Potent stimulation of large‐conductance Ca2+‐activated K+ channels by rottlerin, an inhibitor of protein kinase C‐δ, in pituitary tumor (GH3) cells and in cortical neuronal (HCN‐1A) cells

Abstract

The effects of rottlerin, a known inhibitor of protein kinase C‐δ activation, on ion currents were investigated in pituitary tumor (GH₃) cells. Rottlerin (0.3–100 µM) increased the amplitude of Ca²⁺‐activated K⁺ current (I_K(Ca)) in a concentration‐dependent manner with an EC₅₀ value of 1.7 µM. In intracellular perfusion with rottlerin (1 µM) or staurosporine (10 µM), phorbol 12‐myristate 13‐acetate‐induced inhibition of I_K(Ca) in these cells was abolished. In cell‐attached mode, rottlerin applied on the extracellular side of the membrane caused activation of large‐conductance Ca²⁺‐activated K⁺ (BK_Ca) channels, and a further application of BAPTA‐AM (10 µM) to the bath had no effect on rottlerin‐stimulated channel activity. When cells were exposed to rottlerin, the activation curve of these channels was shifted to less positive potential with no change in the slope factor. Rottlerin increased BK_Ca‐channel activity in outside‐out patches. Its change in kinetic behavior of BK_Ca channels is primarily due to an increase in mean open time. With the aid of minimal kinetic scheme, a quantitative description of rottlerin stimulation on BK_Ca channels in GH₃ cells was also provided. Under current‐clamp configuration, rottlerin (1 µM) decreased the firing of action potentials. I_K(Ca) elicited by simulated action potential waveforms was enhanced by this compound. In human cortical HCN‐1A cells, rottlerin (1 µM) could also interact with the BK_Ca channel to stimulate I_K(Ca). Therefore, rottlerin may directly activate BK_Ca channels in neurons or endocrine cells. J. Cell. Physiol. 210: 655–666, 2007.