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Introduction
The G protein-coupled inwardly rectifying potassium
channel (GIRK) is a member of the inwardly rectifying
potassium (K+) channel family. Five GIRK subunits have been
identified in mammals and Xenopus oocytes, named
GIRK1_5 (also designated Kir3.1_5)[1]. GIRK channels are
homotetra-mers or heterotetramers. In vivo, GIRK1 has to assemble
with the other GIRK family members to form functional
channels[1]. GIRK channels are downstream effectors of G
protein-coupled receptors, and they can be activated by direct
binding of the βγ subunits of G protein to the
channels[2_4]. A large number of neuromodulators are capable of opening
GIRK channels in a G protein-dependent,
membrane-delimited fashion, including adenosine, serotonin, dopamine,
adrenalin, acetylcholine, opioids, and
somatostatin[5_7]. GIRK channels are distributed in the pancreas, heart and brain,
and play pivotal roles in controlling insulin release, vascular
tone, heart rate, neuronal signaling, and membrane
excitability[8]. For example, the activation of the heart muscarinic
m2 receptor slows the heart rate by activating GIRK1/GIRK4
heterotetrameric channels[9,10]. In the hippocampus, baclofen
can activate GIRK channels, which produce a reversible
reduction of EPSP[11].
Dorsal root ganglion (DRG) neurons are the primary
neurons for somatic and visceral afferentation. Their
bodies reside in the DRG, and extend axons
that innervate both peripheral and central targets. A diverse
group of ligand- and voltage-gated ion channels transduce
innocuous and noxious (nociceptive) stimuli into depolarizations that
are conducted along axons and finally converted into
neurotransmitter release[12]. There are many G protein-coupled
receptors on DRG neurons, such as the μ opioid
receptor[13], the GABAB
receptor[14], the oxytocin
receptor[15],
SNSR4/mrgX1[16], etc. The activation of these receptors can affect the
conductance of DRG neurons. For example, the activation of
human sensory neuron-specific G protein-coupled receptors
(SNSR), expressed solely in small diameter primary sensory
neurons, modulates neuronal K+ channels and synaptic
transmission[16]; clonidine, an
α2-adrenoceptor agonist, inhibits hyperpolarization-activated currents in rat DRG
neurons[17].
Although GIRK channels are very important to mediate
signaling through G protein-coupled receptors, no data
concerning the expression of functional GIRK channels in adult
DRG neurons has been reported. In the present report, we
show that 4 subtypes of GIRK channel subunits are
expressed on DRG neurons, and the GIRK channels are
functionally coupled to the GABAB receptor.
Materials and methods
RT-PCR analysis The total RNA from these DRG was
extracted using the RNA-Clean kit (TaKaRa, Dalian, China)
and treated with DNase I (RNase-free from TaKaRa, Dalian,
China). cDNA were synthesized using the M-MLV RTase
cDNA Synthesis Kit (TaKaRa, China). PCR was performed
using the following primer pairs: GIRK1, forward
5'-CGGCA-GCGGTTCGTGGACAAG-3' and reverse
5'-TGGCTGGTGC-TATTAAAGGGGAAGACAT-3'; GIRK2, forward
5'-AGCC-GAGACAGGACCAAAAGGAAAATC-3' and reverse 5'-ACGGGGTGCTGGTCTCATAGGTCTC-3'; GIRK3, forward
5'-CTACCGCTACCTGACCGACCTGTT-3' and reverse 5'-AGCCCCTTCTTCCTCCACCTTCT-3'; GIRK4, forward
5'-GGAGAAGACCGGCAAGTGTAACG-3' and reverse 5'-GCCCCCAAGCAAAGGAGGAC-3'. For GIRK1, PCR
amplification was performed for 35 cycles using Taq polymerase
(TaKaRa, China), each cycle consisted of 30 s at 94 °C, 30 s at
60 °C, and 1 min at 72 °C. For GIRK2, PCR amplification was
performed for 35 cycles using LA Taq polymerase and buffer I
(TaKaRa, China), each cycle consisted of 40 s at 94 °C, 40s at
60 °C, and 1 min at 72 °C. For GIRK3, PCR amplification was
performed for 35 cycles using LA Taq polymerase and
buffer II (TaKaRa, China), each cycle consisted of 40 s at 94
°C, 40 s at 60 °C, and 1 min at 72 °C. For GIRK4, PCR amplification was
performed for 35 cycles using Taq polymerase (TaKaRa,
China), each cycle consisted of 40 s at 94 °C, 40 s at 58 °C,
and 1 min at 72 °C. The amplified samples were analyzed by
standard agarose gel electrophoresis. DL-2000 DNA marker
(TaKaRa, China) or BenchTop 1 kb DNA ladder (Promega,
Madison, Wisconsin, USA) was used. All the products were
further confirmed by DNA sequencing.
