reduction in voltage gated k channel_j neurosci
TRANSCRIPT
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
1/16
,
*Department of Anesthesiology and Perioperative Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas,
USA
Program in Neuroscience, The University of Texas Graduate School of Biomedical Sciences, Houston, Texas, USA
Peripheral neuropathy is one of the most common compli-
cations of diabetes. One of the most prominent features of
diabetic neuropathy is the development of pain that typically
involves the extremities, occurring as an exaggerated
response to either a painful stimulus (hyperalgesia) or a
mild and normally non-painful stimulus (allodynia) (Brown
and Asbury 1984; Clark and Lee 1995). The precise cellular
mechanisms of hyperalgesia and allodynia in diabetic
neuropathic pain remain poorly understood. Increased excit-
ability of primary sensory neurons plays a critical role in
painful diabetic neuropathy (Hong et al. 2004; Jagodic et al.
2007). It has been shown that voltage-gated Na+ channel
currents are significantly increased in both small- and large-
sized dorsal root ganglion (DRG) neurons in diabetic rats
(Hong et al. 2004; Hong and Wiley 2006). There is also
a significant increase in low- and high-voltage-gated
Ca2+channel currents in DRG neurons in diabetic neuropathy
(Hall et al. 1995; Jagodic et al. 2007). However, changes in
other ion channels involved in the increased excitability of
DRG neurons in diabetic neuropathic pain are not fully
known.
Received May 6, 2010; revised manuscript received May 26, 2010;
accepted June 9, 2010.
Address correspondence and reprint requests to Hui-Lin Pan, MD,
PhD, Department of Anesthesiology and Perioperative Medicine, Unit
110, The University of Texas MD Anderson Cancer Center, 1515 Hol-
combe Blvd., Houston, TX 77030, USA.
E-mail: [email protected]
Abbreviations used: 4-AP, 4-aminopyridine; BDNF, brain-derived
neurotrophic factor; DAP, 3,4-diaminopyridine; DRG, dorsal root
ganglion; IB4, isolectin B4; NGF, nerve growth factor; PBS, phosphate-
buffered saline; Kv channels, voltage-gated K+ channels; STZ,
streptozotocin; TEA, tetraethylammonium.
Abstract
Abnormal hyperexcitability of primary sensory neurons plays
an important role in neuropathic pain. Voltage-gated potas-
sium (Kv) channels regulate neuronal excitability by affecting
the resting membrane potential and influencing the repolari-
zation and frequency of the action potential. In this study, we
determined changes in Kv channels in dorsal root ganglion
(DRG) neurons in a rat model of diabetic neuropathic pain.
The densities of total Kv, A-type (IA) and sustained delayed
(IK) currents were markedly reduced in medium- and large-,
but not in small-, diameter DRG neurons in diabetic rats.
Quantitative RT-PCR analysis revealed that the mRNA levelsof IA subunits, including Kv1.4, Kv3.4, Kv4.2, and Kv4.3, in the
DRG were reduced 50% in diabetic rats compared with
those in control rats. However, there were no significant dif-
ferences in the mRNA levels of IK subunits (Kv1.1, Kv1.2,
Kv2.1, and Kv2.2) in the DRG between the two groups.
Incubation with brain-derived neurotrophic factor (BDNF)
caused a large reduction in Kv currents, especially IA currents,
in medium and large DRG neurons from control rats. Fur-
thermore, the reductions in Kv currents and mRNA levels of IA
subunits in diabetic rats were normalized by pre-treatment
with anti-BDNF antibody or K252a, a TrkB tyrosine kinase
inhibitor. In addition, the number of medium and large DRG
neurons with BDNF immunoreactivity was greater in diabetic
than control rats. Collectively, our findings suggest that diabe-
tes primarily reduces Kv channel activity in medium and large
DRG neurons. Increased BDNF activity in these neurons likely
contributes to thereduction in Kv channel function through TrkBreceptor stimulation in painful diabetic neuropathy.
Keywords: diabetic neuropathy, dorsal root ganglion, ion
channels, neuropathic pain, neurotophic factors, voltage-
gated potassium channels.
J. Neurochem. (2010) 114, 14601475.
JOURNAL OF NEUROCHEMISTRY | 2010 | 114 | 14601475 doi: 10.1111/j.1471-4159.2010.06863.x
1460 Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
2010 The Authors
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
2/16
Voltage-gated K+ (Kv) channels are important for the
regulation of the resting membrane potential, the duration
and frequency of the action potential, and the release of
neurotransmitters in neurons (Kim et al. 2005; Catacuzzeno
et al. 2008). The native Kv currents in primary sensoryneurons include two major types based on their inactivation
kinetics and sensitivities to tetraethylammonium (TEA) and
3,4-diaminopyridine (DAP) or 4-aminopyridine (4-AP):
slowly inactivating delayed currents (IK) and rapidly
inactivating transient A-type currents (IA) (Everill et al.
1998; Liu and Simon 2003; Vydyanathan et al. 2005). In the
isolectin B4 (IB4)-positive DRG neurons, the IA is particu-
larly important in the control of the spike onset, the threshold
of the action potential firing, and the firing frequency
(Vydyanathan et al. 2005). The IK is also involved in
determining the threshold of the action potential firing, the
repolarization and after-hyperpolarization phase, and the
resting potential in primary sensory neurons (Safronov et al.
1996; Catacuzzeno et al. 2008). It has been shown that
traumatic nerve injury reduces the mRNA levels of the
Kv1.1, Kv1.2, Kv1.4, Kv2.2, and Kv4.2 subunits in DRG
neurons (Kim et al. 2002) and both the IA and IK in DRG
neurons (Everill and Kocsis 1999; Abdulla and Smith 2001;
Yang et al. 2004). Nevertheless, little is known about the
changes in Kv channel activity in DRG neurons in diabetic
neuropathic pain.
Brain-derived neurotrophic factor (BDNF) is normally
present in some small- and medium-sized DRG neurons
(Zhou and Rush 1996; Thompson et al. 1999). The expres-
sion level of BDNF is increased in small-sized DRG neuronsin response to peripheral inflammation (Karchewski et al.
2002; Obata et al. 2003a). Increased BDNF expression also
occurs in axotomized medium and large DRG neurons
(Tonra et al. 1998; Obata et al. 2003b) and in the DRG in
diabetic rats (Fernyhough et al. 1995). It has been shown that
treatment with BDNF, not nerve growth factor (NGF), can
reduce the mRNA levels of IA subunits in DRG neurons
(Park et al. 2003). However, it is not clear whether BDNF
plays a role in reducing Kv channel function in DRG neurons
in diabetic neuropathy. In this study, we (i) examined the
changes in Kv currents in different sized DRG neurons in a
rat model of painful diabetic neuropathy and (ii) determined
the role of BDNF in reducing Kv channel activity in DRG
neurons in diabetic neuropathic pain.
Methods
Animal model of diabetic neuropathic pain
Male SpragueDawley rats (9 weeks old, Harlan SpragueDawley,
Indianapolis, IN, USA) were used. All experiments were approved
by the Animal Care and Use Committee of the University of Texas
M. D. Anderson Cancer Center and conformed to the guidelines of
the National Institutes of Healths Guide for the Care and Use of
Laboratory Animals. All efforts were made to minimize both the
suffering and number of animals used. Diabetes was induced by a
single intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin
(STZ; Sigma, St. Louis, MO, USA) freshly dissolved in 0.9% sterile
saline (Chen and Pan 2002). Age-matched vehicle-injected rats were
used as the controls. Previous studies have demonstrated that after
STZ injection, most rats display reproducible mechanical allodynia
and hyperalgesia within 3 weeks, lasting for at least 7 weeks
(Courteix et al. 1993; Chen and Pan 2002; Khan et al. 2002). This
model of neuropathic pain mimics the symptoms of neuropathy in
diabetic patients, with alterations in pain sensitivity and poor
responses to l opioid administered systemically or intrathecally
(Courteix et al. 1993; Malcangio and Tomlinson 1998; Zurek et al.
2001; Chen and Pan 2002; Khan et al. 2002). Diabetes was
confirmed in the STZ-injected rats by measurement of the blood
glucose concentration. The glucose level in the blood from the tail
vein was assayed using ACCU-CHEK test strips (Roche Diagnos-
tics, Indianapolis, IN, USA). The blood glucose level was measured
3 weeks after STZ administration, and only the rats with high blood
glucose level (> 300 mg/dL) were used. Neuropathic pain indiabetic rats was confirmed by examination of nociceptive mechan-
ical thresholds by using the paw pressure Analgesy-Meter (Ugo
Basile Biological Research, Comerio, Italy) (Chen and Pan 2002,
2006).
