Korean J Physiol Pharmacol.  2016 Sep;20(5):525-531. 10.4196/kjpp.2016.20.5.525.

DAMGO modulates two-pore domain K⁺ channels in the substantia gelatinosa neurons of rat spinal cord

Affiliations
  • 1Department of Biomedical Science, Graduate School of Biomedical Science; Engineering, Hanyang University, Seoul 04763, Korea. eurijj@hanyang.ac.kr

Abstract

The analgesic mechanism of opioids is known to decrease the excitability of substantia gelatinosa (SG) neurons receiving the synaptic inputs from primary nociceptive afferent fiber by increasing inwardly rectifying K⁺ current. In this study, we examined whether a µ-opioid agonist, [D-Ala2,N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), affects the two-pore domain K⁺ channel (K2P) current in rat SG neurons using a slice whole-cell patch clamp technique. Also we confirmed which subtypes of K2P channels were associated with DAMGO-induced currents, measuring the expression of K2P channel in whole spinal cord and SG region. DAMGO caused a robust hyperpolarization and outward current in the SG neurons, which developed almost instantaneously and did not show any time-dependent inactivation. Half of the SG neurons exhibited a linear I~V relationship of the DAMGO-induced current, whereas rest of the neurons displayed inward rectification. In SG neurons with a linear I~V relationship of DAMGO-induced current, the reversal potential was close to the K⁺ equilibrium potentials. The mRNA expression of TWIK (tandem of pore domains in a weak inwardly rectifying K⁺ channel) related acid-sensitive K⁺ channel (TASK) 1 and 3 was found in the SG region and a low pH (6.4) significantly blocked the DAMGO-induced K⁺ current. Taken together, the DAMGO-induced hyperpolarization at resting membrane potential and subsequent decrease in excitability of SG neurons can be carried by the two-pore domain K⁺ channel (TASK1 and 3) in addition to inwardly rectifying K⁺ channel.

Keyword

DAMGO; K⁺ current; Opioid; SG neuron; TASK

MeSH Terms

Analgesics, Opioid
Animals
Enkephalin, Ala(2)-MePhe(4)-Gly(5)-*
Hydrogen-Ion Concentration
Membrane Potentials
Neurons*
Rats*
RNA, Messenger
Spinal Cord*
Substantia Gelatinosa*
Analgesics, Opioid
Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
RNA, Messenger

Figure

  • Fig. 1 The effect of DAMGO on resting membrane potential (RMP) in SG neuron.(A) In current clamp condition, the application of DAMGO (1 µM) caused a reversible hyperpolarization of RMP in SGN (−61.4±1.2 mV, control RMP; −67.6±0.8 mV, DAMGO; n=10). In voltage clamp condition at −60 mV, DAMGO induced an outward current in SG neurons with time course similar to the hyperpolarization (control value of 10.1±4.1 pA and DAMGO 37.8±3.2 pA, n=10, respectively). This experiment was conducted in normal ACSF. (B) The inhibitory effect of naloxone (1 µM) on DAMGO-induced outward current. Data are presented as mean±SEM and asterisks (*)indicated a significant difference at p<0.05 (paired t-test).

  • Fig. 2 The I-V relationship of DAMGO-induced current.The I~V relationship of the DAMGO-induced current was diverse. Currents were activated by ramp pulses (from −120 to −40 mV). Three neurons exhibited the pure inward rectification (IKir). Half of neurons (n=13) exhibited a linear relationship suggesting DAMGO opens IK(L).

  • Fig. 3 The reversal potential of DAMGO-induced current with linear relationship.The reversal potential of DAMGO-induced current at different [K+]O was plotted on a semilog scale. The slope of a regression line (60.2) is close to the value predicted by the Nernst equation (58.8 at 23℃), indicating that the current is through K+-selective channels.