Preparation of isolated DRG The animal experiments
were carried out in adherence with the National Institutes
of Health Guidelines on the Use of Laboratory Animals and
were approved by Second Military Medical University
Committee on Animal Care. Adult male Sprague-Dawley rats
(150_200 g), from the Experimental Animal Center in
Second Military Medical University, were housed with access
to food and water ad libitum under standard conditions in a
12/12 light dark cycle. The adult rats were deeply
anaesthetized with 40 mg/mL sodium pentobarbital and killed by
decapitation. The DRG were removed and digested in
solution containing 1 mg/mL collagenase type 1A (Sigma, St
Louis, MO, USA), 0.4 mg/mL trypsin type I (Sigma, USA),
and 0.1 mg/mL Dnase I (Sigma, USA) in DMEM at 37 °C
for 40 min, triturated, and plated on coverslips. The cells
were observed with an Olympus IX71 inverted microscope
(Olympus Optical, Tokyo, Japan).
Whole cell patch-clamp recording The cells were bathed
in normal Ringer's solution containing (in mmol/L): NaCl,
140; KCl, 5; CaCl2, 2; MgCl2, 1; glucose, 10 and HEPES,
10; pH 7.4 adjusted with NaOH. When an increase in
[K+]o was needed, NaCl was reduced by equal amounts to keep the
osmolarity unchanged. Patch pipettes had a resistance
of 6_8 MΩ. The pipette solution consisted of (in
mmol/L): KCl, 120; NaCl, 20; HEPES, 10;
glucose·H2O, 10; EGTA, 1;
Na2ATP·3H2O, 3;
MgCl2·6H2O, 3; NaGTP, 0.3; pH 7.2 adjusted
with NaOH. Recordings were made with an Axon-patch 200B
amplifier and pClampex 9.0 software (Axon Instruments,
Sunnyvale, CA, USA). Analogue signals were filtered at 2
kHz, sampled at 10 kHz and stored on a PC hard disk for
further analysis. Baclofen and GTPγS used in the
experiments were from Sigma. Data analysis was performed using
Clampfit 9.0 (Axon Instruments, USA) and Origin 7.0
(OriginLab Corporation, Northampton, MA, USA). Data were
all presented as mean±SD.
Results
GIRK channel subunits in rat DRG In the RT-PCR
analysis of total RNA extracted from DRG, the
oligonucleotide primers for GIRK1-4 showed amplification of
products of expected sizes (Figure 1). These results
demonstrated that the mRNA for the 4 subunits of GIRK were
expressed in the adult DRG.
GIRK currents in DRG neurons To determine whether
the GIRK subunits in the DRG neurons contributed to form
functional channels, voltage-dependent whole-cell currents
from isolated DRG neurons were recorded and characterized.
After dissociation procedures, DRG neurons dendrites might
have been destroyed, and only neurons with a thin axon
were selected to be tested. Neurons retained their active
properties after isolation as demonstrated by the
voltage-clamp protocol. Successive depolarizing voltage steps
evoked typical sequences of fast inward (presumed
Na+) currents followed by slow outward (presumed
K+) currents (Figure 2).
Figure 3A shows the voltage-dependent currents induced
from a DRG neuron when the cell membrane potential was
changed by a ramp protocol from +90 mV to -190 mV, for
900 ms in Ringer's solution containing 5, 10, 20, and 45
mmol/L K+. The currents were clearly nonlinear, showing
inward rectification. They increased in size in a
concentration-dependent manner when
[K+]o was raised. Furthermore,
the reversal potential was shifted as predicted by the Nernst
equation (Figure 3B). These results strongly suggest that
K+ was the dominant current carrier.