Isolation of DRG neurons
Rats were anesthetized with 23% isoflurane and then rapidly
decapitated. The thoracic and lumbar segments of the vertebral
column were surgically removed. The lumbar DRGs and the nerve
roots were quickly dissected out and transferred immediately into
DMEM (Gibco, Carlsbad, CA, USA) on ice. The DRGs were then
dissected free of the attached connective tissues under a microscope
and minced with fine-spring scissors. The minced ganglion
fragments were placed in a flask containing 5 mL of DMEM inwhich trypsin (type I, 0.2 mg/mL; Sigma) and collagenase (type I,
1 mg/mL; Sigma) had been dissolved. After incubation at 34C in a
shaking water bath for 40 min, soybean trypsin inhibitor (type II,
1.25 mg/mL; Sigma) was added to terminate the digestion. The cell
suspension was subsequently plated onto a 35-mm culture dish
containing poly-L-lysine (50 lg/mL) pre-coated coverslips and
incubated in 5% CO2 at 37C for 1 h. The supernatant was then
removed, and fresh DMEM was carefully added. The cells were
then kept in the incubator for at least another hour before they were
used for electrophysiological recordings. The final electrophysio-
logical recordings were performed 26 h after dissociation to allow
recovery from trypsination and mechanical disruption.
Electrophysiological recordings
The electrodes were pulled from GC150TF-10 glass capillaries
(inner diameter, 1.17 mm; outer diameter, 1.5 mm; Harvard
Apparatus, Holliston, MA, USA) using a micropipette puller and
fire-polished. The neurons were visualized using differential
interference contrast optics on an inverted microscope (Olympus,
Tokyo, Japan). Images of the cells were taken with a CCD camera
and displayed on a video monitor. The neurons were recorded in the
whole-cell configuration at a holding potential of)90 mV using an
EPC-10 amplifier (HEKA Instruments, Lambrecht, Germany). After
the whole-cell configuration was established, the cell membrane
capacitance and series resistance were electronically compensated.
2010 The Authors
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
BDNF and K+ channels in diabetic neuropathy | 1461
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
3/16
Leak currents were subtracted using the online P/4 protocol. All
experiments were performed at 25C. Signals were filtered at
1 kHz, digitized at 10 kHz, and acquired using the Pulse software
program.
To selectively record Kv currents and minimize the contribution
from Ca2+and Na+ currents, the extracellular solution contained (in
mM) 150 choline chloride, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES,
1 CdCl2, and 10 D-glucose (pH 7.4 adjusted with KOH, osmolarity
320 mOsm). Because both 4-AP and DAP can directly stimulate
high voltage-gated Ca2+channels (Wu et al. 2009), CdCl2 was used
to block high voltage-gated Ca2+channels. The electrode resistance
was 23 MW when filled with the solution containing (in mM) 120
potassium gluconate, 20 KCl, 2 MgCl2, 10 EGTA, 10 HEPES, 5
Na2-ATP, and 1 CaCl2 (pH 7.2 adjusted with KOH, osmolarity
300 mOsm). To further determine the Kv current subtype in the
DRG neurons, the IA and IK currents were differentiated using
25 mM TEA and 5 mM DAP, blockers of native IK and IA,
respectively (Robertson and Nelson 1994; Safronov et al. 1996;
Everill et al. 1998; Vydyanathan et al. 2005). DAP was used in thisstudy because it shows a more potent inhibition of IA than 4-AP
(Robertson and Nelson 1994). The protocol used to measure Kv
current activation was performed at a holding potential of)90 mV
and consisted of 400-ms depolarization pulses from )70 to 60 mV
in 10-mV increments at 2-s intervals (Vydyanathan et al. 2005).
Drug application
The drugs were dissolved in distilled water at 1000 times the final
concentration and kept frozen in aliquots. The stock solutions were
diluted in the appropriate external solution just before use and held
in a series of independent syringes connected to corresponding fused
silica columns (ID 200 lm). The distance from the column mouth to
the cell being recorded was about 100 lm. Each drug solution was
delivered to the recording chamber by gravity, and rapid solutionexchange (about 200 ms) was achieved by controlling the corre-
sponding valve switch. All drugs and chemicals were purchased
from Sigma-Aldrich except BDNF and the anti-BDNF antibody,
which were purchased from Millipore (Temecula, CA, USA).
Real-time RT-PCR analysis of Kv subunit expression
Total RNA was extracted from rat lumbar DRGs at the L4-L6 level
using the Purelink total RNA purification system (Invitrogen,
Carlsbad, CA, USA) with on-column Dnase I digestion according to
the manufacturers instructions. cDNA was prepared by using the
Superscript III first-strand synthesis kit (Invitrogen).
Quantitative PCR was performed using the iQ5 real-time PCR
detection system with the SYBR Green PCR kit (Bio-Rad,
Hercules, CA, USA). All samples were analyzed in duplicate using
an annealing temperature of 60C, and each experiment was
repeated at least once. The primer pairs used are listed in Table 1.
To calculate the relative Kv subunit mRNA expression levels in
each sample, standard curves were generated using a twofold
dilution of the cDNA from the DRGs as the PCR template. The
relative amount of Kv-subunit mRNA in each sample was first
normalized to the level of the housekeeping gene S18 and was then
normalized to its expression level in control rats. The PCR product
specificity was verified by melting-curve analysis and agarose gel
electrophoresis.
Double immunofluorescence labeling of BDNF and Nissl in the DRG
Four rats in each group were used for the immunofluorescence
labeling. Rats were used 3 weeks after treatment with either STZ or
vehicle control. Under deep anesthesia induced by sodium pento-
barbital (60 mg/kg, i.p.), rats were perfused intracardially with
250 mL of saline, 250 mL of 4% paraformadehyde in 0.1 M
phosphate-buffered saline (PBS), and 150 mL of 10% sucrose in
PBS (pH 7.4). The lumbar DRGs at the L4-L6 levels were removed
quickly and cryoprotected in 30% sucrose in PBS for 24 h at 4C.
To determine the distribution of BDNF in DRG neurons in both
control and diabetic rats, immunofluorescent labeling of BDNF and
Nissl (a neuronal marker) in the DRG sections were performed. The
DRG tissues were cut into 30-lm-thick sections and collected free-floating in 0.1 M PBS. The tissue sections were rinsed in 0.1 M
Tris-buffered saline, and incubated with the primary antibody (rabbit
anti-BDNF, dilution 1 : 500; Millipore) for 2 h at 25C and then
5 days at 4C. The specificity of this antibody has been demon-
Table 1 List of primers used for real-time PCR
Gene name Primer Sequence Location
rat Kv1.1 Kv1.1-p1 5 gga gcg ccc cct acc cga gaa g 3 454475
(NM_173095) Kv1.1-p2 5 ggt gaa tgg tgc ccg tga agt cct 3 644621
rat Kv1.2 Kv1.2-p1 5 tcc cgg atg cct tct ggt g 3 16631683
(NM_012970) Kv1.2-p2 5
ggc ctg ctc ctc tcc ctc tgt 3
18621842rat Kv1.4 Kv1.4-p1 5 ttg tga acg cgt ggt aat aaa tgt gt 3 605630
(NM_012971) Kv1.4-p2 5 ggc ggc ctc ctg act ggt aat aat a 3 804780
rat Kv2.1 Kv2.1-p1 5gcg act gct cag acc cct tag ctc 3 239262
(NM_013186) Kv2.1-p2 5 tct gga atc gtg atc agc gct ttg 3 11361113
rat Kv2.2 Kv2.2-p1 5 cgt gga gaa ggc tgg aga gtc g 3 17271748
(NM_054000) Kv2.2-p2 5 tgg gct gga gga aga agt gtt gtt 3 19191896
rat Kv3.4 Kv3.4-p1 5 cca cgg ggc aat gac cac acc 3 643663
(XM_001070801) Kv3.4-p2 5 aca cag cgc acc cac cag cat tcc t 3 777753
rat Kv4.2 Kv4.2-p1 5 gcc gca gcg cct agt cgt tac c 3 12981319
(NM_031730) Kv4.2-p2 5 tga tag cca ttg tga ggg aaa aga gca 3 15591533
rat Kv4.3 Kv4.3-p1 5 ctc cct aag cgg cgt cct ggt cat t 3 12531277
(NM_031739) Kv4.3-p2 5 ctt ctg tgc cct gcg ttt atc tgc tct c 3 13611334
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
2010 The Authors
1462 | X.-H. Cao et al.
