Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment

Abstract

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than E k . Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector.

Keywords: GABAB receptor; GAT-1; opioid; periaqueductal gray; withdrawal.

Figure 1

Opioid receptors simultaneously couple to a potassium conductance and a GAT-1 conductance. (A) Example trace of currents from a neuron voltage clamped at −56 mV (drug superfusion shown by bars). MENK (30 μM) induced an outward current in neuron from a morphine-treated mouse. (B,C) Currents produced by voltage steps from −56 mV to −136 mV in −10 mV increments in a neuron from (B) a vehicle-treated mouse and (C) a morphine-treated mouse before (left) and during MENK (30 μM) application (right). (D) Subtracted current-voltage relationships for MENK (current in MENK—current during control conditions). Reversal potentials were determined at the point where they cross the abscissa. The MENK current reversed polarity near E_K in neurons from vehicle-treated (n = 4), but not in neurons from morphine-treated mice (n = 5).

Figure 2

GABA_B receptors do not couple to a GAT-1 conductance. (A) Example trace of currents from a neuron voltage clamped at −56 mV (drug superfusion shown by bars). Baclofen (10 μM) induced an outward current in a neuron from a morphine-treated mouse. (B,C) Currents produced by voltage steps from −56 mV to −136 mV in −10 mV increments in a neuron from (B) a vehicle-treated mouse and (C) a morphine-treated mouse before (left) and during baclofen (10 μM) application (right). (D) Subtracted current-voltage relationships for baclofen (current in baclofen–current during control conditions). Reversal potentials were determined at the point where they cross the abscissa. The baclofen current reversed polarity near E_Kin neurons from vehicle-treated (n = 8), and morphine-treated mice (n = 17). (E) Reversal potential of the MENK and baclofen-induced currents in cells from vehicle-treated and chronic morphine-treated mice. Arrows indicate that the average current did not reverse polarity at the most negative potential that could be tested (−136 mV). The number of neurons is shown beside the bar. ^*P-value of 0.034, Students t-test.