Ba2+ can block Kir channel currents in a dose-dependent
manner. Figure 4A shows the effects of 300 µmol/L
Ba2+ on the inwardly rectifying currents in a DRG neuron induced by
a ramp voltage change from 0 mV to -90 mV in 45 mmol/L
K+-containing Ringer's solution.
Ba2+ 300 µmol/L significantly reduced the basal inward currents at 80 mV by 23%±10%
(n=6; Figure 4B). This result implies that the inward currents
mediated by inwardly rectifying K+ channels existed.
GABAB receptors have been reported to be highly
expressed in DRG neurons[14]. To test whether the inwardly
rectifying currents in the DRG were regulated by the G
protein-coupled receptor, we used the
GABAB receptor agonist baclofen to activate the receptor in DRG neurons. Figure
5A, 5B show that the inward currents were markedly increased in magnitude with time following the perfusion of
100 µmol/L baclofen. An increase of 260%±140%
(n=6) in the current obtained at -80 mV was found with 100 µmol/L
baclofen (Figure 5C). The currents enhanced by baclofen
were considerably suppressed by addition of 300 µmol/L
Ba2+ (Figure 5A, 5B). The currents obtained in the presence
of 300 µmol/L Ba2+ were actually smaller in magnitude than
the basal currents (Figure 5A_5C), which may be due to the
fact that Ba2+ also suppressed the intrinsic fraction currents
mediated by the Kir channels.
To further confirm that the activation of G protein can
activate the inwardly rectifying currents of the DRG neurons,
changes in inward currents were studied when guanosine
triphosphate (GTP) in the pipette solution was omitted and
replaced with 300 µmol/L GTPγS, a nonhydrolysable GTP
analogue which can activate the G
protein[18]. Figure 6A shows the inward currents markedly increased in magnitude with
time following the inclusion of 300 µmol/L
GTPγS. An increase of 136%±38%
(n=6) in the current obtained at -80 mV was found with 300 µmol/L
GTPγS included in the pipette (Figure 6B). The currents enhanced by
GTPγS were considerably suppressed by the addition of 300 µmol/L
Ba2+ (Figure 6A, 6B). These data demonstrated that the GIRK channels
expressed in DRG neurons contributed to the inward
currents activated by G proteins.
Discussion
Besides the heart and pancreas, GIRK distributes in the
nervous system. Earlier studies have demonstrated
GIRK1_4 mRNA distribute distinctly in rat
brains[19_21]. In adult mice, GIRK2-like immunostaining was detected in lamina II in all
levels of the spinal cord, but not in the
DRG[22], suggesting that functional GIRK channels of adult mouse DRG neurons
were composed of other GIRK subunits except GIRK2.
Furthermore, Kanjhan et al reported that no functional GIRK
currents were recorded in P0 mouse DRG
neurons[23], and they hypothesized that the functional expression of GIRK
channels might increase with exposure to sensory of painful
stimuli during postnatal development. In rats, it has been
shown that GIRK1_3 mRNA are localized in DRG neurons at
embryonic d 17[24]. In the present study, we demonstrated
the transcription of GIRK1_4 in adult rat DRG neurons. Our
results indicate that GIRK1_3 mRNA may continuously
transcript from embryonic day to adult time in rat DRG neurons,
suggesting that the 4 subunits are all candidates for
functional GIRK channels in the DRG neurons of adult rats, and
also indicating that the subunit compositions of GIRK
channels may be changed with different developmental stages in
different species.
GIRK channels are widely regarded as postsynaptic effectors in the CNS, primarily because of the clear
dissociation of the postsynaptic and presynaptic inhibitory effects
of multiple neurotransmitters in hippocampal pyramidal
neurons[25]. GIRK1 and GIRK2 were also found almost
exclusively in postsynaptic membranes of putative excitatory
synapses in the outer layer of the substantia gelatinosa of the
dorsal horn[26]. The activation of postsynaptic GIRK1
and/or GIRK2-containing channels in the spinal cord dorsal horn
represents a powerful means by which intrathecal µ- and
δ-selective opioid agonists evoke
analgesia[26]. In the present study, GIRK channels are found functionally expressed in
DRG neurons, producing further molecular bases in the
regulation of neuronal excitation which modulates sensory information conveying from the periphery to the spinal cord.
