Dialysis of lactotropes with antisense oligonucleotides assigns guanine nucleotide binding protein subtypes to their channel effectors

Abstract

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 Gα 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 αo, αi1, αi2, αi3, and αs. 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 αo subunit. The typical increase of the voltage-activated potassium current in response to dopamine was abolished by AS directed against αi3 but not αo 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 αo to calcium channels and via αi3 to potassium channels. This approach may be applied to any cultured cell to study the roles of specific members of a protein family. Copyright © 1992 by The Endocrine Society.