Chinese hamster ovary (CHO) cells, stably transfected with the long form of the prolactin (PRL) receptor (PRL-R) cDNA, were used for PRL-R signal transduction studies. Patch-clamp technique in whole cell and cell-free configurations were employed. Exposure of transfected CHO cells to 5 nM PRL led to the increase of Ca- and voltage-dependent K channel (K) activity. The effect was direct as it was observed also in excised patch experiments. A series of tyrosine kinase inhibitors was studied to investigate the possible involvement of protein tyrosine kinases in K functioning and its stimulation by PRL. Genistein, lavendustin A, and herbimycin A decreased in a concentration and time-dependent manner the amplitude of the K current in whole cell and the open probability of K channels in cell-free experiments. The subsequent application of PRL was ineffective. The protein tyrosine phosphatase inhibitor orthovanadate (1 mM) stimulated K channel activity in excised patches, indicating that channels can be modulated in opposite directions by protein tyrosine kinase and protein tyrosine phosphatase. Moreover, in whole cell experiments as well as in excised patch recordings, anti-JAK2 tyrosine kinase antibody decreased the K conductance and the open probability of the K channels. Subsequent application of PRL was no longer able to stimulate K conductance. Immunoblotting studies using the same anti-JAK2 antibody, revealed the constitutive association of JAK2 kinase with PRL-R. Preincubation of anti-JAK2 antibody with the JAK2 Immunizing Peptide abolished the effects observed using anti-JAK2 antibody alone in both electrophysiological and immunoblotting studies.

We conclude from these findings that these K channels are regulated through tyrosine phosphorylation/dephosphorylation; JAK2 tyrosine kinase, constitutively associated with PRL-R, is implicated in PRL stimulation of K channels.

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