GABA can depolarize A- and C-type cells of DRG and
decrease input resistance, mainly due to the activation of
GABAA receptors and anionic ionotropic receptors expressed
in rat DRG neurons[27]. The
GABAB receptor has also been reported to be present in DRG neurons and play important
physiological roles[14,28,29], and to modulate N-type calcium
channels through G(o)
pathways[14,28_30]. GABA inhibits peptide secretion through the activation of
GABAB receptors that are functionally coupled to pertussis
toxin-sensitive G proteins in peripheral sensory neurons from
embryonic chick DRG[31]. In the present study, we found the
presence of GABAB receptor-GIRK channel complexes on DRG
neurons. These findings provide possible mechanism
underlying presynaptic inhibition through the activation of
GABAB receptors in peripheral sensory neurons.
Conclusion
The mRNA for GIRK1_4 were detected in the DRG of
adult rats. GTPγS enhanced inwardly rectifying
K+ currents of the DRG neurons, while
Ba2+ inhibited such currents. The GIRK channels were shown to couple to
GABAB receptors as baclofen increased the inwardly rectifying
K+ currents. These results demonstrate that the expression of GIRK
channels and the presence of GABAB receptor-GIRK channel
complexes in DRG neurons of adult rats.
Acknowledgements
We thank Dr Xu ZHANG and Dr Lan BAO in the
Shanghai Institutes for Biological Science for their helpful
suggestions and comments.
References
1 Mark, MD, Herlitze S. G-protein mediated gating of
inward-rectifier K+ channels. Eur J Biochem 2000; 19: 5830_6.
2 Logothetis DE, Kurachi Y, Galper J, Neer EJ, Clapham DE. The
beta gamma subunits of GTP-binding proteins activate the
muscarinic K+ channel in heart. Nature 1987; 6102: 321_6.
3 He C, Zhang H, Mirshahi T, Logothetis DE. Identification of a
potassium channel site that interacts with G protein beta gamma
subunits to mediate agonist-induced signaling. J Biol Chem 1999;
18: 1251_74.
4 He C, Yan X, Zhang H, Mirshahi T, Jin T, Huang A,
et al. Identification of critical residues controlling G protein-gated
inwardly rectifying K+ channel activity through interactions with
the beta gamma subunits of G proteins. J Biol Chem 2002; 8:
6088_96.
5 Sodickson DL, Bean BP. Neurotransmitter activation of
inwardly rectifying potassium current in dissociated hippocampal
CA3 neurons: Interactions among multiple receptors. J Neurosci
1998; 20: 8153_62.
6 Yamada M, Inanobe A, Kurachi Y. G protein regulation of
potassium ion channels. Pharmacol Rev 1998; 4: 723_57.
7 Karschin A. G protein regulation of inwardly rectifying
K+ channels. News Physiol Sci 1999; 14: 215_20.
8 Hille B. Ionic channels of excitable membranes, 2nd ed.
Sunderland, MA: Sinauer Associates; 1992.
9 Krapivinsky G, Gordon EA, Wickman K, Velimirovic B,
Krapivinsky L, Clapham DE. The G-protein-gated atrial
K+ channel IKACh is a heteromultimer of two inwardly rectifying
K+ channel proteins. Nature 1995; 6518: 135_41.
10 Wickman K, Nemec J, Gendler SJ, Clapham DE. Abnormal heart
rate regulation in GIRK4 knockout mice. Neuron 1998; 1:
103_14.
11 Tomoko T, Christian A. Phasic and tonic attenuation of EPSPs
by inward rectifier K+ channels in rat hippocampal pyramidal
cells. J Physiol 2002; 539 (Pt 1): 67_75.
12 McCleskey EW, Gold MS, Ion channels of nociception. Annu
Rev Physiol 1999; 61: 835_56.
13 Shaqura MA, Zollner C, Mousa SA, Stein C, Schafer M.
Characterization of µ opioid receptor binding and G protein coupling in
rat hypothalamus, spinal cord, and primary afferent neurons
during inflammatory pain. J Pharmacol Exp Ther 2004; 2:
7121_8.
