Dopamine D-2-receptor isoforms expressed in AtT20 cells inhibit Q-type high-voltage-activated Ca2+ channels via a membrane-delimited pathway

Dopamine D-2, receptors both acutely and chronically inhibit high-voltage-a ctivated Ca2+ channels (HVA-CCs). Two alternatively spliced isoforms, D-2L (long) and D-2S (short), are expressed at high levels in rat pituitary inte rmediate lobe melanotropes but are lacking in anterior lobe corticotropes. We stably transfected D-2L and D-2S into corticotrope-derived AtT20 cells. Both isoforms coupled to inhibition of Q-type calcium channels through pert ussis toxin-sensitive G proteins. Thus, we have created a model system in w hich to study the kinetics of D-2-receptor regulation of Ca2+ channels. Rap id inhibition of HVA-CCs was characterized using a novel fluorescence Video imaging technique for the measurement of millisecond kinetic events. We me asured the time elapsed (lag time) between the arrival of depolarizing isot onic 66 mM K+, sensed by fluorescence from included carboxy-X-rhodamine (CX R), and the beginning of increased intracellular Ca2+ levels (sensed by cha nges in indo 1 fluorescence ratio). The lag time averaged 350-550 ms, with no significant differences among cell types. Addition of the D-2-agonist qu inpirole (250 mu M) to the K+/CXR solution significantly increased the lag times for D-2-expressing cells but did not alter the lag time for AtT20 con trols. The increased lag times for D-2L- and D-2S-transfected cells suggest that at least a fraction of the Ca2+ channels was inhibited within the ini tial 350550 ms. As this inhibition time is too fast for a multistep second messenger pathway, we conclude that inhibition occurs via a membrane-limite d diffusion mechanism.