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

• Cho PS
• Lee HK
• Lee SH
• Im JZ
• Jung SJ

• KMID: 2350509
• DOI: http://doi.org/10.4196/kjpp.2016.20.5.525

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).

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