14 Towers S, Princivalle A, Billinton A, Edmunds M, Bettler B,
Urban L, et al. GABAB receptor protein and mRNA distribution
in rat spinal cord and dorsal root ganglia. Eur J Neurosci 2000; 9:
3201_10.
15 Yang Q, Wu ZZ, Li X, Li ZW, Wei JB, Hu QS. Modulation by
oxytocin of ATP-activated currents in rat dorsal root ganglion
neurons. Neuropharmacology 2002; 5: 910_6.
16 Chen H, Ikeda SR. Modulation of ion channels and synaptic
transmission by a human sensory neuron-specific
G-protein-coupled receptor, SNSR4/mrgX1, heterologously expressed in
cultured rat neurons. J Neurosci 2004; 21: 5044_53.
17 Yagi J, Sumino R. Inhibition of a hyperpolarization-activated
current by clonidine in rat dorsal root ganglion neurons. J
Neurophysiol 1998; 3: 1094_104.
18 Andrade R, Malenka RC, Nicoll RA. A G protein couples
serotonin and GABAB receptors to the same channels in hippocampus.
Science 1986; 4781: 1261_5.
19 Karschin CW, Schreibmayer N, Dascal H, Lester N, Davidson A.
Karschin. Distribution and localization of a G protein-coupled
inwardly rectifying K+ channel in the rat. FEBS Lett 1994; 348:
139_44.
20 Li JH, You ZD, Song CY, Lu CL, He C. The expression of
G-protein-gated inwardly rectifying K+ channels GIRK1 and GIRK2
mRNAs in the supraoptic nucleus of the rat and possible role
involved. Neuroreport 2001; 5: 1007_10.
21 Karschin C, Dissmann E, Stuhmer W, Karschin A. IRK(1_3) and
GIRK(1_4) inwardly rectifying K+ channel mRNAs are
differentially expressed in the adult rat brain. J Neurosci 1996; 11:
3559_70.
22 Mitrovic I, Margeta-Mitrovic M, Bader S, Stoffel M, Jan LY,
Basbaum AI. Contribution of GIRK2-mediated postsynaptic
signaling to opiate and alpha2-adrenergic analgesia and analgesic sex
differences. Proc Natl Acad Sci USA 2003; 100: 271_6.
23 Kanjhan R, Coulson EJ, Adams DJ, Bellingham MC. Tertiapin-Q
blocks recombinant and native large conductance
K+ channels in a use-dependent manner. J Pharmacol Exp Ther 2005; 314:
1353_61.
24 Karschin C, Karschin A. Ontogeny of gene expression of Kir
channel subunits in the rat. Mol Cell Neurosci 1997; 10:
131_48.
25 Luscher C, Jan LY, Stoffel M, Malenka RC, Nicoll RA. G protein
coupled inwardly rectifying K+ channels (GIRKs) mediate
postsynaptic but not presynaptic transmitter actions in hippocampal
neurons. Neuron 1997; 19: 687_95.
26 Marker CL, Lujan R, Loh HH, Wickman K. Spinal
G-protein-gated potassium channels contribute in a dose-dependent manner
to the analgesic effect of mu- and delta- but not kappa-opioids. J
Neurosci 2005; 25: 3551_9.
27 Williams J, Zieglgansberger W. Mature spinal ganglion cells are
not sensitive to opiate receptor mediated actions. Neurosci Lett
1981; 21: 211_6.
28 Menon-Johansson AS, Berrow N, Dolphin AC. G(o) transduces
GABAB-receptor modulation of N-type calcium channels in
cultured dorsal root ganglion neurons. Pflugers Arch 1993; 425:
335_43.
29 Tatebayashi H, Ogata N. Kinetic analysis of the
GABAB-mediated inhibition of the high-threshold
Ca2+ current in cultured rat sensory neurons. J Physiol 1992; 447: 391_407.
30 Green KA, Cottrell GA. Actions of baclofen on components of
the Ca-current in rat and mouse DRG neurons in culture. Br J
Pharmacol 1988; 94: 235_45.
31 Holz GG IV, Kream RM, Spiegel A, Dunlap K. G proteins couple
alpha-adrenergic and GABAb receptors to inhibition of peptide
secretion from peripheral sensory neurons. J Neurosci 1989; 9:
657_66.
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