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
4/16
strated previously by pre-adsorption with the immunizing antigens
(Zhou and Rush 1996; Zhou et al. 1999). The sections were rinsed
in Tris-buffered saline and incubated with the secondary antibody
(biotin-conjugated goat anti-rabbit IgG, dilution 1 : 200; Jackson
ImmunoResearch Inc., West Grove, PA, USA) for 2 h at 25C.
Then, the sections were incubated with a 1 : 100 dilution of
streptavidin-horseradish peroxidase for 1 h at25C. Subsequently,
the sections were incubated with fluorescein-tyramide (dilution
1 : 100; PerkinElmer, Waltham, MA, USA) for 10 min at 25C.
The sections were then rinsed and incubated with NeuroTrace red
fluorescent Nissl stain (dilution 1 : 100; Molecular Probes, Eugene,
OR, USA) for 40 min at25C. Finally, the sections were rinsed,
Fig. 1 Reduction in the current densities of total Kv, 3,4-diamino-
pyridine (DAP)-sensitive IA, and tetraethylammonium (TEA)-sensitive
IK in medium dorsal root ganglion (DRG) neurons from diabetic rats.
(a, b) Representative traces showing different types of Kv currents in
medium DRG neurons from a control and diabetic rat. The neurons
were held at )90 mV and depolarized from )70 to 60 mV in 10-mV
increments (inset). (c) IV curves show the current densities of total
Kv, IA, and IK in medium DRG neurons from rats in the control ( n = 22
cells) and diabetic (n = 24 cells) groups. (d) Voltage-dependent
activation kinetics (GV curves) of total Kv, IA, and IK in medium
DRG neurons from control and diabetic rats. The V0.5 values of total
Kv currents (control rats: 2.77 2.11 mV, n = 22; diabetic rats:
10.57 1.71 mV, n = 25, p < 0.05) and IK (control rats: 4.85
2.43 mV, n = 17; diabetic rats: 13.92 3.20 mV, n = 20, p < 0.05),
but not IA (control rats: 10.18 5.59 mV, n = 19; diabetic rats: 1.35
4.51 mV, n = 22, p > 0.05), were significantly different between the
control and diabetic rats (t-test). There was no significant difference in
the k value of total Kv currents (control rats: 17.75 0.75, n = 22;
diabetic rats: 19.39 0.70, n = 25, p > 0.05), IA (control rats:
18.10 2.21, n = 19; diabetic rats: 16.09 0.82, n = 22, p > 0.05),
and IK (control rats: 15.05 0.59, n = 17; diabetic rats: 16.45 0.61,
n = 20, p > 0.05) between the control and diabetic rats (t-test).
*p < 0.05 compared with the corresponding value in the control group
(two-way ANOVA).
2010 The Authors
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
BDNF and K+ channels in diabetic neuropathy | 1463
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
5/16
mounted on slides, dried, and sealed with a coverslip. The negative
controls were processed in the same manner except the primary
antibody was omitted. The sections were examined on a laser
scanning confocal microscope (Carl Zeiss, Jena, Germany), and the
areas of interest were photographed. To quantify the changes in the
BDNF-expressing neurons from the DRG of diabetic rats, the
number of BDNF-immunoreactive neurons and Nissl-positive
neurons in the DRG of control and diabetic rats was counted by
an investigator who was blinded to the experimental groups. The
cell counting was performed using three images per tissue section,
and three tissue sections were randomly selected from the L5 DRG
for each rat (n = 4 rats per group).
(a)
(b)
(c)
(d)
Fig. 2 Reduction in the current densities of total Kv, 3,4-diamino-
pyridine (DAP)-sensitive IA, and 3,4-diaminopyridine (TEA)-sensitive
IK in large dorsal root ganglion (DRG) neurons from diabetic rats. (a, b)
Original traces show different types of Kv currents in large DRG
neurons from a control and diabetic rat. Neurons were held at )90 mV
and depolarized from )70 to 60 mV in 10-mV increments (inset). (c)
IVcurves show differences in the current densities of total Kv, I A, and
IK in large DRG neurons between the control (n = 19 cells) and dia-
betic (n = 17 cells) groups. (d) Voltage-dependent activation kinetics
of total Kv, IA, and IK in large DRG neurons from control and diabetic
rats. The V0.5 values of total Kv currents (control: 1.38 2.21 mV,
n = 19; diabetic: 11.98 2.66 mV, n = 17, p < 0.05) and IK (control:
4.67 2.57 mV, n = 10; diabetic: 17.16 3.71 mV, n = 11, p < 0.05),
but not IA (control:)4.71 3.02 mV, n = 19; diabetic: 3.33 3.97 mV,
n = 17, p > 0.05), were significantly different between control and
diabetic rats (t-test). There was no significant difference in the kvalue
of total Kv currents (control: 19.66 0.95, n = 19; diabetic:
20.47 0.75, n = 17, p > 0.05), IA (control: 16.51 1.56, n = 19;
diabetic: 13.25 0.84, n = 17, p > 0.05), and IK (control: 16.93 1.06,
n = 10; diabetic: 20.19 2.15, n = 11, p > 0.05) between the two
groups (t-test). *p < 0.05 compared with the corresponding value in
the control group (two-way ANOVA).
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
2010 The Authors
1464 | X.-H. Cao et al.
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
6/16
Data analysis and curve fitting
Data are presented as mean SEM. The electrophysiological data
were analyzed using the PulseFit software program (HEKA). The
amplitude of the total Kv currents was measured at the peak, and the
amplitude of the DAP-sensitive IA and TEA-sensitive IK were
obtained by subtracting the amplitude of the DAP- and TEA-
resistant Kv currents from that of the total Kv currents, respectively.
The whole-cell currentvoltage (IV) curves for individual neurons
(a)
(b)
(c)
(d)
Fig. 3 Lack of changes in the total Kv, 3,4-diaminopyridine (DAP)-
sensitive IA, and tetraethylammonium (TEA)-sensitive IK in small dorsal
root ganglion (DRG) neurons from diabetic rats. (a, b) Current traces
showing different types of Kv currents in small DRG neurons from a
control and diabetic rat. Neurons are held at )90 mV and depolarized
from )70 to 60 mV in 10-mV increments (inset). (c) Comparison of the
current densities of total Kv currents, IA, and IK in small DRG neurons
between the control (n = 41 cells) and diabetic (n = 39 cells) group. (d)
Steady-state activation (GV) curvesof total Kv, IA,andIK in small DRG
neurons from control and diabetic rats. There was no significant dif-
ference in the V0.5 value of total Kv currents (control: 3.80 1.98 mV,
n = 36; diabetic: 1.01 2.02 mV, n = 39, p > 0.05), IA (control:
10.40 4.99 mV, n = 36; diabetic: 8.58 3.81 mV, n = 37, p > 0.05),
and IK (control: 17.29 3.06 mV, n = 23; diabetic: 13.12 2.43,
n = 15, p > 0.05) between the control and diabetic rats ( t-test). Also,
the k values showed no difference in total Kv currents (control:
16.22 0.69, n = 36; diabetic: 17.01 0.71, n = 39, p > 0.05), IA
(control: 15.29 1.55, n = 36; diabetic: 16.85 1.47, n = 37,
p > 0.05), and IK (control: 17.53 2.31 mV, n = 23; diabetic:
17.49 1.42, n = 15, p > 0.05) between the two groups (t-test).
2010 The Authors
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
BDNF and K+ channels in diabetic neuropathy | 1465
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
7/16
were generated by calculating the peak outward current at each
testing potential and normalizing to the cell capacitance. The
conductancevoltage (GV) curves were described with the Boltz-
mann equation: G/Gmax = 1/[1 + exp(V0.5 ) Vm/k)], where V0.5 is
the membrane potential at which 50% activation is observed, kis the
slope of the function, and Vm is the membrane potential. Differences
between the means were tested for significance using paired or
unpaired Students t-tests, repeated-measures ANOVA followed by
Dunnetts post hoc test, or two-way ANOVA followed by Bonferronis
post hoc test. A p-value of < 0.05 was considered to be statistically
significant.
