng/ml, this number increased significantly to 70% (P < 0.0001) (Fig. 6A). There was no obvious change on adding different concentrations of NT-3. GDNF increased the number of hSK1-positive cells to 52% (P = 0.01), but only when used at a concentration of 500 ng/ml (data not shown). The same results were achieved when the cells were analysed by size (neurones of small, medium and large diameter).
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hIK1-like immunoreactivity was seen in 71% of all neurones when growth factors were not added. NGF did not show an effect, whereas NT-3 increased the number of positive cells by a small degree, to 82% at 50 ng/ml (P = 0.0001) and to 79% at 500 ng/ml (P = 0.0079). GDNF did not seem to have any significant influence (Fig. B). There was a suggestion that NGF at 100 ng/ml increased the number of large hIK1-positive neurones (P = 0.023), whereas all cell sizes showed an increase with NT-3 at both 50 and 500 ng/ml.
In the absence of additional growth factors, 47% of cells showed immunoreactivity for SNS/PN3, which increased significantly to 77% on addition of 100 ng/ml NGF (P < 0.0001). Also, NT-3 at 50 ng/ml showed an increase to 59% (P = 0.0009), whereas GDNF did not have any influence on levels of SNS/PN3 (Fig. C). The same trend was seen in all cell sizes.
Preincubation
There was no immunoreactivity of hSK1 in human and rat DRG (Fig. A) and control nerve (Fig. B) when the antibodies were preincubated with an excess of the corresponding peptide (1.4 mg/ml for M6, 1.2 mg/ml for L155). The same result was achieved when the hIK1 (Fig. C) and SNS/PN3 antibodies were preincubated with their peptides (1.3 mg/ml for M20, 1.6 mg/ml for M4, 2.0 mg/ml for K104). For all preabsorption experiments, a 100-fold excess of peptide was used.
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Discussion |
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Calcium-activated potassium channels are responsible for part of the AHP in excitable cells. A change in their distribution in injured sensory neurones may therefore contribute to pathophysiology. In this paper, we have demonstrated for the first time the presence of the calcium-activated potassium channel hSK1 in control and injured human sensory neurones and have detected an hIK1-like channel in the same tissues.
SK1 mRNA is widely expressed in the CNS and these channels are known regulators of neuronal excitability (Litt et al., 1999; Stocker and Pedarzani, 2000). In contrast, several groups have failed to detect mRNA for IK channels in brain and spinal cord and the channel is generally considered to be non-neuronal (Ishii et al., 1997; Joiner et al., 1997; Jensen et al., 1998). There are, however, reports of neuronal calcium-activated potassium conductances with pharmacological and biophysical characteristics that most closely resemble those of the recombinant hIK channel (Hay and Kunze, 1994; Greffrath et al., 1998). Although we cannot be absolutely certain of the specificity of the hIK antibody (M20) under the conditions of the immunocytochemistry experiments, the immunostaining was restricted to a subpopulation of DRG neurones and could be blocked by peptide. A specific band was detected with M20 on Western blots from cell lines that stably expressed IK but not those that expressed wild-type or SK1 channels. The detection was blocked by the corresponding peptide. The apparent molecular mass of this band was 200 kDa, which corresponds to four times that of the predicted monomeric channel subunit of 48 kDa (Joiner et al., 1997). As far as is known, all other 6TM potassium channels exist as tetrameric proteins (Kreusch et al., 1998) and our findings suggest that this is probably also the case for hIK. Like some potassium channel complexes, the IK homotetramer protein is heat- and SDS-resistant (Heginbotham et al., 1997; Arkin et al., 1998; Corey and Clapham, 1998). The data thus support the contention that the M20 immunoreactivity represents specific staining of IK channels. One possibility is that hIK1-like immunostaining in our tissues represents a hitherto undescribed neuronal form or splice variant, which requires further characterization.
In the present study, hSK1 and hIK immunoreactivity was detected in a high proportion of small-, medium- and large-diameter human DRG neurones. To our knowledge, there are no electrophysiological studies on human DRG focusing on AHPs with which to compare this expression pattern. In adult rat DRG, Gold et al. (1996) observed calcium-dependent medium duration AHPs (mean 150 ms) following a train of action potentials in cells of all diameters, and a second, slower component (>2 s) in 25% of small-diameter neurones. Amir and Devor (1997) found that >90% of A
fibres exhibited a medium-duration AHP following a burst of spikes. We detected SK1 and IK-like immunoreactivity in 42 and 71% of cultured neonatal DRG neurones, respectively, and this expression was highly dependent on the growth factor environment (see later). More detailed studies that include the other SK channels are required to correlate the presence of specific calcium-activated potassium channels with AHP phenotypes in sensory neurones.
The expression of hSK-1 decreased significantly in both small and large cell bodies of injured human DRG. There was also a decrease in hIK1-like immunoreactivity in predominantly large cells, but also in small and medium cells. In five (out of 23) of the nerve lesions examined there was a lack of hIK-1 immunoreactivity distally, but this immunoreactivity was observed proximally. These five nerves, unlike the others, had all previously undergone Wallerian degeneration (they were collected 
3 weeks after injury). This indicates a decrease in hIK1-like immunoreactivity distal to the injury site, possibly resulting from decreased expression and/or axonal transport across the injury site.