  • Fig. 4 RT-PCR analysis and electrophysiological property of K2P channels in the SG region.(A) The mRNA expression of two pore domain K channels (TREK1, TREK2, TRAAK, and TASK3) was observed in SG. Of TASK family, mRNA expression of TASK1 and 3 was only detected in SG, while TASK2 and 5 were not found. Arrow heads indicate 200 and 300 bp, and each size of RT-PCR products is as follows; β-actin 252 bp, TRAAK 198 bp, TREK1 194 bp, TREK2 201 bp, TASK1 244 bp, TASK2 157 bp, TASK3 194 bp, and TASK5 176 bp. (B) The effect of acidic pH on DAMGO-induced current. Low pH (pH 6.4) blocked DAMGO-induced current and increment of slope conductance by DAMGO was reduced in pH 6.4 condition. (C) The effect of Zn2+ on DAMGO-induced current. In some SG neurons (n=4/7), application of ZnCl2 (100 µM) blocked DAMGO-induced current, while others were not altered by ZnCl2 (n=3/7).


Reference

1. Kumazawa T, Perl ER. Excitation of marginal and substantia gelatinosa neurons in the primate spinal cord: indications of their place in dorsal horn functional organization. J Comp Neurol. 1978; 177:417–434. PMID: 412881.
Article
2. Yoshimura M, Jessell TM. Membrane properties of rat substantia gelatinosa neurons in vitro. J Neurophysiol. 1989; 62:109–118. PMID: 2754464.
Article
3. Merchenthaler I, Maderdrut JL, Altschuler RA, Petrusz P. Immunocytochemical localization of proenkephalin-derived peptides in the central nervous system of the rat. Neuroscience. 1986; 17:325–348. PMID: 3517687.
Article
4. Besse D, Lombard MC, Zajac JM, Roques BP, Besson JM. Pre- and postsynaptic distribution of mu, delta and kappa opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord. Brain Res. 1990; 521:15–22. PMID: 2169958.
5. Gouardères C, Beaudet A, Zajac JM, Cros J, Quirion R. High resolution radioautographic localization of [125I]FK-33-824-labelled mu opioid receptors in the spinal cord of normal and deafferented rats. Neuroscience. 1991; 43:197–209. PMID: 1717884.
Article
6. Faull RL, Villiger JW. Opiate receptors in the human spinal cord: a detailed anatomical study comparing the autoradiographic localization of [3H]diprenorphine binding sites with the laminar pattern of substance P, myelin and nissl staining. Neuroscience. 1987; 20:395–407. PMID: 2438589.
Article
7. Yoshimura M, North RA. Substantia gelatinosa neurones hyperpolarized in vitro by enkephalin. Nature. 1983; 305:529–530. PMID: 6621700.
Article
8. Grudt TJ, Williams JT. mu-Opioid agonists inhibit spinal trigeminal substantia gelatinosa neurons in guinea pig and rat. J Neurosci. 1994; 14:1646–1654. PMID: 8126561.
Article
9. Schneider SP, Eckert WA 3rd, Light AR. Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons. J Neurophysiol. 1998; 80:2954–2962. PMID: 9862898.
Article
10. Rusin KI, Moises HC. mu-Opioid receptor activation reduces multiple components of high-threshold calcium current in rat sensory neurons. J Neurosci. 1995; 15:4315–4327. PMID: 7540671.
11. Taddese A, Nah SY, McCleskey EW. Selective opioid inhibition of small nociceptive neurons. Science. 1995; 270:1366–1369. PMID: 7481826.
Article
12. Seol GH, Kim J, Cho SH, Kim WK, Kim JW, Kim SJ. The inhibitory effect of opioid on the hyperpolarization-activated cation currents in rat substantia gelatinosa neurons. Korean J Physiol Pharmacol. 2001; 5:373–380.
13. Enyedi P, Czirják G. Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol Rev. 2010; 90:559–605. PMID: 20393194.
14. Goldstein SA, Bockenhauer D, O'Kelly I, Zilberberg N. Potassium leak channels and the KCNK family of two-P-domain subunits. Nat Rev Neurosci. 2001; 2:175–184. PMID: 11256078.