Results
Three weeks after diabetic induction, the diabetic rats
showed a large reduction in their paw withdrawal thresh-
olds in response to the pressure stimulus applied to the
hindpaw (Chen and Pan 2002; Chen et al. 2009), as
compared with age-matched control rats (control rats:
121.11 3.51 g; diabetic rats: 77.78 3.64 g, p < 0.05,
t-test).
The DRG neurons were divided into three groups
according to their cell diameters, which were measured
with a calibrated eyepiece reticule: small (< 30 lm),
medium (3040 lm), and large (> 40 lm). To determine
the whole-cell Kv currents in these three groups of DRG
neurons, we normalized the peak outward current to the
cell capacitance. There were no significant differences in
the capacitance of the three groups of DRG neurons
between the diabetic rats (small, 26.23 1.08 pF, n = 39;
medium, 62.98 3.75 pF, n = 24; large, 114.51 6.35 pF,
n = 17) and age-matched control rats (small, 25.77
1.99 pF, n = 41; medium, 60.33 2.42 pF, n = 22; large,
121.94 7.36 pF, n = 19; p < 0.05, two-way ANOVA).
Fig. 4 Lack of differences in the total Kv,
3,4-diaminopyridine (DAP)-sensitive IA, and
tetraethylammonium (TEA)-sensitive IK in
isolectin B4 (IB4)-positive and IB4-negative
small dorsal root ganglion (DRG) neurons
between the control and diabetic group. (a,
b) IV curves show the similar amplitudes
of the Kv current density in IB4-positive and
IB4-negative neurons at different potentialsin the control (IB4-positive, n = 13 cells; IB4-
negative, n = 5 cells) and diabetic (IB4-po-
sitive, n = 31 cells; IB4-negative, n = 5
cells) groups.
Fig. 5 Changes in the mRNA levels of individual Kv subunits in the
dorsal root ganglion (DRG) from diabetic rats. (a) Differences in the
mRNA levelsof IA subunits(Kv1.4,Kv3.4, Kv4.2, andKv4.3) in the DRG
between control and diabetic rats. (b) Lack of differences in the mRNA
levels of IK subunits (Kv1.1, Kv1.2, Kv2.1, and Kv2.2) in the DRG
between control and diabetic rats (n = 8 rats, in each group). *p < 0.05
compared with the corresponding value in the control group (t-test).
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
2010 The Authors
1466 | X.-H. Cao et al.
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
8/16
(a)
(b)
(c)
(d)
(e)
Fig. 6 Effects of brain-derived neurotrophic
factor (BDNF) treatment on the total Kv,3,4-diaminopyridine (DAP)-sensitive IA, and
tetraethylammonium (TEA)-sensitive IK
currents in dorsal root ganglion (DRG)
neurons from control rats. (a) IV curves
show that BDNF treatment had no effect on
the current densities of total Kv, IA, and IK in
small DRG neurons (n = 12). (b) IVcurves
show that BDNF treatment reduced the
current densities of total Kv, IA, and IK in
medium DRG neurons (n = 13). (c) IV
curves show that reduced the current den-
sities of total Kv, IA, and IK in large DRG
neurons (n = 12). (d) K252a, but not K252b,
abolished the BDNF effects on total Kv, IA,
and IK in medium DRG neurons (n = 7 in
each group). (e) K252a, but not K252b,
blocked the BDNF effects on total Kv, IA,
and IK in large DRG neurons (n = 8 in each
group). *p < 0.05 compared with the corre-
sponding value in the control or vehicle
(K252b) group (two-way ANOVA). [Correction
after online publication 27 July 2010: Figure
6 was replaced with the correct version of
the figure.]
2010 The Authors
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
BDNF and K+ channels in diabetic neuropathy | 1467
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
9/16
Reduction in Kv currents in medium and large DRG neurons
from diabetic rats
The total Kv current density was significantly reduced in
diabetic rats in both medium and large DRG neurons, as
compared with the total Kv current density in the DRGneurons from the control group (Figs 1 and 2). The peak
current density of both the IA and IK in the medium and large
DRG neurons was also significantly smaller in the diabetic
than in the control group (Figs 1 and 2).
Steady-state activation is an important property of Kv
currents and can influence the excitability of DRG neurons.
Thus, we determined the steady-state activation of total Kv,
IA, and IK in medium and large DRG neurons. There was a
significant depolarizing shift in the total Kv and IK currents
in medium DRG neurons from diabetic rats (total Kv,
V0.5 = 10.57 1.71 mV; IK, V0.5 = 13.92 3.20 mV), as
compared with those from the control rats (total Kv,
V0.5 = 2.77 2.11 mV; IK, V0.5 = 4.85 2.43 mV; t-test)
(Fig. 1d). Also, a similar depolarizing shift was found in
the IK in the large DRG neurons from the diabetic rats (total
Kv, V0.5 = 11.98 2.66 mV; IK, V0.5 = 17.16 3.71 mV),
as compared with those from the control rats (total Kv,
V0.5 = 1.38 2.21 mV; IK, V0.5 = )4.67 2.57 mV; t-test)
(Fig. 2d). However, there was no significant difference in the
steady-state activation of IA in the medium and large DRG
neurons between the diabetic and control groups (Figs 1dand 2d).
Lack of changes in Kv currents in small DRG neurons in
diabetic rats
The current densities of the total Kv, IA, and IK in small DRG
neurons from diabetic rats were not significantly different
from the current densities in small DRG neurons from control
rats (Fig. 3). In addition, the activation kinetics of the total,
IA, and IK from small DRG neurons did not differ signifi-
cantly between the control and diabetic rats (Fig. 3d).
Because the phenotypes of the small DRG neurons are
heterogenous, we further examined the Kv currents in IB4-
positive and IB4-negative small DRG neurons in diabetic and
control rats. Immediately before recording, the neurons were
labeled with IB4Alexa Fluor 594 (3 lg/mL) in a Tyrode
solution for 10 min and then rinsed for at least 3 min
Fig. 7 Effects of the anti-brain-derived
neurotrophic factor (BDNF) antibody on the
current densities of total Kv, 3,4-diamino-
pyridine (DAP)-sensitive IA, and tetraethy-
lammonium (TEA)-sensitive IK currents in
dorsal root ganglion (DRG) neurons from
diabetic rats. (a) IV curves show that
treatment with the anti-BDNF antibody
(50 ng/mL) slightly increased the current
densities of total Kv, IA, and IK in small DRG
neurons (n = 8). (b) IV curves show that
treatment with the anti-BDNF antibody
profoundly increased the current densities
of total Kv, IA, and IK in medium DRG neu-
rons (n = 13). (c) IV curves show that
treatment with the anti-BDNF antibody
substantially increased the current densities
of total Kv, IA, and IK in large DRG neurons
(n = 9). Note that the boiled anti-BDNF
antibody had no effects on the Kv current
density in small, medium, or large DRG
neurons. *p < 0.05 compared with the cor-
responding value in the control group (two-
way ANOVA).
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
2010 The Authors
1468 | X.-H. Cao et al.
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
10/16
(Vydyanathan et al. 2005). There were no significant differ-
ences in the Kv current densities in the IB4-positive or IB4-
negative neurons between the control and diabetic groups
(Fig. 4).
Changes in mRNA levels of IA and IK subunits in the DRG
from diabetic rats
We next measured the mRNA levels of Kv a-subunits in the
DRG from control and diabetic rats. Because the Kv1.4,
Kv3.4, Kv4.2, and Kv4.3 subunits contribute to the IA
channels in DRG neurons (Stuhmer et al. 1989; Oliveret al.
2004; Chien et al. 2007), we measured the mRNA levels of
Kv1.4, Kv3.4, Kv4.2, and Kv4.3 in the DRG from control
and diabetic rats. The mRNA levels of the Kv1.4, Kv3.4,
Kv4.2, and Kv4.3 subunits were all reduced approximately
50% in the diabetic rats, as compared with the levels in the
control rats (Fig. 5a). Kv1.1, Kv1.2, Kv2.1, and Kv2.2 are
important subunits of the IK channels (Murakoshi and
Trimmer 1999; Malin and Nerbonne 2002; Beekwilder et al.
2003). However, there were no significant differences in the
mRNA levels of the Kv1.1, Kv1.2, Kv2.1, and Kv2.2
subunits in the DRG between the control and diabetic rats
(Fig. 5b).
Role of BDNF in diabetes-induced reduction in Kv currents
in medium and large DRG neuronsIt has been shown that BDNF expression is increased in the
rat DRG after diabetic induction (Fernyhough et al. 1995).