The key neurotrophic factors for sensory fibres are NGF, brain-derived neurotrophic factor, NT-3 and GDNF. It is already known that BK channels in the rat brain are activated by both NT-3 and NGF (Holm et al., 1997). In the present study, we have demonstrated, using cultured neonatal rat DRG neurones, that NGF significantly increases the number of cells expressing hSK1, whilst NT-3 stimulates hIK1 to some extent. The effects of these neurotrophins on SNS/PN3 were included in our culture studies and were in accord with previous observations (Wood et al., 2000). Decreased retrograde transport of these neurotrophic factors in injured sensory neurones may thus reduce expression of these ion channels and increase excitability.
There appears to be remarkable plasticity of voltage and ligand-gated ion channels in injured human sensory neurones (Coward et al., 2000, 2001a, b; Yiangou et al., 2000a, b, 2001), some of which appear to be related to changes in retrograde transport and local release of neurotrophic factors and cytokines (Anand et al., 1997; Bär et al., 1998; Saldanha et al., 2000). A similar translocation mechanism has been described for both voltage-gated potassium channels and voltage-gated sodium channels (Ishikawa et al., 1999). Whereas the accumulation of sodium channels at the site of injury or proximal to it may lead to hyperexcitability, in the case of potassium channels this may result from their decrease in neuronal soma and in fibres distal to the injury, particularly in regenerating fibres. Calcium-activated potassium channels have already been implicated in ectopic spontaneous discharges of injured rat DRG neurones (Amir and Devor, 1997; Xing and Hu, 1999) and in the chronic constriction injury model of neuropathic pain in rats (Honma et al., 1999). Blockade of IK1 and other potassium channels by aminopyridines, such as 4-AP (4-aminopyridine) and 3,4-DAP (3,4-diaminopyridine), may also explain the paraesthesiae induced by these medications. Treatment of Lambert–Eaton myasthenic syndrome with 3,4-DAP, for example, is commonly known to result in circumoral or digital paraesthesiae (Sanders et al., 2000). Furthermore, animal data also indicate that the sensory dysfunction reported in trials of 4-AP may result from the selective response characteristics of sensory fibres (Bowe et al., 1987).
Studies on rodent potassium currents in cutaneous sensory afferents showed a marked decrease after axotomy (Everill and Kocsis, 1999). These currents were mainly voltage-activated, and the underlying ion channels have not been characterized (Everill et al., 1998). The currents are maintained when NGF is applied to the nerve terminals after axotomy (Everill and Kocsis, 2000), suggesting a supportive effect of this neurotrophin on the underlying channel(s), similar to our in vitro findings for hSK1.
It is not possible to relate the changes in avulsed DRG to pain mechanisms in patients with spinal cord root avulsion injury, as the pain in such patients is related to deafferentation, with generation of abnormal impulses and other mechanisms within the dorsal spinal cord (Berman et al., 1998). A number of these patients will also have injuries distal to the DRG at other spinal levels, which may contribute to their pain. However, spinal nerve root injuries occur commonly in patients with prolapsed intervertebral discs, with consequent radiating pain, which may be intractable even after surgery; our findings may be relevant to the mechanism of pain in such cases. Furthermore, the nature of the DRG avulsion injury is different in some respects from a surgical rhizotomy and the changes observed may share features with DRG following peripheral axotomy: there is marked displacement of the DRG, associated vascular disturbance and an acute inflammatory response. It is known that axotomy of the central processes of sensory ganglia leads to different changes within DRG cells, in comparison with injuries distal to the DRG. For example, the expression of GAP-43 and of c-Jun in DRG may not be upregulated in rodents following central axotomy, but is upregulated following peripheral axotomy (Chong et al., 1994; Broude et al., 1997). As it is not possible to obtain human DRG at operation with peripheral nerve injuries, we are limited to future post-mortem studies to clarify the relationship of central with peripheral axotomy in human sensory neurones. Studies of SK1 and IK1 in rodent models of neurogenic pain will also help clarify these mechanisms.
Selective potassium channel openers are therefore likely to represent novel therapies for pain following nerve injury. The actions of a number of known potassium channel openers, including those of SK1 and IK1, also deserve pharmacological investigation in pain mechanisms, from the molecular to the clinical level. Previous studies have demonstrated that calcium-activated potassium channels can be stimulated by agonists such as somatostatin (White et al., 1991), natriuretic peptides (White et al., 1993), prolactin (Prevarskaya et al., 1995) and ß-amyloid-precursor protein (Furukawa et al., 1996), but the significance of these findings for pain mechanisms is not known.
This paper clearly demonstrates temporal and spatial changes in the distribution of hSK1 and hIK1-like immunoreactivity in injured human sensory neurones. These changes may contribute to sensory neurone hypersensitivity and the development of paraesthesiae, pain and allodynia. These channels are therefore important targets for the development of novel therapeutic agents.
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Received May 21, 2001. Revised September 4, 2001. Accepted September 24, 2001.
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