Use of Antisense-Generating Plasmids to Probe the Function of Signal Transduction Proteins in Primary Neurons

Abstract

Investigation of the function of intracellularly located protein components of receptor-effector transduction pathways has been hampered not only by their inaccessibility, but also by the lack of specific tools. The problem is particularly acute when the effect of interest must be measured at the single cell level, for example, receptor modulation of ion channels. Assignment of different G protein subunits to roles in ion channel modulation by receptors progressed greatly with the availability of antibodies raised against peptide sequences corresponding to the carboxyl-terminus of Gα-subunits. This is the part of the a-subunit molecule thought to interact with effector, so these antibodies were predicted to indirectly occlude receptor-effector interaction. Indeed, we found that injection of an antibody against Gα₀, into sympathetic neurons inhibited α₂-adrenoceptor effects on Ca²⁺ current, and antibody against Gα_q/11 inhibited muscarinic and bradykinin receptor effects on the M-type K⁺ current (1,2). However, this approach is seriously limited by the close sequence homologies between members of G protein a-subunit subfamilies (3), which results in the antibodies being unable to discriminate between members in many cases.