Article
15. Blankenship ML, Coyle DE, Baccei ML. Transcriptional expression of voltage-gated Na+ and voltage-independent K+ channels in the developing rat superficial dorsal horn. Neuroscience. 2013; 231:305–314. PMID: 23219908.
16. Duprat F, Lesage F, Fink M, Reyes R, Heurteaux C, Lazdunski M. TASK, a human background K+ channel to sense external pH variations near physiological pH. EMBO J. 1997; 16:5464–5471. PMID: 9312005.
17. Ribeiro-da-Silva A, Pioro EP, Cuello AC. Substance P- and enkephalin-like immunoreactivities are colocalized in certain neurons of the substantia gelatinosa of the rat spinal cord: an ultrastructural double-labeling study. J Neurosci. 1991; 11:1068–1080. PMID: 1707094.
Article
18. Arvidsson U, Dado RJ, Riedl M, Lee JH, Law PY, Loh HH, Elde R, Wessendorf MW. delta-Opioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin. J Neurosci. 1995; 15:1215–1235. PMID: 7532700.
Article
19. Arvidsson U, Riedl M, Chakrabarti S, Lee JH, Nakano AH, Dado RJ, Loh HH, Law PY, Wessendorf MW, Elde R. Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord. J Neurosci. 1995; 15:3328–3341. PMID: 7751913.
Article
20. Kemp T, Spike RC, Watt C, Todd AJ. The mu-opioid receptor (MOR1) is mainly restricted to neurons that do not contain GABA or glycine in the superficial dorsal horn of the rat spinal cord. Neuroscience. 1996; 75:1231–1238. PMID: 8938756.
21. Kerchner GA, Zhuo M. Presynaptic suppression of dorsal horn inhibitory transmission by mu-opioid receptors. J Neurophysiol. 2002; 88:520–522. PMID: 12091574.
22. Glaum SR, Miller RJ, Hammond DL. Inhibitory actions of delta 1-, delta 2-, and mu-opioid receptor agonists on excitatory transmission in lamina II neurons of adult rat spinal cord. J Neurosci. 1994; 14:4965–4971. PMID: 8046463.
Article
23. Talley EM, Lei Q, Sirois JE, Bayliss DA. TASK-1, a two-pore domain K+ channel, is modulated by multiple neurotransmitters in motoneurons. Neuron. 2000; 25:399–410. PMID: 10719894.
24. Berg AP, Talley EM, Manger JP, Bayliss DA. Motoneurons express heteromeric TWIK-related acid-sensitive K+ (TASK) channels containing TASK-1 (KCNK3) and TASK-3 (KCNK9) subunits. J Neurosci. 2004; 24:6693–6702. PMID: 15282272.
25. Kim Y, Bang H, Kim D. TASK-3, a new member of the tandem pore K+ channel family. J Biol Chem. 2000; 275:9340–9347. PMID: 10734076.
26. Morton MJ, O'Connell AD, Sivaprasadarao A, Hunter M. Determinants of pH sensing in the two-pore domain K+ channels TASK-1 and -2. Pflugers Arch. 2003; 445:577–583. PMID: 12634929.
27. Devilliers M, Busserolles J, Lolignier S, Deval E, Pereira V, Alloui A, Christin M, Mazet B, Delmas P, Noel J, Lazdunski M, Eschalier A. Activation of TREK-1 by morphine results in analgesia without adverse side effects. Nat Commun. 2013; 4:2941. PMID: 24346231.
Article
28. Clarke CE, Veale EL, Green PJ, Meadows HJ, Mathie A. Selective block of the human 2-P domain potassium channel, TASK-3, and the native leak potassium current, IKSO, by zinc. J Physiol. 2004; 560:51–62. PMID: 15284350.
Article
29. Kang D, Han J, Talley EM, Bayliss DA, Kim D. Functional expression of TASK-1/TASK-3 heteromers in cerebellar granule cells. J Physiol. 2004; 554:64–77. PMID: 14678492.
Article
30. Torborg CL, Berg AP, Jeffries BW, Bayliss DA, McBain CJ. TASK-like conductances are present within hippocampal CA1 stratum oriens interneuron subpopulations. J Neurosci. 2006; 26:7362–7367. PMID: 16837582.
Article
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