Dialysis of lactotropes with antisense oligonucleotides assigns guanine nucleotide binding protein subtypes to their channel effectors.
This article describes a new approach for determining the role of endogenous guanine nucleotide binding (G) protein subunits in signal transduction. Sequential patch-clamping was applied to BSA gradient-enriched cultured lactotropes from lactating rats, first to dialyze antisense oligodeoxyribonucleotides (AS) directed against G alpha protein mRNAs and 48 h later to record ion-current responses to the PRL release inhibitor, dopamine. The effectiveness and specificity of action of six types of AS were determined by their effects on the in vitro translation of alpha o, alpha i1, alpha i2, alpha i3, and alpha s. The specificity of AS could be enhanced by replacing guanine by cytosine bases within the center core of AS and by maximizing the number of mismatches against nontargeted mRNAs within the extremities of AS. A total of 59 out of 240 cells could be investigated using the sequential patch clamp procedure in the absence of antibiotics. The typical decrease of the voltage-activated calcium current in response to 10 nM dopamine was diminished or abolished by AS, in correlation with the inhibition of in vitro translation of the alpha o subunit. The typical increase of the voltage-activated potassium current in response to dopamine was abolished by AS directed against alpha i3 but not alpha o mRNA. Control experiments showed that culture conditions or loss of receptor affinity for dopamine were not responsible for the loss of response. The results suggest that dopamine D2 receptors are linked via alpha o to calcium channels and via alpha i3 to potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Baertschi
A J
AJ
Department of Physiology, University of Virginia, Charlottesville 22908.
Audigier
Y
Y
Lledo
P M
PM
Israel
J M
JM
Bockaert
J
J
Vincent
J D
JD
D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D003956:Dialysis; D004298:Dopamine / Q000494:pharmacology*; D005260:Female; D019204:GTP-Binding Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010903:Pituitary Gland, Anterior / Q000166:cytology* / Q000378:metabolism; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D014176:Protein Biosynthesis; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects
D015220:Calcium Channels; D016376:Oligonucleotides, Antisense; D015221:Potassium Channels; D004247:DNA; D019204:GTP-Binding Proteins; D004298:Dopamine