To determine whether increased BDNF contributes to the
reduction in Kv currents in the DRG in diabetic neuropathy,
we first determined whether BDNF treatment affects Kv
currents in DRG neurons from control rats. Treatment of
DRG neurons from control rats with 50 ng/mL of BDNF for
24 h (Youssoufian and Walmsley 2007) significantly
reduced the peak current densities of the total, IA, and IK
in medium- and large-diameter neurons (Fig. 6ac). How-
ever, BDNF treatment had no significant effect on the Kv
current density in the small DRG neurons from control rats.
TrkB is the high-affinity BDNF receptor and is primarily
present in medium and large DRG neurons (McMahon et al.
1994; Wetmore and Olson 1995; Karchewski et al. 1999).
We next determined whether the effect of BDNF on Kv
Fig. 8 Effects of K252a on the current
densities of total Kv, 3,4-diaminopyridine
(DAP-sensitive) IA, and tetraethylammoni-
um (TEA)-sensitive IK currents in dorsal root
ganglion (DRG) neurons from diabetic rats.
(a) IV curves show that treatment with
K252a (300 nM) slightly increased the cur-
rent densities of total Kv, IA, and IK in small
DRG neurons (n = 11). (b) IVcurves show
that treatment with K252a substantially in-
creased the current densities of total Kv, IA,
and IK in medium DRG neurons (n = 10).
(c) IV curves show that treatment with
K252a profoundly increased the current
densities of total Kv, IA, and IK in large DRG
neurons (n = 9). *p < 0.05 compared with
the corresponding value in the diabetic
control group (two-way ANOVA).
2010 The Authors
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
BDNF and K+ channels in diabetic neuropathy | 1469
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
11/16
currents in DRG neurons was mediated by TrkB. Pre-
treatment of DRG neurons with K252a (300 nM) (Tapley
et al. 1992; Bhave et al. 1999), a TrkB receptor inhibitor,
blocked the BDNF effect on the Kv current density in DRG
neurons from control rats (Fig. 6d and e). The inactive
analogue of K252a, K252b (300 nM), did not significantly
alter the BDNF effect on the Kv current density in these
neurons.
To examine the role of BDNF in the diabetes-induced
reduction in the Kv current density, we next tested the effect
of the anti-BDNF antibody on the Kv currents in DRG
neurons from diabetic rats. Incubation of DRG neurons from
diabetic rats with the BDNF antibody (1 : 50) for 24 h
caused a large increase in the peak current densities of the
total Kv in all sizes of DRG neurons (Fig. 7). Also, there was
a large increase in the IA and IKdensities in medium and large
DRG neurons after treatment with the anti-BDNF antibody
(Fig. 7b and c). Treatment with the boiled BDNF antibody
had no effect on the Kv current density in DRG neurons.
To test the hypothesis that BDNF reduces the Kv
currents through TrkB receptor stimulation in diabetic
neuropathy, we assessed the effect of K252a on the Kv
current density of DRG neurons from diabetic rats.
Incubation of DRG neurons with K252a (300 nM), for
24 h profoundly increased the total Kv current density in
all sizes of DRG neurons from diabetic rats (Fig. 8).
Treatment with K252a also increased the IA and IK
densities in all three groups of DRG neurons from diabetic
rats (Fig. 8).
Effects of anti-BDNF antibody and K252a on Kv currents in
DRG neurons from control rats
To estimate whether BDNF and its receptors (TrkB) are up-
regulated in DRG neurons in diabetic neuropathy, we also
examined the effects of anti-BDNF antibody and K252a on
Kv current density in DRG neurons from control rats. In
contrast to the evident effects of anti-BDNF antibody and
K252a on Kv currents of DRG neurons from diabetic rats,
treatment with BDNF antibody (1 : 50) or K252a
(300 nM), for 24 h had little effects on the Kv current
density in all sizes of DRG neurons obtained from control
rats (Fig. 9).
Fig. 9 Effects of K252a and anti-brain-
derived neurotrophic factor (BDNF) anti-
body on the current densities of total
Kv, 3,4-diaminopyridine (DAP)-sensitive IA,
and tetraethylammonium (TEA)-sensitive Ik
currents in dorsal root ganglion (DRG)neurons from control rats. (a) IV curves
show that treatment with K252a (300 nM,
n = 11) and anti-BDNF antibody (50 ng/mL,
n = 9) only had a small effect on the current
density of IA in small DRG neurons. (b) IV
curves show the effect of treatment with
K252a (n = 9) and anti-BDNF antibody
(n = 10) on the current densities of total Kv,
IA, and Ik in medium DRG neurons. (c) IV
curves show the lack of effect of treatment
with K252a (n = 8) and anti-BDNF antibody
(n = 8) on the current densities of total Kv,
IA, and Ik in large DRG neurons. *p < 0.05
compared with the corresponding value in
the control group (two-way ANOVA).
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
2010 The Authors
1470 | X.-H. Cao et al.
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
12/16
Role of BDNF in diabetes-induced reduction in the
expression levels of IA subunits in DRG neurons
Because we found a large reduction in the mRNA levels of IA
subunits in the DRG tissue of diabetic rats, we further
determined the role of BDNF in diabetes-induced decreases
in the expression level of Kv1.4, Kv3.4, Kv4.2, and Kv4.3
subunits in DRG neurons using the real-time PCR technique.
Incubation of BDNF (50 ng/mL) for 4 h in DRG neurons
from control rats caused a large decrease in the mRNA levels
of Kv1.4, Kv3.4, Kv4.2, and Kv4.3 subunits (Fig. 10a).
Furthermore, in DRG neurons from diabetic rats, treatment
with the anti-BDNF antibody (1 : 50) or K252a (300 nM)
for 4 h reversed the decrease in the mRNA levels of Kv1.4,
Kv3.4, Kv4.2, and Kv4.3 subunits (Fig. 10b).
Altered distribution patterns of BDNF-immunoreactive
DRG neurons in diabetic rats
Additionally, we determined whether diabetic neuropathy
affects the distribution of BDNF in different sized DRG
neurons. In control rats, BDNF immunoreactivity was
distributed in some small and medium DRG neurons. In
contrast, BDNF immunoreactivity was present in most
medium and large DRG neurons from diabetic rats
(Fig. 11a). The total number of BDNF-immunoreactive
neurons in the DRG was much greater in the diabetic
(2487/4808, 51.72%) than in the control (1108/5040,21.98%; chi-squared test) rats. There were more medium
and large neurons with BDNF immunoreactivity in the DRG
from diabetic rats than control rats (Fig. 11b).
Discussion
In the present study, we found that the density of Kv currents,
especially the IA, in medium and large DRG neurons was
significantly reduced in a rat model of painful diabetic
neuropathy. Quantitative PCR analysis showed that the
mRNA levels of IA subunits, including Kv1.4, Kv3.4, Kv4.2,
and Kv4.3, were significantly reduced in the DRG of diabetic
rats. However, there were no significant differences in the
mRNA levels of the IK subunits (Kv1.1, Kv1.2, Kv2.1, and
Kv2.2) in the DRG between the diabetic and control rats.
Furthermore, the large reduction in the Kv current density
observed in the diabetic rats was mimicked by treatment of
DRG neurons from control rats with BDNF. Treatment with
either the anti-BDNF antibody or a TrkB inhibitor reversed
the changes in the Kv current density of DRG neurons from
diabetic rats but had little effect on Kv currents in all sizes of
DRG neurons from control rats. In addition, the number of
medium and large DRG neurons with BDNF immunoreac-
tivity was markedly increased in diabetic rats. Therefore, our
parallel biochemical and electrophysiological results provideimportant new information that diabetic neuropathy reduces
Kv activity, particularly the IA, in medium and large DRG
neurons. Increased BDNF activity likely contributes to the
reduction in the Kv current density through TrkB receptor
stimulation in diabetic neuropathy.
Kv channels are crucial in the control of neuronal
excitability, and their down-regulation can increase neuronal
excitability (Pongs 1999; Vydyanathan et al. 2005; Chi and
Nicol 2007; Chien et al. 2007; Catacuzzeno et al. 2008).
