Slob, a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium channel, was identified with a yeast two-hybrid screen. Slob and dSlo coimmunoprecipitate from Drosophila heads and heterologous host cells, suggesting that they interact in vivo. Slob also coimmunoprecipitates with the Drosophila EAG potassium channel but not with Drosophila Shaker, mouse Slowpoke, or rat Kv1.3. Confocal fluorescence microscopy demonstrates that Slob and dSlo redistribute in cotransfected cells and are colocalized in large intracellular structures. Direct application of Slob to the cytoplasmic face of detached membrane patches containing dSlo channels leads to an increase in channel activity. Slob may represent a new class of multi-functional channel-binding proteins.

Neurotransmitter and hormone regulation of cellular function can result from a concomitant stimulation of different signaling pathways. Signaling cascades are strongly regulated during disease and are often targeted by commonly used drugs. Crosstalk of different signaling pathways can have profound effects on the regulation of cell excitability. Members of all the three main structural families of potassium channels: inward-rectifiers, voltage-gated and 2-P domain, have been shown to be regulated by direct phosphorylation and Gq-coupled receptor activation. Here we test members of each of the three families, Kir3.1/Kir3.4, KCNQ1/KCNE1 and TREK-1 channels, all of which have been shown to be regulated directly by phosphatidylinositol bisphosphate (PIP2). The three channels are inhibited by activation of Gq-coupled receptors and are differentially regulated by protein kinase A (PKA). We show that Gq-coupled receptor regulation can be physiologically modulated directly through specific channel phosphorylation sites. Our results suggest that PKA phosphorylation of these channels affects Gq-coupled receptor inhibition through modulation of the channel sensitivity to PIP2.

A large number of ion channels maintain their activity through direct interactions with phosphatidylinositol bisphosphate (PIP 2 ). For such channels, hydrolysis of PIP 2 causes current inhibition. It has become controversial whether the inhibitory effects on channel activity represent direct effects of PIP 2 hydrolysis or of downstream PKC action. We studied Phospholipase C (PLC)-dependent inhibition of G protein-activated inwardly rectifying K + (Kir3) channels. By monitoring simultaneously channel activity and PIP 2 hydrolysis, we determined that both direct PIP 2 depletion and PKC actions contribute to Kir3 current inhibition. We show that the PKC-induced effects strongly depend on PIP 2 levels in the membrane. At the same time, we show that PKC destabilizes Kir3/PIP 2 interactions and enhances the effects of PIP 2 depletion on channel activity. These results demonstrate that PIP 2 depletion and PKC-mediated effects reinforce each other and suggest that both of these interdependent mechanisms contribute to Kir3 current inhibition. This mechanistic insight may explain how even minor changes in PIP 2 levels can have profound effects on Kir3 activity. We also show that stabilization of Kir3/PIP 2 interactions by Gbg attenuates both PKC and Gq-mediated current inhibition, suggesting that diverse pathways regulate Kir3 activity through modulation of channel interactions with PIP 2 .

Cyclin D1 repression of STAT3 expression may explain the association between cyclin D1 overexpression and improved outcome in breast cancer. In addition, bortezomib can amplify the proapoptotic function of cyclin D1, raising the possibility that cyclin D1 levels may be a marker for predicting the response to this novel drug.