Phe303in TMVI of the α1B-Adrenergic Receptor Is a Key Residue Coupling TM Helical Movements to G-protein Activation†

The effectiveness of β2-agonists in preterm delivery is reduced by several factors. The aim of this study was to determine the influence of late pregnancy in the uterus-relaxing effect of terbutaline in the rat in vitro. Rat uterine tissues from late pregnancy (days 15, 18, 20 and 22) were used. In vitro electrical field-stimulation (EFS) was used to evoke contractions. The radioligand-binding technique, reverse transcription-polymerase chain reaction and radioimmunoassay technique were used to determine the β-adrenergic receptor density and mRNA level and the plasma sex hormone level, respectively. The activated G-protein level of the β-adrenergic receptors was investigated by a radiolabelled GTP binding assay. EFS-induced contractions were inhibited by terbutaline. This effect decreased towards term with respect to both the EC50 and maximal inhibition values. A drop in plasma progesterone level was also detected. Binding studies revealed an increase in β-adrenergic receptor number on the last day of pregnancy, which correlated with the change in receptor mRNA level. The G-protein-activating effect of terbutaline decreased continuously between days 15 and 20. Surprisingly, terbutaline decreased the G-protein activation to below the basal level on day 22. However, progesterone pretreatment set back the uterine action of terbutaline, increased the density of the β2-adrenergic receptors and their mRNA level and increased the G-protein-activating property of terbutaline. These data provide evidence of a pregnancy-induced decrease in activated G-protein level after β2-agonist stimulation. The decrease in plasma progesterone level has a crucial role in this process. The effects of β2-adrenergic receptor agonists in tocolytic therapy may possibly be potentiated with progesterone.

Download Full-text

Faculty Opinions recommendation of Re-examining the 'Dissociation Model' of G protein activation from the perspective of Gβγ signaling.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738875770.793579982 ◽

2020 ◽

Author(s):

Philip Wedegaertner

Keyword(s):

G Protein ◽

Protein Activation ◽

G Protein Activation ◽

Dissociation Model

Download Full-text

Opioid-Activated Postsynaptic, Inward Rectifying Potassium Currents in Whole Cell Recordings in Substantia Gelatinosa Neurons

Journal of Neurophysiology ◽

10.1152/jn.1998.80.6.2954 ◽

1998 ◽

Vol 80 (6) ◽

pp. 2954-2962 ◽

Cited By ~ 49

Author(s):

S. P. Schneider ◽

W. A. Eckert ◽

A. R. Light

Keyword(s):

Membrane Potential ◽

G Protein ◽

Potassium Currents ◽

Substantia Gelatinosa ◽

Opioid Agonist ◽

Membrane Hyperpolarization ◽

Whole Cell ◽

Protein Activation ◽

G Protein Activation ◽

Whole Cell Recordings

Schneider, S. P., W. A. Eckert III, and A. R. Light. Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons. J. Neurophysiol. 80: 2954–2962, 1998. Using tight-seal, whole cell recordings from isolated transverse slices of hamster and rat spinal cord, we investigated the effects of the μ-opioid agonist (d-Ala2, N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO) on the membrane potential and conductance of substantia gelatinosa (SG) neurons. We observed that bath application of 1–5 μM DAMGO caused a robust and repeatable hyperpolarization in membrane potential ( V m) and decrease in neuronal input resistance ( R N) in 60% (27/45) of hamster neurons and 39% (9/23) of rat neurons, but significantly only when ATP (2 mM) and guanosine 5′-triphosphate (GTP; 100 μM) were included in the patch pipette internal solution. An ED50 of 50 nM was observed for the hyperpolarization in rat SG neurons. Because G-protein mediation of opioid effects has been shown in other systems, we tested if the nucleotide requirement for opioid hyperpolarization in SG neurons was due to G-protein activation. GTP was replaced with the nonhydrolyzable GTP analogue guanosine-5′- O-(3-thiotriphosphate) (GTP-γ-S; 100 μM), which enabled DAMGO to activate a nonreversible membrane hyperpolarization. Further, intracellular application of guanosine-5′- O-(2-thiodiphosphate) (GDP-β-S; 500 μM), which blocks G-protein activation, abolished the effects of DAMGO. We conclude that spinal SG neurons are particularly susceptible to dialysis of GTP by whole cell recording techniques. Moreover, the depletion of GTP leads to the inactivation of G-proteins that mediate μ-opioid activation of an inward-rectifying, potassium conductance in these neurons. These results explain the discrepancy between the opioid-activated hyperpolarization in SG neurons observed in previous sharp electrode experiments and the more recent failures to observe these effects with whole cell patch techniques.

Download Full-text