We found here that there was a profound decrease in the
density of total Kv, IA, and IK in medium and large DRG
neurons from diabetic rats. In addition, the reduction in the
IA density was greater than the reduction in the IK density in
these DRG neurons from diabetic rats. Consistent with our
electrophysiological data, the mRNA levels of IA subunits,
including Kv1.4, Kv3.4, Kv4.2, and Kv4.3, were signifi-
cantly reduced in the DRG from diabetic rats. Thus, reduced
expression of IA subunits in diabetes could account for the
reduced IA density seen in the medium and large DRG
neurons from diabetic rats. Inhibition of the IA can increase
the firing frequency and broadening of the action potential,
leading to increased Ca2+influx and neurotransmitter release
(Hoffman et al. 1997; Vydyanathan et al. 2005; Catacuzz-
eno et al. 2008). For example, knockout of the Kv4.2
Fig. 10 Reduction in the mRNA levels of IA subunits by brain-derived
neurotrophic factor (BDNF) in dorsal root ganglion (DRG) neurons. (a)
Effects of BDNF treatment on the mRNA levels of IA subunits (Kv1.4,
Kv3.4, Kv4.2, and Kv4.3) in DRG neurons from control rats (n = 4
samples in each group; t-test). (b) Effects of treatment with the anti-
BDNF antibody (50 ng/mL) or K252a (300 nM) on the mRNA levels of
Kv1.4, Kv3.4, Kv4.2, and Kv4.3 subunits in DRG neurons from diabetic
rats (n = 5 samples in each group; repeated measures ANOVA).*p < 0.05 compared with the corresponding value in the control group.
2010 The Authors
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
BDNF and K+ channels in diabetic neuropathy | 1471
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
13/16
subunit reduces the IA and increases the excitability of DRG
neurons, resulting in enhanced sensitivity to tactile and
thermal stimuli (Hu et al. 2006). Furthermore, down-
regulation of IA subunits in DRG neurons induces mechan-
ical hypersensitivity (Chien et al. 2007). It has been shown
that nerve ligation injury decreases the mRNA levels of the
Kv1.1, Kv1.2, Kv1.4, Kv2.2, and Kv4.2 subunits in DRG
neurons (Rasband et al. 2001; Kim et al. 2002). However,
we found that although the mRNA level of Kv1.4 was
significantly reduced in the diabetic group, the mRNA levels
of Kv1.1 and Kv1.2 did not differ significantly between the
control and diabetic groups. This discrepancy is likely
caused by the difference in the peripheral nerve damage
caused by traumatic nerve ligation and diabetic neuropathy.
Data from our present study suggest that the reduced IA in
medium and large DRG neurons could result from reduced
expression of IA subunits and contributes to the abnormal
hyperexcitability of DRG neurons in diabetic neuropathic
pain.
IK channels shape action potentials by keeping the single
action potential short and elevating the firing adaptation
(Safronov et al. 1996; Lien and Jonas 2003; Catacuzzeno
et al. 2008). For instance, suppression of Kv1.1 by dendro-
toxin-K or siRNA enhances the firing activity of DRG
neurons (Chi and Nicol 2007). In addition, Kv1.1 mutant
mice show increased pain responses (Clark and Tempel
1998). Thus, reduction of IK channels may also contribute to
the abnormal hyperexcitability of DRG neurons in painful
diabetic neuropathy. We found that the IK density was
reduced mainly in the medium and large DRG neurons from
diabetic rats. We also observed a depolarizing shift in the IK
in these neurons, which suggests that it is unfavorable to
open IK channels on primary sensory neurons in diabetic
neuropathy. However, the mRNA levels of the IK subunits,
including Kv1.1, Kv1.2, Kv2.1, and Kv2.2, did not differ
significantly between the control and diabetic rats. The
mechanisms underlying the reduction in the IK in DRG
neurons in diabetic neuropathy are not clear. The depolar-
izing shift in the IK alone is not sufficient to explain the large
reduction in the IK density in the medium and large DRG
neurons from diabetic rats. Post-translational regulation,
such as phosphorylation, may play a role in this reduction in
the IK density in DRG neurons in diabetic rats. For example,
the Kv channel activity reconstituted by Kv1.1 or Kv2.1 is
controlled by phosphorylation (Boland and Jackson 1999;
Park et al. 2006). It has been shown that diabetes increases
the activity of protein kinase C (Ishii et al. 1998) and that
increased protein kinase C activation can inhibit Kv1.1
(a)
(b)
Fig. 11 Differences in the distribution pat-
tern of brain-derived neurotrophic factor
(BDNF) immunoreactive neurons in the
dorsal root ganglion (DRG) between control
and diabetic rats. (a) Representative con-
focal images show a greater number of
BDNF immunoreactive neurons in mediumand large neurons from a diabetic rat. Col-
ocalization of BDNF and Nissl (a neuronal
marker) is indicated in yellow when the two
images are digitally merged. Images are
single confocal optical sections. (b) The
histogram shows the distinct differences in
the distribution of BDNF immunoreactive
neurons in different sized DRG neurons
between control and diabetic rats. *p < 0.05
compared with the corresponding value for
the non-treated diabetic group (chi-squared
test).
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
2010 The Authors
1472 | X.-H. Cao et al.
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
14/16
(Boland and Jackson 1999). Thus, increased protein kinase C
activity in DRG neurons could reduce the IK density through
phosphorylation of certain IK subunits in diabetic neuro-
pathy.
Previous studies of sensory neurons in diabetic neuro-pathic pain have largely focused on small DRG neurons,
because normal nociception is thought to be mediated
primarily by small-sized DRG neurons and unmyelinated
afferent fibers. We have shown previously that transient
receptor potential vanilloid 1 receptors (TRPV1)-expressing
small sensory neurons are not involved in the development
of allodynia in this rat model of diabetic neuropathic pain
(Khan et al. 2002). In addition, damage to large myelinated
afferent fibers is well known in diabetic neuropathy
(Ochodnicka et al. 1995). We found that the diabetes-
induced reduction in Kv currents was limited predominantly
to medium and large DRG neurons. These findings provide
further evidence that medium- and large-sized DRG neurons,
which are typically associated with myelinated primary
afferent fibers, are important in the development of diabetic
neuropathic pain. These results indicate that the reduced Kv
current activity in these primary sensory neurons could
contribute to abnormal hyperexcitability of these primary
sensory neurons and the mechanical allodynia seen in
diabetic neuropathy.
Another important finding of this study is that BDNF
plays a critical role in the reduction in the Kv currents in
DRG neurons from diabetic rats. We found that acute BDNF
treatment reduced the mRNA levels of IA subunits in DRG
neurons. Furthermore, BDNF treatment mainly reduced theKv current density, especially the IA current density, in
medium and large DRG neurons from control rats. The role
of BDNF in the reduction of Kv currents in DRG neurons
from diabetic rats was also supported by our finding that
treatment with the anti-BDNF antibody or a TrkB inhibitor,
K252a, reversed the changes in the Kv currents and the
mRNA levels of IA subunits in DRG neurons from diabetic
rats. Our data suggest that BDNF reduces the IA current
density by inhibiting the expression of IA subunits in DRG
neurons in diabetic rats. Although it remains uncertain how
BDNF suppresses the expression of IA subunits in DRG
neurons, BDNF may inhibit the expression of IA subunits
through the transcriptional repressor neuron restrictive
silencing factor/repressor element-1 Silencing transcription
factor (NRSF/REST). It has been shown that NRSF/REST is
involved in epigenetic silencing of Kv4.3 expression by
nerve injury in DRG neurons (Uchida et al. 2010). It is not
clear to what degree the paradigm of BDNF application used
in this study mimics the BDNF production in the DRG in
diabetes. It has been suggested that the manner of BDNF
application can have a very different effect on functional
outcomes (Greenberg et al. 2009). Because there is no
information about the time course of changes in BDNF
concentrations in the DRG in diabetes, we did not compare
whether slow and rapid increases in BDNF concentrations
produce different effects on Kv currents in DRG neurons. It
should be noted that K252a can block TrkA, TrkB, and
TrkC. It has been shown that treatment with NGF (acting via
TrkA and p75 neurotrophin receptors) can maintain Kvchannel activity after nerve ligation injury in sensory
neurons (Everill and Kocsis 2000). However, because NGF
does not affect the mRNA levels of IA current subunits in
DRG neurons (Park et al. 2003), it is less likely that the
observed effect of K252a on Kv channel activity in diabetic
DRG neurons in this study is mediated by TrkA. We found
that in contrast to the evident effects of anti-BDNF antibody
and K252a on Kv currents of DRG neurons from diabetic
rats, treatment with anti-BDNF antibody or K252a had little
effect on the Kv current density in all sizes of DRG neurons
from control rats. These data suggest that the functional
activity of BDNF is increased, which reduces Kv channel
activity through TrkB receptor stimulation in DRG neurons
of diabetic rats.
