EARLY EFFECTS OF PRL ON ION CONDUCTANCES IN CHO CELLS EXPRESSING PRL RECEPTOR

Chinese hamster ovary (CHO-K1) cells were stably transfected with prol actin (PRL) receptor cDNA. These cells (CHO-E32) expressed the long fo rm of functional PRL receptor. Using microfluorimetric and patch-clamp techniques, we have investigated the effects of PRL on intracellular Ca2+ concentration ([Ca2+](i)) and membrane ion conductances. Exposure of CHO-E32 cells to 5 nM PRL resulted in an increase in [Ca2+](i). Tw o types of response were observed: 1) a stimulation of Ca2+ entry and 2) an intracellular Ca2+ mobilization. As PRL inhibited voltage-activa ted Ca2+ current, the PRL-induced Ca2+ increase does not involve volta ge-activated Ca2+ channels. PRL also increased a charybdotoxin-sensiti ve Ca2+-dependent K+ conductance. Simultaneous measurements showed tha t PRL hyperpolarized the membrane potential before increasing intracel lular Ca2+ levels. In voltage clamp, hyperpolarizing voltage steps wer e associated with increased Ca2+ concentrations, whereas depolarizing voltage steps decreased [Ca2+](i). Cell-free patch-clamp experiments s howed that PRL directly stimulates K+ channel activity. Our results su ggest the existence of a regulatory complex involving a protein kinase tightly associated with the Ca2+-activated K+ channels and that PRL s timulates these channels by means of the activation of protein kinase. The resulting hyperpolarization stimulates Ca2+ entry, probably throu gh voltage-insensitive nonspecific channels.