In addition, we found that the number of medium and large
DRG neurons that were immunoreactive to BDNF was
increased in diabetic rats. Our findings are consistent with the
results from studies of rats subjected to nerve ligation injury
(Tonra et al. 1998; Zhou et al. 1999). We found that BDNF
treatment had little effect on the Kv currents in small DRG
neurons in control rats. However, the Kv current density in
small DRG neurons was slightly increased after treatment
with the anti-BDNF antibody or a TrkB receptor inhibitor in
the diabetic rats. Because BDNF and TrkB receptors were
normally present in a subpopulation of small DRG neurons(McMahon et al. 1994; Zhou and Rush 1996), it is possible
that BDNF may tonically inhibit the expression of certain Kv
channels through TrkB receptors in these neurons in diabetic
rats. It is not completely clear how BDNF leads to increased
TrkB activation in DRG neurons of diabetic rats. We propose
that BDNF released from diabetic DRG neurons activates
neuronal TrkB through an autocrine mechanism. Therefore,
increased BDNF activity could contribute to the large
reduction in Kv channel function in medium and large
DRG neurons in diabetic neuropathy through augmented
TrkB receptor activation.
In conclusion, this study provides novel information that
diabetic neuropathy reduces Kv currents, particularly the IA,
in medium and large DRG neurons. BDNF likely plays an
important role in the reduction in the Kv channel activity of
these neurons in painful diabetic neuropathy through TrkB
receptor stimulation. Because reduction in Kv currents can
enhance neuronal excitability, increased BDNF activity may
enhance the excitability of DRG neurons by down-regulating
the Kv channels in diabetic neuropathy. This new informa-
tion is important for our understanding of the mechanisms
underlying the hyperactivity of primary sensory neurons and
the increased afferent input to the spinal dorsal horn in
painful diabetic neuropathy.
2010 The Authors
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
BDNF and K+ channels in diabetic neuropathy | 1473
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
15/16
Acknowledgements
This study was supported by the National Institutes of Health grants
GM64830 and NS45602 and by the N.G. and Helen T. Hawkins
Endowment to H.L.P.
References
Abdulla F. A. and Smith P. A. (2001) Axotomy- and autotomy-induced
changes in Ca2+ and K+ channel currents of rat dorsal root ganglion
neurons. J. Neurophysiol. 85, 644658.
Beekwilder J. P., OLeary M. E., van den Broek L. P., van Kempen G. T.,
Ypey D. L. and van den Berg R. J. (2003) Kv1.1 channels of dorsal
root ganglion neurons are inhibited by n-butyl-p-aminobenzoate, a
promising anesthetic for the treatment of chronic pain. J. Phar-
macol. Exp. Ther. 304, 531538.
Bhave S. V., Ghoda L. and Hoffman P. L. (1999) Brain-derived neuro-
trophic factor mediates the anti-apoptotic effect of NMDA in cer-
ebellar granule neurons: signal transduction cascades and site of
ethanol action. J. Neurosci.19
, 32773286.Boland L. M. and Jackson K. A. (1999) Protein kinase C inhibits Kv1.1
potassium channel function. Am. J. Physiol. 277, C100110.
Brown M. J. and Asbury A. K. (1984) Diabetic neuropathy. Ann. Neurol.
15, 212.
Catacuzzeno L., Fioretti B., Pietrobon D. and Franciolini F. (2008) The
differential expression of low-threshold K+ currents generates
distinct firing patterns in different subtypes of adult mouse tri-
geminal ganglion neurones. J. Physiol. 586, 51015118.
Chen S. R. and Pan H. L. (2002) Hypersensitivity of spinothalamic tract
neurons associated with diabetic neuropathic pain in rats. J. Neu-
rophysiol. 87, 27262733.
Chen S. R. and Pan H. L. (2006) Blocking mu opioid receptors in the
spinal cord prevents the analgesic action by subsequent systemic
opioids. Brain Res. 1081, 119125.
Chen S. R., Samoriski G. and Pan H. L. (2009) Antinociceptive effectsof chronic administration of uncompetitive NMDA receptor
antagonists in a rat model of diabetic neuropathic pain. Neuro-
pharmacology 57, 121126.
Chi X. X. and Nicol G. D. (2007) Manipulation of the potassium channel
Kv1.1 and its effect on neuronal excitability in rat sensory neurons.
J. Neurophysiol. 98, 26832692.
Chien L. Y., Cheng J. K., Chu D., Cheng C. F. and Tsaur M. L. (2007)
Reduced expression of A-type potassium channels in primary
sensory neurons induces mechanical hypersensitivity. J. Neurosci.
27, 98559865.
Clark C. M., Jr and Lee D. A. (1995) Prevention and treatment of the
complicationsof diabetesmellitus.N. Engl. J. Med. 332, 12101217.
Clark J. D. and Tempel B. L. (1998) Hyperalgesia in mice lacking the
Kv1.1 potassium channel gene. Neurosci. Lett. 251, 121124.
Courteix C., Eschalier A. and Lavarenne J. (1993) Streptozocin-induceddiabetic rats: behavioural evidence for a model of chronic pain.
Pain 53, 8188.
Everill B. and Kocsis J. D. (1999) Reduction in potassium currents in
identified cutaneous afferent dorsal root ganglion neurons after
axotomy. J. Neurophysiol. 82, 700708.
Everill B. and Kocsis J. D. (2000) Nerve growth factor maintains
potassium conductance after nerve injury in adult cutaneous
afferent dorsal root ganglion neurons. Neuroscience 100, 417
422.
Everill B., Rizzo M. A. and Kocsis J. D. (1998) Morphologically
identified cutaneous afferent DRG neurons express three different
potassium currents in varying proportions. J. Neurophysiol. 79,
18141824.
Fernyhough P., Diemel L. T., Brewster W. J. and Tomlinson D. R. (1995)
Altered neurotrophin mRNA levels in peripheral nerve and skeletal
muscle of experimentally diabetic rats. J. Neurochem. 64, 1231
1237.
Greenberg M. E., Xu B., Lu B. and Hempstead B. L. (2009) New
insights in the biology of BDNF synthesis and release: implicationsin CNS function. J. Neurosci. 29, 1276412767.
Hall K. E., Browning M. D., Dudek E. M. and Macdonald R. L. (1995)
Enhancement of high threshold calcium currents in rat primary
afferent neurons by constitutively active protein kinase C.
J. Neurosci. 15, 60696076.
Hoffman D. A., Magee J. C., Colbert C. M. and Johnston D. (1997)
K+ channel regulation of signal propagation in dendrites of
hippocampal pyramidal neurons. Nature 387, 869875.
Hong S. and Wiley J. W. (2006) Altered expression and function of
sodium channels in large DRG neurons and myelinated A-fibers in
early diabetic neuropathy in the rat. Biochem. Biophys. Res.
Commun. 339, 652660.
Hong S., Morrow T. J., Paulson P. E., Isom L. L. and Wiley J. W. (2004)
Early painful diabetic neuropathy is associated with differential
changes in tetrodotoxin-sensitive and -resistant sodium channels indorsal root ganglion neurons in the rat. J. Biol. Chem. 279, 29341
29350.
Hu H. J., Carrasquillo Y., Karim F., Jung W. E., Nerbonne J. M.,
Schwarz T. L. and Gereau R. W. t. (2006) The kv4.2 potas-
sium channel subunit is required for pain plasticity. Neuron 50,
89100.
Ishii H., Koya D. and King G. L. (1998) Protein kinase C activation and
its role in the development of vascular complications in diabetes
mellitus. J. Mol. Med. 76, 2131.
Jagodic M. M., Pathirathna S., Nelson M. T., Mancuso S., Joksovic
P. M., Rosenberg E. R., Bayliss D. A., Jevtovic-Todorovic V. and
Todorovic S. M. (2007) Cell-specific alterations of T-type calcium
current in painful diabetic neuropathy enhance excitability of
sensory neurons. J. Neurosci. 27, 33053316.
Karchewski L. A., Kim F. A., Johnston J., McKnight R. M. and VergeV. M. (1999) Anatomical evidence supporting the potential for
modulation by multiple neurotrophins in the majority of adult
lumbar sensory neurons. J. Comp. Neurol. 413, 327341.
Karchewski L. A., Gratto K. A., Wetmore C. and Verge V. M. (2002)
Dynamic patterns of BDNF expression in injured sensory neurons:
differential modulation by NGF and NT-3. Eur. J. Neurosci. 16,
14491462.
Khan G. M., Chen S. R. and Pan H. L. (2002) Role of primary afferent
nerves in allodynia caused by diabetic neuropathy in rats. Neuro-
science 114, 291299.
Kim D. S., Choi J. O., Rim H. D. and Cho H. J. (2002) Downregulation
of voltage-gated potassium channel alpha gene expression in dorsal
root ganglia following chronic constriction injury of the rat sciatic
nerve. Brain Res. Mol. Brain Res. 105, 146152.
Kim J., Wei D. S. and Hoffman D. A. (2005) Kv4 potassium channelsubunits control action potential repolarization and frequency-
dependent broadening in rat hippocampal CA1 pyramidal neuro-
nes. J. Physiol. 569, 4157.
Lien C. C. and Jonas P. (2003) Kv3 potassium conductance is necessary
and kinetically optimized for high-frequency action potential
generation in hippocampal interneurons. J. Neurosci. 23, 2058
2068.
Liu L. and Simon S. A. (2003) Modulation of IA currents by capsaicin in
rat trigeminal ganglion neurons. J. Neurophysiol. 89, 13871401.
Malcangio M. and Tomlinson D. R. (1998) A pharmacologic analysis of
mechanical hyperalgesia in streptozotocin/diabetic rats. Pain 76,
151157.
Journal Compilation 2010 International Society for Neurochemistry, J. Neurochem. (2010) 114, 14601475
2010 The Authors
1474 | X.-H. Cao et al.
-
8/2/2019 Reduction in Voltage Gated K Channel_J Neurosci
16/16
Malin S. A. and Nerbonne J. M. (2002) Delayed rectifier K+ currents,
IK, are encoded by Kv2 alpha-subunits and regulate tonic firing in
mammalian sympathetic neurons. J. Neurosci. 22, 1009410105.
McMahon S. B., Armanini M. P., Ling L. H. and Phillips H. S. (1994)
Expression and coexpression of Trk receptors in subpopulations of
adult primary sensory neurons projecting to identified peripheraltargets. Neuron 12, 11611171.
Murakoshi H. and Trimmer J. S. (1999) Identification of the Kv2.1 K+
channel as a major component of the delayed rectifier K+ current in
rat hippocampal neurons. J. Neurosci. 19, 17281735.
Obata K., Yamanaka H., Dai Y., Tachibana T., Fukuoka T., Tokunaga A.,
Yoshikawa H. and Noguchi K. (2003a) Differential activation of
extracellular signal-regulated protein kinase in primary afferent
neurons regulates brain-derived neurotrophic factor expression
after peripheral inflammation and nerve injury. J. Neurosci. 23,
41174126.
Obata K., Yamanaka H., Fukuoka T., Yi D., Tokunaga A., Hashimoto N.,
Yoshikawa H. and Noguchi K. (2003b) Contribution of injured and
uninjured dorsal root ganglion neurons to pain behavior and the
changes in gene expression following chronic constriction injury of
the sciatic nerve in rats. Pain 101, 6577.Ochodnicka E., Ochdnicky M., Belej K., Fusekova E. and Boselova L.
(1995) Quantitative analysis of myelinated nerve fibers of periph-
eral nerve in streptozotocin-induced diabetes mellitus. Mol. Chem.
Neuropathol. 25, 225233.
Oliver D., Lien C. C., Soom M., Baukrowitz T., Jonas P. and Fakler B.
(2004) Functional conversion between A-type and delayed rectifier
K+ channels by membrane lipids. Science 304, 265270.
Park S. Y., Choi J. Y., Kim R. U., Lee Y. S., Cho H. J. and Kim D. S.
(2003) Downregulation of voltage-gated potassium channel alpha
gene expression by axotomy and neurotrophins in rat dorsal root
ganglia. Mol. Cells 16, 256259.
Park K. S., Mohapatra D. P., Misonou H. and Trimmer J. S. (2006)
Graded regulation of the Kv2.1 potassium channel by variable
phosphorylation. Science 313, 976979.
Pongs O. (1999) Voltage-gated potassium channels: from hyperexcit-ability to excitement. FEBS Lett. 452, 3135.
Rasband M. N., Park E. W., Vanderah T. W., Lai J., Porreca F. and
Trimmer J. S. (2001) Distinct potassium channels on pain-sensing
neurons. Proc. Natl Acad. Sci. USA 98, 1337313378.
Robertson B. E. and Nelson M. T. (1994) Aminopyridine inhibition and
voltage dependence of K+ currents in smooth muscle cells from
cerebral arteries. Am. J. Physiol. 267, C1589C1597.
Safronov B. V., Bischoff U. and Vogel W.. (1996) Single voltage-gated
K+ channels and their functions in small dorsal root ganglion
neurones of rat. J. Physiol. 493 (Pt 2), 393408.
Stuhmer W., Ruppersberg J. P., Schroter K. H., Sakmann B., Stocker M.,
Giese K. P., Perschke A., Baumann A. and Pongs O. (1989)
Molecular basis of functional diversity of voltage-gated potassium
channels in mammalian brain. EMBO J. 8, 32353244.
Tapley P., Lamballe F. and Barbacid M. (1992) K252a is a selective
inhibitor of the tyrosine protein kinase activity of the trk
family of oncogenes and neurotrophin receptors. Oncogene 7,
371381.Thompson S. W., Bennett D. L., Kerr B. J., Bradbury E. J. and
McMahon S. B. (1999) Brain-derived neurotrophic factor is an
endogenous modulator of nociceptive responses in the spinal cord.
Proc. Natl Acad. Sci. USA 96, 77147718.
Tonra J. R., Curtis R., Wong V., Cliffer K. D., Park J. S., Timmes A.,
Nguyen T., Lindsay R. M ., Ac heson A. and D iStefano P. S . (19 98)
Axotomy upregulates the anterograde transport and expression of
brain-derived neurotrophic factor by sensory neurons. J. Neurosci.
18, 43744383.
Uchida H., Sasaki K., Ma L. and Ueda H. (2010) Neuron-restrictive
silencer factor causes epigenetic silencing of Kv4.3 gene after
peripheral nerve injury. Neuroscience 166, 14.
Vydyanathan A., Wu Z. Z., Chen S. R. and Pan H. L. (2005) A-type
voltage-gated K+ currents influence firing properties of isolectin
B4-positive but not isolectin B4-negative primary sensory neurons.J. Neurophysiol. 93, 34013409.
Wetmore C. and Olson L. (1995) Neuronal and nonneuronal expression
of neurotrophins and their receptors in sensory and sympathetic
ganglia suggest new intercellular trophic interactions. J. Comp.
Neurol. 353, 143159.
Wu Z. Z., Li D. P., Chen S. R. and Pan H. L. (2009) Aminopyridines
potentiate synaptic and neuromuscular transmission by targeting
the voltage-activated calcium channel beta subunit. J. Biol. Chem.
284, 3645336461.
Yang E. K., Takimoto K., Hayashi Y., de Groat W. C. and Yoshimura N.
(2004) Altered expression of potassium channel subunit mRNA
and alpha-dendrotoxin sensitivity of potassium currents in rat
dorsal root ganglion neurons after axotomy. Neuroscience 123,
867874.
Youssoufian M. and Walmsley B. (2007) Brain-derived neurotrophicfactor modulates cell excitability in the mouse medial nucleus of
the trapezoid body. Eur. J. Neurosci. 25, 16471652.
Zhou X. F. and Rush R. A. (1996) Endogenous brain-derived neuro-
trophic factor is anterogradely transported in primary sensory
neurons. Neuroscience 74, 945953.
Zhou X. F., Chie E. T., Deng Y. S., Zhong J. H., Xue Q., Rush R. A. and
Xian C. J. (1999) Injured primary sensory neurons switch pheno-
type for brain-derived neurotrophic factor in the rat. Neuroscience
92, 841853.
Zurek J. R., Nadeson R. and Goodchild C. S. (2001) Spinal and sup-
raspinal components of opioid antinociception in streptozotocin
induced diabetic neuropathy in rats. Pain 90, 5763.
2010 The Authors
Journal Compilation 2010 International Society for Neurochemistry J Neurochem (2010) 114 14601475
BDNF and K+ channels in diabetic neuropathy | 1475